Embed Size (px)
Citation preview
Mag.rer.nat. Hannes Schleifer
Molecular Mechanisms involved in
TRPC/NFAT-mediated Gene Expression
Dissertation
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
an der Naturwissenschaftlichen Fakultät
der Karl-Franzens-Universität Graz
Betreut von ao.Univ.-Prof. Mag.pharm. Dr. Klaus Groschner
Durchgeführt am
Institut für Pharmazeutische Wissenschaften
Department Pharmakologie und Toxikologie
der Karl-Franzens-Universität Graz
von Oktober 2007 bis März 2012
eingereicht 2012
Mein besonderer Dank gilt …
… ao.Univ.-Prof. Mag. Dr. Klaus Groschner, vor allem für die kompetente Betreuung, die
Bereitstellung des Themas und die wissenschaftliche Anleitung.
… Herrn o.Univ.-Prof. Dr. Bernhard-Michael Mayer für die Bereitstellung des Arbeitsplatzes.
… Petra, Annarita, „Mike“, Renate, „Michi S.“, „Michi L.“, Sarah, Zora, Bernhard sowie allen
nicht genannten Kolleginnen und Kollegen der Arbeitsgruppe für Ionenkanal-Pharmakologie
und -Physiologie für ihre Unterstützung, Ratschläge und Hilfe, anregende Diskussionen, ihre
Motivation und die gute Zusammenarbeit.
… allen weiteren Institutsangehörigen, Kolleginnen und Kollegen für das gute Arbeitsklima.
… dem FWF für die Finanzierung meiner Dissertation (Projekt P21925-B19 & P21118-B09).
… all meinen Freunden und jenen Menschen, die das Leben in Graz und an allen anderen
Orten und Gelegenheiten - besonders auch abseits der Universität - lebenswert gemacht
haben.
sowie vor allem
… meiner Familie für den bedingungslosen Rückhalt und die Unterstützung während meiner
Ausbildung
und
… meiner Partnerin Birgit für unzählige wunderschöne Stunden.
No, try not. Do or do not.
There is no try.
(Yoda in Star Wars Episode V – The Empire Strikes Back)
Table of contents
Introduction ................................................................................................................................ 8
Calcium as cellular (second) messenger .............................................................................................. 8
TRPC channels ..................................................................................................................................... 8
TRPC3 a prototypical example of TRPC channels .............................................................................. 11
CRAC – from TRPs to STIM1/Orai1 .................................................................................................... 14
Blockers of non-voltage gated Ca2+ channel ..................................................................................... 17
Aim of the thesis....................................................................................................................... 19
Applied methods ...................................................................................................................... 20
Results ...................................................................................................................................... 21
PKC-dependent coupling of calcium permeation through transient receptor potential
canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes. ....................................................... 21
Novel pyrazole compounds for pharmacological discrimination between receptor-operated and
store-operated Ca2+ entry pathways. ................................................................................................ 38
TRPC3 links Orai1-mediated Ca2+ entry to CaN/NFAT activation in RBL mast cells .......................... 73
Discussion, conclusions & outlook ........................................................................................... 94
Insights into key determinants of TRPC3 structure/function relationship ....................................... 94
New pyrazole derivatives as TRPC3 and Orai1 blockers ................................................................... 96
Effects of changes in TRPC3 function on downstream cellular signalling ......................................... 97
Abbreviations ........................................................................................................................... 99
List of Figures .......................................................................................................................... 100
References to database entries and software ....................................................................... 100
References .............................................................................................................................. 101
VI
Abstract
Ca2+ is involved in the control of numerous cellular processes, ranging from secretion and
generation of mediators to gene transcription and cell proliferation. Ca2+ entry can be
initiated via lipid messenger-dependent receptor-operated entry (ROCE) or an intracellular
Ca2+ store filling state-dependent, store-operated entry pathway (SOCE). Aside voltage-gated
channels, TRPCs and Orai-channels are key proteins mediating these processes.
Aberrancies in Ca2+ entry can lead to disorders like nephritic diseases, progressing
malfunctions of motoric coordination, cardiac pathologies or immune deficiencies. TRPC3
represents a prototypical TRPC cation channel that is potentially involved in ROCE as well as
SOCE. Thus, understanding its function and specifically its crosstalk with Orai1, the key pore
forming protein mediating SOCE, is expected to lead to novel, efficient strategies for the
treatment of pathological conditions based on dysfunction of cellular Ca2+ handling.
An approach combining molecular modelling with site-directed mutagenesis of TRPC3 led to
the identification of a single amino acid residue in the putative pore region, which is
essential for Ca2+ permeation. The TRPC3E630Q mutation reduced divalent permeability of the
channel by more than two orders of magnitude and a TRPC3E630K mutation was found to
eliminate cation permeability completely. Both mutations were found to act in a dominant-
negative way on endogenous TRPCs.
Pharmacological modulation of TRPC3 and Orai1 was characterized for several pyrazoles. A
compound designated as Pyr6, potently and selectively blocked Orai1-mediated SOCE while
a newly synthesised structure, termed Pyr10, displayed selectivity for block of TRPC3. Using
these novel pharmacological and genetic tools, evidence was obtained for a microdomain
signalplex, which includes TRPC3 either as a key scaffold protein or as a signalling
component essential for both Ca2+ entry and targeting of essential Ca2+ signalling
components into regulatory membrane and transcriptionally active microdomains of RBL-
2H3 mast cells as well as HL-1 cardiac myocytes. The identified scaffold function of TRPC3
was found to be dynamically regulated by reversible phosphorylation by PKC.
These results shed light on the molecular function and the (patho)physiological role of
TRPC3.
VII
Kurzzusammenfassung
Ca2+ kontrolliert eine Anzahl zellulärer Prozesse, die von Sekretion über Produktion von Botenstoffen
bis hin zu Gentranskription und Zellproliferation reichen. Der Ca2+-Einstrom kann als ein durch Lipid-
Mediatoren ausgelöster, rezeptorabhängiger (ROCE) oder als ein speicherregulierter, vom
Füllungszustand intrazellulärer Ca2+-Speicher abhängiger (SOCE) Einstrom erfolgen. Neben
spannungsabhängigen Kanälen sind TRPCs und die Orai-Kanäle die Schlüsselproteine für diese
Prozesse.
Anomalien im Ca2+-Einstrom führen zu verschiedensten Krankheitsbildern, wie Nierenerkrankungen,
fortschreitenden motorischen Koordinationsstörungen, kardialen Pathologien und Immunschwäche.
TRPC3 repräsentiert einen sowohl im ROCE als auch möglicherweise im SOCE involvierten TRPC
Kationen-Kanal. Das Verständnis seiner Funktion und im Speziellen der wechselseitigen
gegenseitigen Beeinflussung von Orai1, dem für SOCE zuständigen Porenprotein, sollte zu
effektiveren Behandlungsstrategien für Erkrankungen führen, welche auf einer Dysfunktion des
zellulären Ca2+-Handlings beruhen.
Eine Kombination aus molekularer Modellierung und zielgerichteter TRPC3-Mutagenese identifizierte
eine einzelne Aminosäure in der putativen Porenregion als für die Ca2+-Permeation verantwortlich.
Diese TRPC3E630Q-Mutation reduzierte die Permeation divalenter Ionen um mehr als zwei
Größenordnungen und die TRPC3E630K-Mutation blockierte die Kationen-Permeation völlig. Beide
Mutationen wirken dominant-negativ auf endogene TRPCs.
Pyrazolderivate wurden hinsichtlich der Modulation von TRPC3 und Orai1 charakterisiert. Dabei
konnte eine als Pyr6 bezeichnete Substanz identifiziert werden, die den speicherabhängigen Orai1-
Einstrom selektiv blockiert. Pyr10, eine neu synthetisierte Verbindung, wirkte in ähnlicher Weise
selektiv auf den TRPC3-Kanal. Mittels dieser neuen pharmakologischen aber auch der entwickelten
genetischen Werkzeuge konnten wichtige Hinweise für die Existenz eines in Mikrodomänen
organisierten Signalplexus gefunden werden. Dieser Signalkomplex scheint TRPC3 entweder als
Schlüsselprotein eines funktionellen Gerüsts oder als Signalkomponente zu beinhalten und ist von
entscheidender Bedeutung für die Entstehung lokaler, transkriptionell aktiver Ca2+-Signale durch
Rekrutierung von Ca2+-Signalkomponenten in die Plasmamembran von Herzmuskelzellen (HL-1) oder
Mastzellen (RBL-2H3). Diese Gerüstfunktion von TRPC3 wird dynamisch durch reversible PKC-
Phosphorylierung reguliert.
Die Ergebnisse dieser Arbeit zeigen somit neue Aspekte der molekularen Funktion und
(patho)physiologische Rolle von TRPC3 auf.
8
Introduction
Calcium as cellular (second) messenger
Ca2+ is a key mediator of a broad range of fundamental cellular processes. Changes in
cytosolic free Ca2+ govern a multitude of cellular activities, which range from B-cell
activation, mast cell degranulation, muscle cell contraction, cardiac pathologies and cell
proliferation to gene transcription. (Berridge et al., 2000; Machaca, 2011) Dependent on the
organism (Jammes et al., 2011) and cell type, a diverse array of channels and channel
regulating stimuli control the entry of Ca2+ ions across the plasma membrane into the
cytosol. Mammalian cells express several important Ca2+ entry pathways including voltage-
gated channels like the L- or T-type channel families as well as predominantly receptor-
regulated (/-operated) Ca2+ entry (ROCE) or store-operated Ca2+ entry (SOCE). ROCE is
considered to be mediated in large part through members of the ion channel super family of
TRP channels, while SOCE is predominantly governed by STIM/Orai protein complexes.
TRPC channels
The „canonical“ family of TRP proteins forms one of 6 subfamilies1 of a roughly 30 member
comprising superfamily of cation channels genetically conserved in organisms from yeast,
plants and invertebrates to vertebrates (see figure 1). (Denis et al., 2002; Moiseenkova-Bell
et al., 2011; Nilius et al., 2011; Nilius et al., 2007; Wolstenholme et al., 2011)
Figure 1: Phylogenetic tree of the 6 mammalian
TRP-superfamily subfamilies (Nilius et al., 2007).
1 Not accounting for a TRPY-annotated channel family postulated in fungi, single homologues in plants or a
TRPN family found only in drosophila, worms and zebrafish.
9
The trp-channel was initially described in drosophila melanogaster (fruit fly) by the group of
Arnold (Cosens et al., 1969; Pak et al., 1970), characterized as membrane conductance
defect by B. Minke (Minke et al., 1975), cloned by Montell and co-workers (Montell et al.,
1989) and identified as cation transporter and later as channel by (Hardie et al., 1993; Hardie
et al., 1992; Hu et al., 1995; Minke, 2010). A further important step was the cloning of the
first human homologue by the group of L. Birnbaumer (Zhu et al., 1995). Sharing an overall
homology of about 20-60%, the members of the TRP-superfamily additionally possess
common features such as formation of homo- or heterotetramers to build up complete
channel structures comprised of six TM segments in each subunit, an N- and C-terminus
located intracellularly, localization of a putative pore-forming region between TM 5 and 6 as
well as all constituting cation channels with nonetheless highly variable ion selectivity
(Owsianik et al., 2006). Subfamilies share a so-called TRP-box sequence motif (EWKAFR) C-
terminally of the pore-forming region (Montell, 2001; Padinjat et al., 2004). Ankyrin repeats
and a CaM-binding domain are as well common (see figure 2). (Abramowitz et al., 2007;
Gaudet, 2007; Hardie, 2007; Schindl et al., 2007)
Figure 2: Overview of structural features of TRP channels (Moran et al., 2011)
TRP channel function is used in many tissues to sense and transmit diverse, predominantly
external stimuli, ranging from physical, chemical and osmotic changes to as well the filling
state of calcium stores. This sensory function is directly or indirectly conducted via signalling
10
cascades from the PM to intracellular effectors (Clapham, 2003; Corey, 2003; Corey et al.,
2004; O'Neil et al., 2005; Su et al., 2011; Tominaga, 2007).
The so-called “canonical” family of TRP channels (TRPCs), with 7 members, of which 6 are
expressed endogenously in humans2, is most closely related to the original drosophila trp-
channel and expressed in a wide variety of tissues in the body (Abramowitz et al., 2009;
Garcia et al., 1997).
Observations from knock-out and knock-in mouse models as well as experiments in cell
lines, suggest a tight link between TRPC channels and various physiological functions.
TRPC1-deficiency led to a defective immune response in mice, affecting B- and T-cells
activation, possibly establishing a link to SOCE (Quintana et al., 2005). Changes in the TRPC1
protein levels in salivary gland cells affected calcium-dependent saliva secretion (Liu et al.,
2007b; Singh et al., 2001). TRPC2-lacking mice show changes in respect to social and sexual
behaviour (Leypold et al., 2002; Stowers et al., 2002). TRPC3 has been connected with
motor-neuron coordination, as a point mutation in the murine channel from threonine to
alanine at AA 5733 leads to progressing cerebellar dysfunction and motor coordination
problems characterized as ataxia in the animals. This neuronal dysfunction was apparently
caused by sustained channel activity (Becker et al., 2011; Becker et al., 2009; Trebak, 2010).
Expression levels of the TRPC3 protein seem to be important for pathological changes in the
heart as a consequence of hypertensive states. This pathological function was found
associated with gene expression of hypertrophy effectors proteins as well as the channel
itself leading to cardiac hypertrophy and heart failure via the CaM/CaN/NFAT-signalling
pathway (Brenner et al., 2007; Bush et al., 2006; Nakayama et al., 2006; Nishida et al., 2008).
TRPC4 was suggested to be associated with vascular endothelial function especially
relaxation and microvascular permeability (Tiruppathi et al., 2002), as well as endothelial cell
proliferation and differentiation (Graziani et al., 2010). TRPC5 appears to be important in
hippocampal neurons (Greka et al., 2003). For TRPC6 a connection between the late-onset
state type of proteinuric kidney disease focal and segmental glomeruosclerosis and gain of
function point mutations in the gene sequence is well established (Reiser et al., 2005; Winn
2 TRPC2 is a pseudogene in humans and expressed in rodents
3 Unless indicated otherwise aminoacid numbering is according to the UniProt entry Q13507 version 106 of
2011/04/05, due to changes in the entry the current version current numbers change by a value of -12.
11
et al., 2005). In TRPC6 knock-out animal models TRPC3 overexpression as possible mode of
compensation was observed (Dietrich et al., 2005). (Freichel et al., 2004; Freichel et al.,
2005; Nilius et al., 2007)
All 7 TRPC channels form homo- and heterotetrameric cation-permeable pores with
different selectivity for Ca2+ and can be combined into subgroups of TRPC1, TRPC4 & 5 and
TRPC3, 6 & 7 depending on activation characteristics. The general mechanism of activation
for TRPCs involves PLC as crucial upstream signalling element. Channel opening occurs either
after Gq-receptor-dependent PLCβ-activation or tyrosine-kinase-dependent PLCƔ-activation,
leading to hydrolysis of PIP2 to DAG and IP3. These lipid messengers activate the channels
directly or indirectly after IP3-receptor driven ER-Ca2+ depletion (see schematic
representation in figure 3). The difference between the TRPC 3, 6 & 7 and the TRPC 4 & 5
group is, that the later isoforms are non-responsive to direct DAG (or analogue) stimulation,
whereas TRPC3, 6 & 7 can easily be activated by DAG and analogues alone (Hofmann et al.,
1999; Lemonnier et al., 2008; Okada et al., 1999; Trebak et al., 2003a; Trebak et al., 2003b;
Venkatachalam et al., 2003).
Figure 3: Scheme of typical TRPC channel activation, a more detailed one can be
found in a review of D.E. Clapham (Clapham, 2003). (Putney, 2004)
TRPC3 a prototypical example of TRPC channels
TRPC3 is a prototypical, well-characterised member of the TRPC family (Eder et al., 2007a).
TRPC channels were long considered as pore proteins involved in SOCE phenomena even
extending this to a highly Ca2+-selective current called ICRAC (whose molecular components
are introduced later) (Parekh et al., 2005).
12
An expression level-dependent change in mode of activation for TRPC3 shall be mentioned
(Salido et al., 2009; Zitt et al., 2002). Aside the above described role of PIP2-hydrolysis and
therefore DAG- and PIP3-dependent receptor activation (Zhu et al., 1998), a store-dependent
activation has been observed in various studies with immune cells by the group of James
Putney. This is leading to the idea of a switch of the predominant activation mode
dependent on the channel protein’s expression level from a SOCE mode with low and a
ROCE mode with high expression levels. (Putney, 2004; Trebak et al., 2002; Vazquez et al.,
2001; Vazquez et al., 2003; Yildirim et al., 2005) In historic review, this duality and the
involvement of other TRPCs in SOCE phenomena might explain the earlier identification of
these channels as proteins relevant for CRAC.
The (patho)physiological role of TRPC3 in cellular systems is not only limited to the
mentioned effect on gene expression via the CaM/CaN/NFAT-pathway in cardiac
hypertrophy (Eder et al., 2011) or a function of the protein itself as a scaffold for these
signalling factors in cardiac and immune cells (Poteser et al., 2011 and Schleifer et al., 2012 -
in preparation). Association of TRPC3 with PKCβ in B-cells after channel activation (Numaga
et al., 2010) or the binding of TRPC3 to immunophilins (Sinkins et al., 2004) was shown. In
addition, our lab could demonstrate protein-protein interaction of TRPC3 with other TRPC-
family members like TRPC4 or TRPC6 (Poteser et al., 2006) and the direct interaction of
TRPC3 with other ion-transporting proteins, even modulating their mode of action (Eder et
al., 2007b; Rosker et al., 2004).
Recent mutagenesis studies on other TRP- as well as voltage-gated cation channels
combined with information on channel structures by X-ray crystallography give rise to the
possibility of modelling the structural features of TRPC3. A model based on a proposal of
Owsianik and colleagues (Owsianik et al., 2006) and the K+-channel KcsA to identify key
properties like the selectivity filter, binding sites for agonists or regulatory phosphorylation
sites was generated (Poteser et al., 2011).
The model for TM 1 to 6 fits data of cryo-electronmicroscopy data, revealing a compact
structure in the center of a tetramer around an internal chamber surrounded by a looser
outer part (see figure 4A) (Mio et al., 2005; Mio et al., 2007). For electrophysiological studies
a pore model of the TRPC putative pore region (TM5-TM6) based on available potassium or
sodium channel structures has been elaborated. Here a pore helix, which is folding back and
13
reaching up to TM 6 after the narrowest part can be assumed (Gaudet, 2008). Experimental
data revealed the negatively charged glutamate at AA 630 is responsible for dehydrating
divalent ions within the channel´s pore (Poteser et al., 2011). This corresponds to the
findings of R. Hardie for the original drosophila trp (Liu et al., 2007a).
Various studies present other key features of the TRPC3 channel. Aa 573 (threonine) and 712
(serine) were identified as PKC-phosphorylation sites controlling the activity of the channel.
“Deleting” either leads to more spontaneously or constitutively active channels, implicating
increased inward currents or even non-tolerable and therefore lethal levels of Ca2+ in the
cells (electrophysiological data on both is unpublished) (Becker et al., 2009; Trebak et al.,
2005). In the N-terminus of the protein two PKG-phosphorylation sites, found at AA 11
(threonine) and 263 (serine), were postulated to regulate inhibition of channel function after
store-depletion. This connects TRPC3 channel activity modulation to phospholipase-
dependent regulation by PK-phosphorylation. (Becker et al., 2009; Kwan et al., 2006; Trebak
et al., 2005)
For downstream signalling, a link between TRPC3 and NFAT via the immunophilin FKBP12
and the NFAT-signalling activator CaM/CaN was shown. Changing one proline to glutamine
at AA 704 in a LPPPFSLVPSPK-motif4 found in all TRPCs and drosophila trp inhibited protein
co-immunoprecipitation in sf9 cells co-expressing TRPC3 and FKPB12. Administration of
rapamycin, a known immunesuppressant blocked immunophilin interaction with TRPCs as
well, establishing TRPC as a scaffold for NFAT activation. (Sinkins et al., 2004)
Another study specifically identifies two charged aminoacids at aa 697 and 698 as STIM1-
binding sites, relating TRPC channels once more to the STIM1/Orai1-pair of proteins relevant
for the CRAC- or STIM1-dependent SOCE phenomena (Zeng et al., 2008).
Little is currently known about structural features outside the actual pore region adjacent to
the end of TM 5. As other studies with ion channels showed that single AA residues can pose
activation sites for lipid agonists (Branstrom et al., 2007) or that even the outer pore
architecture of the channel was able to affect selectivity and permeation (Voets et al., 2004)
this stretch of aa is of great interest. Differences in the sequence of the TRPC3, 6 & 7 and
TRPC4 & 5 subfamily suggest the activation site for DAG activation in this region. See figure
4B for a comprehensive illustration of published TRPC3 key features.
4 Mutated AA printed bold.
14
Figure 4: (A) 3D reconstruction of TRPC3 cryo-electron microscopy
(grey bar represents PM)(Mio et al., 2007). (B) illustration of TRPC3 key features
CRAC – from TRPs to STIM1/Orai1
Named SOCE or in its original definition “capacitive Ca2+ entry” (Putney, 1986), Ca2+ influx
from extracellular space to refill beforehand depleted intracellular stores like the ER is an
important physiological signal for activation of immune cells (Feske et al., 2005; Lewis,
2001). Until the discovery of the two key proteins - STIM1 and Orai1 (Prakriya et al., 2006;
Zhang et al., 2005) - mediating the clearly defined ICRAC, a highly Ca2+-selective inward
current, TRPC channels have for more than 20 years been considered as responsible for
SOCE and also the CRAC phenomena (Parekh et al., 2005).
The currently accepted model for CRAC activation is that STIM1 is an at basal conditions ER-
residing Ca2+ sensor, which upon store-depletion transmits the changed filling state of these
stores to PM-localised, tetrameric Orai1-channels. These are activated by STIM1-clustering, -
translocation to PM-near ER and direct protein-protein interaction with the channel
proteins. (Fahrner et al., 2009; Lewis, 2007; Varnai et al., 2009).
Initially, both proteins were identified in large-scale RNAi studies and until present functional
key domains of each could be annotated.
Human STIM1 is a single TM protein spanning 685 AA. A functional homologue STIM2 with
presumably lower Ca2+ affinity is therefore probably exhibiting a modulatory feedback role in
CRAC (Brandman et al., 2007). Functionally important STIM1-regions are an N-terminally and
therefore within the ER space located EF-hand motif, sensing the changes in Ca2+ in this
B A
15
compartment (down to ≈200 µM from a resting condition of ≈600 µM in the ER) and a SAM,
where the deletion of which blocks oligomerisation and CRAC activation (Brandman et al.,
2007; Luik et al., 2008). Three differently designated and localised regions bearing the
necessary domains for Orai activation, with a combined overlap of 100 aminoacids beginning
from AA 340 were identified in the cytosolic part of the protein. The Orai activating small
fragment (OASF, aa 233-450) (Muik et al., 2009), CRAC activating domain (CAD, aa 342-448)
(Park et al., 2009) and STIM/Orai activating region (SOAR, 344-442) (Yuan et al., 2009), all
contain the second cytosolic CC-domain and the following aminoacids. Further a CRAC
modulatory domain (CMD), a serine/proline-rich region and a C-terminally located polybasic
cluster, all to some extent contributing to and regulating the CRAC and therefore Orai
activation cascade, were discovered (Huang et al., 2006; Muik et al., 2009; Park et al., 2009).
For an overview of STIM1 functional domains see figure 5A.
Human Orai1 (or CRACM1) is generally accepted as the molecular basis of the CRAC pore. Its
homologues Orai2 and Orai3 show similar but lowered reaction upon co-expression with
STIM1 in store-depleted states, different inactivation characteristics, permeability properties
and reaction to inhibitors. (Gwack et al., 2007; Lis et al., 2007) The 4 TM-spanning protein
contains 301 AA. A single point mutation at AA 91 from arginine to tryptophan leads to a
SCID syndrome in humans, accompanied by impaired T-cell function, presumably caused by
defective channel gating. Corresponding mutations in Orai2 and Orai3 lead to similar effects.
(Derler et al., 2009; Feske et al., 2006; Muik et al., 2008; Vig et al., 2008)
Structurally different to TRP(C) channels, the pore is formed by TM 1 and 3 and therefore
not by two adjacent TM, where mutations in AA 106 (E to D) and 190 (E to Q) lead to
reduced Ca2+ selectivity and higher Na+ permeability. An E106Q charge neutralisation
completely eliminates permeability in a dominant negative way upon endogenous Orai1.
The selectivity filter is considered to be located between AA 105 and 114. (Prakriya et al.,
2006; Vig et al., 2006a; Vig et al., 2006b) A recent study showed that one AA in the
extracellular pore region of Orai1 can confer to constitutively, STIM1-independently active
non-Ca2+-selective channels (AA 102 mutated from V to C), where STIM1 coupling
reconstitutes Ca2+ selectivity. This lead to the conclusion of an incomplete intrinsic Ca2+
selectivity of Orai1 and/or the addition and support of this distinctive ICRAC property by
structurally modulating effects of STIM1 binding to the pore protein. (McNally et al., 2012)
16
Considering functional domains within Orai1, the putative CC-domains in the C-terminus of
the protein appear to mediate the physical interaction during CRAC activation interacting
with the second CC-domain in STIM1 (Calloway et al., 2009; Muik et al., 2008). Contrary to
this, studies show that the N-terminally located proline/serine-rich regions and polybasic
stretches of AA seem to negatively modulate STIM/Orai-binding (Li et al., 2007; Takahashi et
al., 2007). A brief overview of human Orai1 domain organisation is presented in figure 5B.
Figure 5: Depiction key functional domain organisation of human (A) STIM1 and (B) Orai1.
Abbreviations used are explained in the abbreviation’s list. (Fahrner et al., 2009)
Activation of the CRAC is mediated by a cascade of steps, which have been studied
extensively. STIM1 is distributed equally in the ER-membrane in resting state, co-localising to
some extent to cellular structures like microtubli ends (Baba et al., 2006; Smyth et al., 2007).
Upon Ca2+ depletion of this organelle, sensed by the EF-hand of STIM1, the proteins cluster
in punctae in PM-near ER-regions. (Luik et al., 2008; Zhang et al., 2005). This causes the
localisation of Orai1 in punctae as well, leading to activation of the CRAC-channels and Ca2+
influx into the cytosol.
Despite the discovery of STIM and Orai as key components of store-operated Ca2+ entry,
TRPC-channels have not completely become out of focus in terms of protein-protein-
interactions of CRAC-relevant proteins (Salido et al., 2009). The group of S. Muallem
showed that STIM1 interaction with TRPC1 and TRPC3 is abrogated by changes of only two
charged aminoacids (aa 684 & 685 in STIM1 and 697 & 698 in TRPC3) (Zeng et al., 2008). This
interaction is in line with studies from the group of I. Ambudkar connecting STIM1, Orai1 and
TRPC1 in various SOCE situations (Ambudkar, 2007; Cheng et al., 2008; Cheng et al., 2011;
A
B
17
Ong et al., 2007). Controversial to the accepted CRAC activation model, involving exclusively
STIM1 and Orai1, L. Birnbaumer proposed an Orai1/TRPC3 interaction model, involving Orai1
only as regulatory β-subunit of a potentially even additionally lipid-raft- or non-lipid-raft-
localisation-dependent TRPC3 ROCE- or SOCE-mode of function. They show increase of TRPC
ROCE (and SOCE) upon moderate Orai1 expression or high level of STIM1 and Orai1 co-
expression, which is not blocked by application of lanthanides. (Liao et al., 2008; Liao et al.,
2007; Liao et al., 2009) Furthermore an indirect interaction of TRPC3 and Orai1 via the
protein Rack1 has been shown recently as well (Bandyopadhyay et al., 2008; Woodard et al.,
2010).
Blockers of non-voltage gated Ca2+ channel
Contrary to voltage-gated Ca2+ channels with numerous experimentally and therapeutically
suitable blockers like nifedepin, verapamil or benzothiazepine, neither for most TRPC-
channels nor for Orai1 are such molecules available at the moment. Nonetheless, a number
of recent studies characterized the inhibitory effects of compounds on these two different
channel families (figure 6). (Birnbaumer, 2009; Harteneck et al., 2011; Parekh, 2010; Varnai
et al., 2009)
Trivalent ions like lanthanides, specifically La3+ or Gd3+, are commonly used as inhibitors of
Ca2+ entry channels but lack sufficient selectivity. In the low micromolar range, Gd3+ seems
to block CRAC/SOCE channels and a Gd3+-resistant residual Ca2+ influx after receptor
stimulation in TRPC3 expressing cells is considered TRPC3 ROCE. (Pang et al., 2011; Trebak et
al., 2002) Nonetheless the group of L. Birnbaumer showed a Gd3+-resistant Ca2+ entry into
HEK cells expressing low amounts of Orai1, a combination of STIM1 and Orai1 or even co-
expressing these proteins with TRPCs in SOCE and ROCE protocols (Liao et al., 2009).
A broad range of organic compounds is applied to affect TRPC and CRAC channel function. 2-
APB initially an IP3 receptor blocker (Maruyama et al., 1997) has been identified to block
TRPC (Lievremont et al., 2005; Xu et al., 2005) as well as Orai1 SOCE in low molecular ranges.
High concentrations lead to an activating effect on Orai3 and additionally change Orai3 pore
properties (DeHaven et al., 2008; Lis et al., 2007; Schindl et al., 2008). SKF-96365, which is
blocking TRPCs in both functional modes and inhibits SOCE and ICRAC in a micromolar range,
is as well not specific affecting other Ca2+ channels too (Boulay et al., 1997; Marumo et al.,
2001; Merritt et al., 1990; Zhu et al., 1996). Myosin light chain kinase inhibitors like ML-9,
18
ML-7 and wortmanin can inhibit SOCE in various cell lines and have been shown to
negatively affect different TRP channels, e.g. TRPC6 (Shi et al., 2007; Watanabe et al., 1996).
Studies indicate effects on STIM1 PM-representation and punctae formation upon CRAC
activation (Smyth et al., 2008).
The so-called “syntha compound” SK-66 synthesised by GlaxoSmithKline has recently been
shown to inhibit CRAC channels in mast cells, as well suppressing interleukin production (Ng
et al., 2008). The group of D.J. Beech showed nanomolar blocking potency on CRAC in
human vascular smooth muscle cells without affecting TRPC1-ROCE or STIM1 function (Li et
al., 2011).
Another highly promising family of compounds are derivatives of 3,5-
bis(trifluoromethyl)pyrazole (BTPs) according to their prominent structural feature termed
“pyrazoles”, initially synthesised and characterised as immunesuppressants affecting NFAT
translocation (Djuric et al., 2000; Trevillyan et al., 2001). BTP2 is commonly considered as a
potent CRAC blocker, however a blocking effect of BTP2 on TRPC3 has been published as
well (He et al., 2005). A number of studies with BTP2 derivatives’ evaluating effects of
pyrazoles on other channels and structural requirements for potent blockers were carried
out (Djuric et al., 2000; Law et al., 2011; Steinckwich et al., 2007; Sweeney et al., 2009).
Recently a BTP2/pyrazole variant named Pyr3 was postulated to specifically block TRPC3
channels and further derivatives were shown to potently affect other TRPC family members
as well (Kiyonaka et al., 2009; Kiyonaka et al., 2010).
Figure 6: Structures of Ca2+
channel inhibitors (Harteneck et al., 2011; Parekh, 2010)
19
Aim of the thesis
The main focus of this thesis was to elucidate the role of TRPC3 in activating and
orchestrating downstream signalling processes, specifically cellular events controlling NFAT
activated gene expression. Ca2+ entry into cells is an important cellular signalling process,
which governs gene transcription, generally referred to as Ca2+/transcription coupling.
Although a link between TRPC3 function and NFAT translocation along with an interaction of
the channel with NFAT-binding proteins has recently been proposed, the molecular
mechanisms involved in TRPC3-mediated Ca2+/transcription coupling, specifically the role of
TRPC3 permeation, dynamic protein-protein interactions with other channels or reversible
protein modifications remain still unclear.
To shed light on these aspects, I set out to generate channel mutants that specifically lack
certain signalling properties and to develop tools for selective pharmacological modulation
of TRPC3 function. The TRPC3 mutants were characterized in a native cellular setting of HL-1
cardiac myocytes and RBL-2H3 mast cells, employing a combined approach of Fura2-Ca2+-
imaging, conventional fluorescence and TIRF microscopy as well as patch clamp
experiments. The mutations were designed based on a computational homology model of
the TRPC3 channel structure and were intended to target the channels selectivity filter as
well as its ability to dynamically associate with scaffold proteins and the Ca2+ sensor CaN.
This approach was expected to provide new insights into:
1. The pore structure and molecular determinants of TRPC3 channel functions
2. The impact of selective block or modulation of TRPC3 and Orai1 function on signalling
events downstream of Ca2+ entry through these channels and
3. The crosstalk of TRPC3 with STIM1/Orai1 SOCE-phenomena and contribution of the
TRPC protein in Ca2+/transcription coupling.
The experiments and conclusions from this work are presented in form of published or for
publication submitted and prepared manuscripts.
20
Applied methods
The following experimental methods were applied in this thesis work. Detailed information
on the procedures is given within the included manuscripts (Poteser et al., 2011) (Schleifer
et al., 2012 - British Journal of Pharmacology - submitted, Schleifer et al., 2012 - in
preparation):
Standard molecular biology techniques, PCR, molecular cloning, site directed
mutagenesis, sequencing and preparation of plasmid DNA for cell culture,
Heterologous expression of proteins in e.coli,
Cell culture and handling of cell lines, mainly HEK-293, RBL-2H3 and HL-1 cells,
including normal passaging and transfection by lipofection or electroporation to
heterologously express proteins,
Application of microscopy techniques, specifically fluorescence and TIRF microscopy
including data evaluation,
Cellular calcium imaging using Fura2 applying receptor-activation and store-depletion
protocols including large scale data analysis and
Initial generation of a TRPC3 homology model and further bioinformatic
methodology (sequence alignments, phylogenetic and homology analysis …) for in-
silico studies of TRPC3 pore properties.
21
Results
PKC-dependent coupling of calcium permeation through transient
receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-
1 myocytes.
Proc Natl Acad Sci USA 108(26): 10556-10561
Michael Potesera, Hannes Schleifera, Michaela Lichteneggera, Michaela Schernthanera,
Thomas Stocknerb, C. Oliver Kappec, Toma N. Glasnovc, Christoph Romanind and Klaus
Groschnera
a
Institute of Pharmaceutical Sciences, University of Graz, 8010 Graz, Austria;
b Institute of Pharmacology, Medical University of Vienna, 1090 Vienna, Austria;
c Institute of Chemistry, University of Graz, 8010 Graz, Austria and
d Institute of Biophysics, University of Linz, 4040 Linz, Austria
PKC-dependent coupling of calcium permeationthrough transient receptor potential canonical 3(TRPC3) to calcineurin signaling in HL-1 myocytesMichael Potesera, Hannes Schleifera, Michaela Lichteneggera, Michaela Schernthanera, Thomas Stocknerb,C. Oliver Kappec, Toma N. Glasnovc, Christoph Romanind, and Klaus Groschnera,1
aInstitute of Pharmaceutical Sciences, University of Graz, 8010 Graz, Austria; bInstitute of Pharmacology, Medical University of Vienna, 1090 Vienna, Austria;cInstitute of Chemistry, University of Graz, 8010 Graz, Austria; and dInstitute of Biophysics, University of Linz, 4040 Linz, Austria
Edited* by Lutz Birnbaumer, National Institute of Environmental Health Sciences, Research Triangle Park, NC, and approved May 17, 2011 (received for reviewApril 21, 2011)
Cardiac transient receptor potential canonical (TRPC) channels arecrucial upstream components of Ca2+/calcineurin/nuclear factor ofactivated T cells (NFAT) signaling, thereby controlling cardiac tran-scriptional programs. The linkage between TRPC-mediated Ca2+ sig-nals and NFAT activity is still incompletely understood. TRPCconductances may govern calcineurin activity and NFAT transloca-tion by supplying Ca2+ either directly through the TRPC pore intoa regulatory microdomain or indirectly via promotion of voltage-dependent Ca2+ entry. Here, we show that a point mutation in theTRPC3 selectivity filter (E630Q), which disrupts Ca2+ permeabilitybut preserves monovalent permeation, abrogates agonist-inducedNFAT signaling in HEK293 cells as well as in murine HL-1 atrial myo-cytes. The E630Q mutation fully retains the ability to convert phos-pholipase C-linked stimuli into L-type (CaV1.2) channel-mediatedCa2+ entry in HL-1 cells, thereby generating a dihydropyridine-sensitive Ca2+ signal that is isolated from the NFAT pathway. Pre-vention of PKC-dependentmodulation of TRPC3 by either inhibitionof cellular kinase activity or mutation of a critical phosphorylationsite in TRPC3 (T573A), which disrupts targeting of calcineurin intothe channel complex, converts cardiac TRPC3-mediated Ca2+ signal-ing into a transcriptionally silent mode. Thus, we demonstrate a di-chotomy of TRPC-mediated Ca2+ signaling in the heart constitutingtwo distinct pathways that are differentially linked to gene tran-scription. Coupling of TRPC3 activity to NFAT translocation requiresmicrodomain Ca2+ signaling by PKC-modified TRPC3 complexes.Our results identify TRPC3 as a pivotal signaling gateway in Ca2+-dependent control of cardiac gene expression.
Ca2+ homeostasis | NFATc1 transactivation | transient receptor potentialcanonical | divalent permeation
As a universal and versatile second messenger, calcium (Ca2+)governs a multitude of cellular effector functions in the
heart including transcriptional programs and cellular remodelingprocesses (1). Coordinated control of cardiac functions by Ca2+ re-quires efficient segregation of Ca2+ signals into regulatory micro-domains, resulting in specificity of coupling between Ca2+ sourcesand Ca2+-dependent effector systems. So far, the molecular com-position and architecture of Ca2+ signaling microdomains forcontrol of cardiac transcriptional programs is incompletely un-derstood. Cation channels of the transient receptor potential ca-nonical (TRPC) family constitute a ubiquitous signal transductionmachinery for Ca2+ entry and have recently been identified as ionchannels that trigger pathophysiological activation of nuclear factorof activated T-cell (NFAT)-mediated gene transcription and hy-pertrophic remodeling in the heart (2–4). TRPC proteins formCa2+ permeable plasma membrane channels that are typically ac-tivated in response to hormonal stimuli linked to phospholipaseC signaling (5). These channels lack, or display only modest, se-lectivity for Ca2+ over monovalent cation (6) and are able to gen-erate increases in cytosolic Ca2+ viamultiplemechanisms includingindirect initiation ofCa2+ entry via voltage-gatedCa2+ channels (7)or the sodium calcium exchanger (NCX) because of modulation of
membrane potential and/or local Na+ gradients (8). TRPC chan-nels are expected to contribute divergently to Ca2+ signaling innonexcitable and in excitable cells, which provide a certain reper-toire of voltage-dependent Ca2+ transport systems.TRPC3 is a lipid-regulated member of the TRPC subfamily and
a potential player in cardiac pathophysiology (9). For homomericTRPC3 channels, a Ca2+/Na+ permeability ratio of ≈1.6 wasdetermined (10) and functional crosstalk of TRPC3 channels withcardiac voltage-gated Ca2+ channels and NCX1 has been sug-gested (7, 11). Representing a typical nonselective cation channel,TRPC3 controls cellular processes by either Ca2+ permeationthrough its pore and generation of a local Ca2+ signal at the TRPchannel signalplex or by remote effects on voltage-gated Ca2+
channels or electrogenic transporters. According to a paradigm ofcardiac (patho)physiology, beat-to-beat Ca2+ cycling (E-C cou-pling) in the heart is separated from Ca2+ signaling events thatcontrol gene expression (12). Interestingly, TRPC3 has beensuggested to govern gene transcription by mechanisms involvinga linkage to voltage-dependent Ca2+ entry (7), which is also es-sential for E-C coupling. However, the Ca2+ entry mechanism,which links cardiac TRPC3 activity to gene expression is elusiveand it is unclear whether nonselective TRPC channels serve asdual Ca2+ signaling units that control segregated Ca2+ pools. Toaddress these questions, we set out to engineer the TRPC3 cationpermeation pathway and generated a single point mutation(E630Q) that lacks divalent- but retains monovalent permeabilityand, thus, the potential to control voltage-dependent Ca2+ entry.This mutant was found capable of functional coupling to cardiacCaV1.2 signaling but not to NFAT activation. We present evi-dence for a tight link between TRPC3 channel activity and NFATnuclear translocation based on Ca2+ permeation through theTRPC3 pore, generating a local Ca2+ signaling event that issensed by the downstream effector calcineurin (CaN), which istargeted to cardiac TRPC3 channels. Moreover, we demonstratethat protein kinase C-dependent modulation of the channelenables switching between transcriptionally active and inactiveTRPC3-signaling modes.
Results and DiscussionIdentification of E630 as a Critical Residue in the TRPC3 SelectivityFilter. In an attempt to obtain TRPC mutants with altered ionselectivity, we initially generated a structural model of the TRPC3pore region by using a recently developed alignment strategy (13)
Author contributions: M.P. and K.G. designed research; M.P., H.S., M.L., M.S., and T.S.performed research; C.O.K., T.N.G., and C.R. contributed new reagents/analytic tools;M.P., H.S., M.L., M.S., and T.S. analyzed data; and M.P., H.S., and K.G. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106183108/-/DCSupplemental.
10556–10561 | PNAS | June 28, 2011 | vol. 108 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1106183108
and structure information available for KcsA as well as a Kv1.2-Kv1.3 chimeric pore. Our hypothetical pore model is illustrated inFig. S1A along with the sequence comparison of TRPC3 andtemplate pore structures (Fig. S1B). Three of the five negativeresidues were predicted to be accessible and exposed to the per-meation pathway. According to our molecular model, only oneglutamate (E630) was localized within the central part of thepermeation pathway, whereas the other two residues (E616 andD639) were predicted as part of the extracellular vestibule. Mu-tagenesis and functional analysis confirmed a critical role of thecentral glutamate in position 630. Charge inversion (E630K)yielded a nonfunctional channel (Fig. S2), whereas neutralization(E630Q) produced a channel that displayed moderately alteredcurrent to voltage (I-V) relation in normal extracellular (Na+ plusCa2+ containing; Fig. 1 A and C) solution with a relative increasein the conductance at neutral potential. The I-V relation of theTRPC3-E630Q mutant was virtually insensitive to changes inextracellular Ca2+ (Fig. S3). Inspection of I-V relations with Ca2+as the sole extracellular cationic charge carrier and BAPTA in thepipette solution to eliminate indirect, Ca2+-mediated currentsrevealed that the E630Q mutation profoundly reduced Ca2+permeation although the channel complex (Fig. 2 B and D). In-ward currents were essentially small or lacking with Ca2+ asa charge carrier even at large hyperpolarizing potentials, and re-versal potentials were difficult to determine in most experiments.Nonetheless, from six experiments a mean of −79.6 ± 6.3 mV wascalculated, demonstrating a substantial shift in reversal potentialcompared with wild-type channels (1.9 ± 1.4; n = 7). The Ca2+/Cs+ permeability ratio was reduced from 4.2 to less than 0.02 inthe mutant. Thus, our experiments identified a key amino acidwithin the cation permeation structure of TRPC3. The negativecharge in position 630 is apparently essential for divalent per-meation but not for transition of monovalent cations through theTRPC3 pore. Our finding is in line with previous reports on therole of negatively charged residues in Ca2+ transition throughTRP pores (14–17) and, specifically, with a prediction of criticalresidues in the TRPC selectivity filter obtained by Liu et al. in astudy with the prototypical Drosophila TRP channel (14). It is ofnote that the identified negatively charged residue is conservedin the TRPC3/6/7 subfamily but absent in more distant relatives ofTRPC3. Therefore, Ca2+ permeation in within the TRPC familyof cation channels may involve distinctly different molecular
mechanisms. Our results support the notion that monovalent anddivalent permeation through nonselective TRP cation channelsmay involve separate, specific interaction sites within the pore,which combine to a “nonselective” pathway that conducts bothtypes of charge carriers. Based on our observation that a singlepoint mutation within the TRPC3 pore causes specific eliminationof Ca2+ permeation, we set out to use this genetically engineeredcation channel to explore the cellular impact of the TRPC3-mediated monovalent conductance and to identify downstreamsignaling pathways that are specifically linked to either themonovalent transport or to Ca2+ entry through the channel pore.Because the TRPC monovalent conductance is considered ofparticular importance in excitable cells, and because recent studieshave demonstrated the relevance of TRPC channels in cardiacpathophysiology (2, 4, 7, 18) we focused on the cardiac system,using the HL-1 murine atrial cell line. Initially, we compared basicproperties of TRPC3 signaling in HL-1 cells with those in thewell characterized electrically nonexcitable HEK293 system.
TRPC3 Mediates Agonist-Induced Ca2+ Signals in HEK293 and HL-1Cells by Divergent Mechanisms. So far, the relative contributionof direct Ca2+ permeation through the TRPC3 pore and indirectmechanisms involving TRPC-mediated changes in membranepotential and voltage-dependent signaling partners such as NCX1has not been evaluated in HEK293 cells. Expression levels ofendogenous voltage-gated Ca2+ channels are below the detectionthreshold, and NCX1 expression is typically moderate to low.Hence, only a minor fraction of the TRPC3-mediated Ca2+ signalis expected to involve indirect mechanisms in HEK293. Pharma-cological characterization of the TRPC3-mediated Ca2+ entrypathway in HEK293 and HL-1 cells, determined by using a clas-sical Ca2+ readdition protocol, revealed that global Ca2+ signalswere based on distinctly different mechanisms (Fig. S4). Elec-trophysiological experiments confirmed that TRPC3 channelswere active when Ca2+ was elevated after activation by agonistadministration in Ca2+-free solution (Fig. S5). TRPC3 over-expressing HEK293 as well as HL-1 displayed Ca2+ entry thatwas highly sensitive to inhibition by the TRPC3 blocker Pyr3 (19).KB-R7943, an inhibitor of NCX reverse mode operation, sup-pressed Ca2+ entry only moderately in HEK293 cells and lackedinhibitory effects in HL-1 cells. Block of voltage-gated, L-typeCa2+ channels by nifedipine strongly suppressed Ca2+ entryinto endothelin-stimulated HL-1 cells but had no effect on theCa2+ signal in HEK293 cells (Fig. S4). These results indicate thatTRPC3 is effectively linked to voltage-gated Ca2+ signaling incardiac cells and are able to produce large global cytosolic Ca2+rises via promotion of Ca2+ entry through CaV1.2 channels. Theobserved lack of NCX-mediated Ca2+ entry into HL-1 cells maybe explained by predominant forward mode operation in thesecardiac cells, based on a tight functional coupling to voltage-gatedCa2+ entry channels as well as more negative membrane poten-tials compared with HEK293 cells. Thus, HL-1 myocytes repre-sent an electrically excitable cell type that displays functionalcross-talk and signaling partnership between nonselective TRPCconductances and voltage-gated Ca2+ channels. Thereby, TRPC3signaling in HL-1 cells is distinctly different from that in thenonelectrically excitable HEK293 cell system.As a next step, we aimed to delineate the cellular role of direct
Ca2+ permeation through TRPC3 channels in these cells by char-acterizing coupling between Ca2+ entry and the NFAT down-stream effector system for wild type and poremutants (E630Q andE630K) of TRPC3.
Ca2+ Permeation Through the Pore of TRPC3 Channels Is Essential forActivation of the NFAT Pathway. Carbachol-induced Ca2+ entry aswell as NFAT translocation was strongly reduced by expression ofeither the Ca2+ permeation-deficient mutant (E630Q) or a pore-deadmutant (E630K) (Fig. 2). Basal Ca2+ entry into nonstimulatedcells was reduced when expressing either of the TRPC3 poremutants to the level of vector-transfected controls (Fig. 2B). Asexpected from the proposed NCX1-mediated Ca2+ entry contri-
Fig. 1. Neutralization of E630 in TRPC3 (E630Q) eliminates Ca2+ but notmonovalent permeability. Representative ramp protocol recordings fromHEK293 cells transfected by either TRPC3-WT (A and B) or the mutantchannel E630Q (C and D) in the presence of 140 mM extracellular Na+ and2 mM Ca2+ (A and C), or in absence of extracellular Na+ and presence of10 mM Ca2+ using 10 mM BAPTA in the pipette solution (B and D), before(black) and after stimulation with 100 μM carbachol (+CCh, red).
Poteser et al. PNAS | June 28, 2011 | vol. 108 | no. 26 | 10557
CELL
BIOLO
GY
bution,TRPC3-E630Qdidnot fully eliminate theCa2+entry signal.Ca2+ entry into cells expressing TRPC3-E630Q remained signifi-cantly higher than in cells transfected to express TRPC3-E630K.Nonetheless, NFAT translocation in HEK293 cells was completelysuppressed with either pore mutation of TRPC3 (Fig. 2 C and D).This finding indicated that Ca2+ permeation through the pore isessential to initiate NFAT translocation, whereas indirect NCX-mediated signaling was barely involved because the NCX inhibitorKB-R7943 (5 μM) failed to prevent NFAT translocation (Fig. S6).A possible dual Ca2+ signaling function of TRPC3 was further
investigated in the electrically excitable cardiac HL-1 cell line. Asillustrated in Fig. 3, Ca2+ entry into endothelin-stimulated cellswas slightly enhanced compared with vector-transfected controls(Fig. 3A) by expression of either wild-type TRPC3 (Fig. 3B) orTRPC3-E630Q (Fig. 3C) but reduced down to basal (non-stimulated) level with TRPC3-E630K (Fig. 3D). Expression ofwild-type TRPC3 (Fig. 3B) or TRPC3-E630Q (Fig. 3C) generated aCa2+ signal that was mainly based on voltage-gated CaV1.2channels as evident by its sensitivity to nifedipine (3 μM). In clearcontrast to the observed Ca2+ signals, cells expressing TRPC3-E630Q lacked endothelin-stimulated NFAT translocation (Fig.3C). Thus, the TRPC3mutant with impaired divalent conductance(E630Q) is able to initiate a large global Ca2+ signal via promotionof voltage-gated Ca2+ entry, but this signal is not translated intoNFATactivation. Importantly, even endogenous TRPC3 channelsappear sufficient to exert a significant impact on NFAT trans-location as evident from vector-transfected controls displayinghigher translocation than cells expression the pore-dead domi-nant-negative E630K mutant. It is of note that, in contrast toHEK293 cells, NFAT translocation in HL-1 cells was not signifi-cantly promoted in response to depletion of intracellular storeswith thapsigargin (Fig. S7), indicating that the channels involved inNFAT signaling of HL-1 are not classical store-operated Ca2+
entry channels. Opening of TRPC3 channels resulted in NFATactivation, but also generation of an additional intracellular Ca2+signal mediated by voltage-gated Ca2+ channels that was fairlywell segregated from the NFAT pathway. Notably, TRPC3-mediated NFAT activation can occur at barely detectable globalCa2+ changes such as in the presence of nifedipine (3 μM) incontrol cells or cells overexpressing TRPC3 (Fig. 3 A and B),therefore we hypothesized that the triggering Ca2+ elevation islikely to take place in a restricted signaling microdomain at theTRPC3 channel complex, containing essential downstream sig-naling components such as calmodulin and calcineurin (CaN) toallow specific transduction of this local Ca2+ signal. Indeed, directassociation of TRPC3 with CaN have been demonstrated (20, 21)
and the existence of a dynamic TRPC/CaN signaling complexeshave been proposed.
Coupling of Cardiac TRPC3 Signaling to Activation of the NFATPathway Involves PKC-Dependent Phosphorylation. Previous inves-tigations demonstrated assembly of CaN along with immuno-phyllins (FKBP12) into TRPC6 signalplexes and dependency ofthis process on protein kinase C-mediated phosphorylation (21).PKC-mediated phosphorylation of TRPC3 appears essential forboth recruitment of CaN into TRPC complexes and inhibitoryregulation (22, 23). This result prompted us to hypothesize thatsuppression of PKC phosphorylation may disrupt the functionalTRPC3/CaN signaling unit without preventing channel function.Consequently, we set out to test whether TRPC3 linkage toNFATnuclear translocation depends on regulation of the channelcomplex by PKC. To suppress TRPC3 phosphorylation by PKCisoenzymes, we performed experiments with GF109203X, a com-pound that inhibits conventional PKC isoforms including PKC-γ,as one essential player in the control of TRPC3 channels (23).Because CaN has been shown to associate with TRPC3/6 channelsin a manner dependent on phosphorylation by PKC (21), wespeculated that prevention of PKC phosphorylation may disruptTRPC/CaN complexes. Indeed, immunoprecipitation experi-ments in HEK293 cells confirmed that the PKC inhibitor preventsassociation of CaN into TRPC3 complexes along with reductionof threonine phosphorylation of the channel protein (Fig. 4).Alternatively, a mutant that is defective in PKC-γ–mediated in-hibitory modulation, i.e., T573A corresponding to the murineTRPC3-moonwalker (Mwk) mutation, was expressed in HEK293and HL-1 cells. It is of note that overexpression of the phos-phorylation-deficient TRPC3-Mwk by itself was barely toleratedby the cells, presumably due to a gain in function leading to Ca2+
overload. Therefore, we transfected cells to overexpress theT573Amutant along with wild-type TRPC3 (DNA ratio 3:1). Thistransfection resulted in a heteromeric TRPC3 conductance largerthan that generated by wild-type TRPC3 alone (Fig. S8) but es-sentially tolerated by the host cells. The derived heteromersappeared correctly targeted into the membrane as indicated byfluorescence microscopy. Interestingly, currents through TRPC3-Mwk/TRPC3-WT channels were rather stable during continuousagonist stimulation, most likely due to lack of inhibitory regula-tion of the channels by PKC and recordings in Na+-free extra-cellular solution confirmed Ca2+ permeability of the channels.Either treatment of cells expressing TRPC3-WT with
GF109203X (2 μM) or transfection of cells with TRPC3-Mwk/TRPC3-WT produced a similar cellular phenotype, displaying
B C DA
Fig. 2. Receptor-stimulated Ca2+ influx as well as NFAT translocation are impaired in HEK293 cells expressing the Ca2+ impermeable TRPC3-E630Q or theimpermeant TRPC3-E630K, compared with cells expressing wild-type TRPC3. (A) Representative traces of fura-2 Ca2+-imaging experiments. Cells were stim-ulated by 100 μM carbachol (arrow). (B) Mean Δ ratio values (± SEM, n > 40) derived from fura-2 Ca2+-imaging. Black bars indicate the basal (unstimulated)Ca2+ entry at indicated transfections. (C) Mean nuclear/cytosol fluorescence intensity ratio (± SEM, n > 11) of HEK293 cells expressing GFP-NFAT and therespective channel protein after stimulation and application of the same protocol as used in fura-2 experiments. Black bar (basal) represents mean nuclear/cytosol fluorescence intensity ratio in HEK293 cells transfected with GFP-NFAT only. (B and C) Asterisks indicate statistically significant of difference to TRPC3-WT–expressing cells. (D) Representative fluorescence images recorded in GFP-NFAT translocation experiments shown at Left. Positions of nuclei are indicatedby arrowheads. Nucleus/cytosol fluorescence ratios of example images are indicated.
10558 | www.pnas.org/cgi/doi/10.1073/pnas.1106183108 Poteser et al.
large-agonist–induced Ca2+ entry signals that were not accom-panied by significant NFAT nuclear accumulation (Fig. 5). This“transcriptionally silent”Ca2+ entry intoHL-1 cells was for a large
part mediated by voltage-gated L-type Ca2+ channels as indicatedby sensitivity to nifedipine (3 μM). Our results demonstrate dis-ruption of transcriptional TRPC3 signaling in response to reduced
A
B
C
D
Fig. 3. Ca2+ entry through TRPC3 is critical for activation of the calcineurin/NFAT pathway in HL-1 atrial myocytes. HL-1 cells were transfected with vectorcontrol (A), TRPC3-WT (B), or the indicated pore mutant (C and D). (Left) Representative traces of fura-2 imaging in cells at basal conditions (unstimulated +Ca2+ readdition) and stimulated with 100 nM endothelin (+ ET-1, arrow) in the absence or presence of 3 μM nifedipine (+ Nif). (Center Left) Mean fura-2 Δratio values (± SEM, n > 20). (Center Right) Mean nuclear/cytosolic NFAT-GFP fluorescence ratio (± SEM, n > 8) in unstimulated HL-1 cells (basal), HL-1 cellsstimulated with 100 nM endothelin (+ ET-1) in the absence and presence of 3 μM nifedipine (+ Nif) and application of the same protocol as used in fura-2experiments. Asterisks indicate statistically significant inhibition by nifedipine. (Right) Representative images of NFAT-localization before stimulation andCa2+ readdition (control), after Ca2+ readdition (basal), and after stimulation by endothelin and subsequent Ca2+ readdition (+ ET-1) in the absence andpresence of 3 μM nifedipine (+ Nif). Positions of nuclei are indicated by arrows. Nucleus/cytosol fluorescence ratios of example images are indicated.
Poteser et al. PNAS | June 28, 2011 | vol. 108 | no. 26 | 10559
CELL
BIOLO
GY
PKC-dependent phosphorylation of the channel or as a conse-quence of the TRPC3-Mwk mutation. This fact may be consid-ered as part of the molecular mechanism underlying the moon-walker pathophysiology (23).Impaired PKC regulation of cardiac TRPC3 is shown to result in
uncoupling from the NFAT pathway without disrupting the link-age between TRPC3 and global myocyte Ca2+ via voltage-gatedCa2+ entry. We provide evidence that TRPC3-CaN/NFATsignaling takesplace in a restrictedmicrodomain and requiresbothdirect Ca2+ permeation through the TRPC3 pore as well as CaNtargeting into the signal complex. Cardiac TRPC3 complexes areshown to produce Ca2+ signals both via direct Ca2+ transport andby control of voltage-dependent Ca2+ entry. Our results demon-strate the ability of cardiac TRPC3 channels to switch in a phos-phorylation-dependent manner between a transcriptionally activeand a transcriptionally silent signaling mode (Fig. 6). BecauseTRPC3 is likely to change in expression along with other TRPCspecies during pathophysiologic stress, the formation of divergent
heteromeric TRPC3 channel complexes may be anticipated. Theobserveddominantnegative effect of thephosphorylation-deficientmoonwalker mutation on CaN/NFAT activation suggests a prom-inent role of phosphorylation in maintenance of transcriptionallyactive TRPC complexes in the heart. Situations of hamperedPKC phopshorylation or promoted dephosphorylation may con-vert TRPC complexes into a cardiac Ca2+ signaling unit that isfunctionally isolated from the CaN/NFAT activation pathway.Our findings highlight the key role of nonselective TRPC
channels in the control of transcriptional programs and extendthis concept by demonstrating a pivotal role of Ca2+ transporttrough the TRP pore structure along with a unique phosphory-lation-dependent molecular switch that allows efficient control ofcardiac gene transcription by neurotransmitters and hormones.
Materials and MethodsHomology Modeling. For details on sequence alignment, homology modelingand model evaluation, see SI Materials and Methods.
A
B C
Fig. 4. GF109203X inhibits phosphorylation of TRPC3and its association with calcineurin. HEK-293 cellsexpressing HA-tagged TRPC3 were incubated with orwithout 2 μM GF109203X (GFX) and lysed and sub-jected to SDS/PAGE and immunoprecipitation. (A Left)Total HEK-cell lysates were immunoprecipitated byusing an anti-HA antibody and immunoblotted withan anti-phospho-threonine antibody. (A Right) Barsrepresenting the densitometric analysis of phospho-threonine-immunoreactivity. Mean values are givenfor carbachol-stimulated cells in the absence and pres-ence of GFX (± SEM, n = 4). Asterisk indicates statisti-cally significant differences. (B) Stripped membraneswere immunoblotted again by using an anti-HA anti-body (Lower). Proteins detected in total cell lysates(lane 1, Input), immunocomplexes (lane 2, IP-HA-C3)and lysates precipitated only with beads (lane 3, Ctrl.).(C) Coimmunoprecipitations of cell homogenates usingantibodies against the HA-tag and calcineurin, andimmunoblotted against calcineurin. Proteins detectedin total cell lysates (lane 1, Input), immunocomplexes(lane 2, IP-HA-C3; lane 3, IP-CN), and lysates pre-cipitated only with beads (lane 4, Ctrl.).
A B C D
Fig. 5. Phosphorylation of threonine 573 of the TRPC3 channel protein is essential for the activation of the calcineurin/NFAT pathway in HL-1 atrial myocytes. (A)Representative traces of fura-2 Ca2+-imaging experiments in cells transfected with either TRPC3-WT or TRPC3-T573A and TRPC3-WT (DNA ratio 3:1, MWK/TRPC3-WT), stimulated by 100nMendothelin (arrow) and in the absence or presence of 3 μMnifedipine (+Nif) or 2 μMGF109203X (+GFX), as indicated. (B)MeanΔ ratiovalues (± SEM, n > 30) of fura-2 Ca2+-imaging experiments. Asterisks indicate statistically significant nifedipine-induced inhibition. (C) Mean nucleus/cytosolfluorescence intensity ratio (± SEM, n> 9) in HL-1 cells expressingGFP-NFAT and the respective (mutant) channel protein after stimulation and application of thesame protocol as used in fura-2 experiments. (B and C) Asterisks indicate statistically significant difference to TRPC3-WT–expressing cells in the absence ofinhibitors (white bar). (D) Representative fluorescence images recorded in GFP-NFAT translocation experiments. Individual nucleus/cytosol fluorescence ratiosare given, and positions of nuclei are indicated by arrows.
10560 | www.pnas.org/cgi/doi/10.1073/pnas.1106183108 Poteser et al.
DNA and Mutagenesis. Site-directed mutagenesis was performed with stan-dard protocols. Details on cDNA constructs and cloning procedures areprovided in SI Materials and Methods.
Cell Culture and Transfection. Cell lines were cultured at 37 °C and 5%CO2. ForHEK293 cells DMEM (Invitrogen) supplied with 10% FCS and for HL-1 atrialmyocytes Claycomb medium (Sigma) supplied with 100 μM norepinephrin,4mM L-glutamin and 10%FCSwere used. Lipofectionwas used for gene trans-fer; for details on DNA amounts and reagents, see SI Materials and Methods.
Electrophysiology. Standard patch clamp protocols were used (SI Materialsand Methods). Standard bath solutions contained 140 or 0 mM NaCl, 0 or140 mM NMDG, 2 mM MgCl2, 10 mM glucose, 10 mM Hepes, 2 or 0 mMCaCl2, and 0 or 2 mM BaCl2 at pH adjusted to 7.4 with NaOH or NMDG. Pi-pette solution contained 120 mM cesium methanesulfonate, 20 mM CsCl,15 mM Hepes, 5 mM MgCl2, and 3 mM EGTA, at pH adjusted to pH 7.3 with
CsOH. For delineation of Ca2+ permeability of TRPC3 mutants, a bath solu-tion containing 132 mM NMDG, 2 mM MgCl2, 10 mM Glucose, 10 mM Hepes,3 mM CaCl2, 7 mM Ca-Gluconate, at pH adjusted to 7.4 with methanesulfonicacid and a pipette solution composed of 140 mM cesium methanesulfonate,15 mM Hepes, 5 mM MgCl2, and 10 mM BAPTA at pH 7.3 was used.
Measurement of NFAT-Translocation. Cells were transfected to express an N-terminally GFP-tagged NFATc1 fusion (15) and plated on coverslips. Forbuffers and solutions see SI Materials and Methods. Agonists as well asinhibitors (Pyr3, GFX109203, nifedipine, or KB-R7943) remained presentcontinuously after administration. GFP-NFAT translocation was monitored(488 nm excitation) with standard fluorescence microscopy (Zeiss Axiovertequipped with Coolsnap HQ). GFP-NFAT and YFP-TRPC3-WT/-mutant fluo-rescence were discriminated by specific cellular localization. Nuclear/cytosolfluorescence intensity ratios of cells were calculated with ImageJ software.
Measurement of Intracellular Ca2+ Signaling. For details on fura-2 calciumimaging experiments see SI Materials and Methods.
Immunoprecipation. In short, protein-A- or protein-G-bead-preclearedsupernatants of lysates from stimulated HEK293 cells were incubated withprecipitating antibody overnight. After the addition of respective protein-A-or protein-G-beads, washing, and denaturation in Lämmli buffer, the im-munocomplexes were separated by SDS/PAGE and subjected to Westernblotting. See SI Materials and Methods for details.
Reagents. Chemicals, reagents, and antibodies were purchased from SigmaAldrich. KB-R7943 and GF109203X were from Tocris Biosciences. The TRPC3pore blocker Pyr3 was synthesized as published (16).
Statistics. Data are presented as mean values ± SEM and was tested forstatistical significance by using the Student t test (*P < 0.05).
ACKNOWLEDGMENTS. We thank Dr. R. Kehlenbach for providing the GFP-NFAT construct and Mrs. R. Schmidt for excellent technical assistance. Thiswork was supported by FWF (Austrian Science Fund) Grant P21925-B19(to K.G.), P22565 (to C.R.), and DK+Metabolic and Cardovascular DiseaseGrant W2126-B18.
1. Kranias EG, BersDM(2007) Calciumandcardiomyopathies. Subcell Biochem45:523–537.2. Bush EW, et al. (2006) Canonical transient receptor potential channels promote
cardiomyocyte hypertrophy through activation of calcineurin signaling. J Biol Chem
281:33487–33496.3. Kuwahara K, et al. (2006) TRPC6 fulfills a calcineurin signaling circuit during
pathologic cardiac remodeling. J Clin Invest 116:3114–3126.4. Nakayama H, Wilkin BJ, Bodi I, Molkentin JD (2006) Calcineurin-dependent cardio-
myopathy is activated by TRPC in the adult mouse heart. FASEB J 20:1660–1670.5. Montell C, Birnbaumer L, Flockerzi V (2002) The TRP channels, a remarkably
functional family. Cell 108:595–598.6. Owsianik G, Talavera K, Voets T, Nilius B (2006) Permeation and selectivity of TRP
channels. Annu Rev Physiol 68:685–717.7. Onohara N, et al. (2006) TRPC3 and TRPC6 are essential for angiotensin II-induced
cardiac hypertrophy. EMBO J 25:5305–5316.8. Rosker C, et al. (2004) Ca(2+) signaling by TRPC3 involves Na(+) entry and local
coupling to the Na(+)/Ca(2+) exchanger. J Biol Chem 279:13696–13704.9. Eder P, Groschner K (2008) TRPC3/6/7: Topical aspects of biophysics and
pathophysiology. Channels (Austin) 2:94–99.10. Kamouchi M, et al. (1999) Properties of heterologously expressed hTRP3 channels in
bovine pulmonary artery endothelial cells. J Physiol 518:345–358.11. Eder P, et al. (2007) Phospholipase C-dependent control of cardiac calcium
homeostasis involves a TRPC3-NCX1 signaling complex. Cardiovasc Res 73:111–119.12. Molkentin JD (2006) Dichotomy of Ca2+ in the heart: Contraction versus intracellular
signaling. J Clin Invest 116:623–626.
13. Notredame C, Higgins DG, Heringa J (2000) T-Coffee: A novel method for fast andaccurate multiple sequence alignment. J Mol Biol 302:205–217.
14. Liu CH, et al. (2007) In vivo identification and manipulation of the Ca2+ selectivityfilter in the Drosophila transient receptor potential channel. J Neurosci 27:604–615.
15. Nilius B, et al. (2005) The selectivity filter of the cation channel TRPM4. J Biol Chem280:22899–22906.
16. Voets T, Janssens A, Droogmans G, Nilius B (2004) Outer pore architecture of aCa2+-selective TRP channel. J Biol Chem 279:15223–15230.
17. Garcia-Martinez C, Morenilla-Palao C, Planells-Cases R, Merino JM, Ferrer-Montiel A(2000) Identification of an aspartic residue in the P-loop of the vanilloid receptor thatmodulates pore properties. J Biol Chem 275:32552–32558.
18. Seth M, et al. (2009) TRPC1 channels are critical for hypertrophic signaling in theheart. Circ Res 105:1023–1030.
19. Kiyonaka S, et al. (2009) Selective and direct inhibition of TRPC3 channels underliesbiological activities of a pyrazole compound. Proc Natl Acad Sci USA 106:5400–5405.
20. Sinkins WG, Goel M, Estacion M, Schilling WP (2004) Association of immunophilinswith mammalian TRPC channels. J Biol Chem 279:34521–34529.
21. Kim JY, Saffen D (2005) Activation of M1 muscarinic acetylcholine receptorsstimulates the formation of a multiprotein complex centered on TRPC6 channels.J Biol Chem 280:32035–32047.
22. Trebak M, et al. (2005) Negative regulation of TRPC3 channels by protein kinase C-mediated phosphorylation of serine 712. Mol Pharmacol 67:558–563.
23. Becker EB, et al. (2009) A point mutation in TRPC3 causes abnormal Purkinje celldevelopment and cerebellar ataxia in moonwalker mice. Proc Natl Acad Sci USA 106:6706–6711.
Fig. 6. Phosporylation of TRPC3 at position 573 (via PLC) enables Ca2+/calmodulin/calcineurin (Ca2+, Cm, CaN)-dependent activation upon receptor-activated Ca2+ entry through TRPC3. Dephosporylation of position 573 turnsTRPCtranscriptionally silentwhileenhancing its general activity. L-type channelsare controlled by TRPC3-induced changes in membrane potential, providingonly negligible effects on Ca2+-induced NFAT activation in HL-1 atrial myocytes.
Poteser et al. PNAS | June 28, 2011 | vol. 108 | no. 26 | 10561
CELL
BIOLO
GY
Correction
CELL BIOLOGYCorrection for “PKC-dependent coupling of calcium permeationthrough transient receptor potential canonical 3 (TRPC3) tocalcineurin signaling in HL-1 myocytes,” by Michael Poteser,Hannes Schleifer, Michaela Lichtenegger, Michaela Schern-thaner, Thomas Stockner, C. Oliver Kappe, Toma N. Glasnov,Christoph Romanin, and Klaus Groschner, which appeared in
issue 26, June 28, 2011, of Proc Natl Acad Sci USA (108:10556–10561; first published June 8, 2011; 10.1073/pnas.1106183108).The authors note that Figs. 2, 3, and 5 appeared incorrectly.
The corrected figures and their corresponding legends appearbelow.
A B C D
Fig. 2. Receptor-stimulated Ca2+ influx as well as NFAT translocation are impaired in HEK293 cells expressing the Ca2+ impermeable TRPC3-E630Q or theimpermeant TRPC3-E630K, compared with cells expressing wild-type TRPC3. (A) Representative traces of fura-2 Ca2+-imaging experiments. Cells were stimu-lated by 100 μM carbachol (arrow). (B) Mean Δ ratio values (±SEM, n > 40) derived from fura-2 Ca2+-imaging. Black bars indicate the basal (unstimulated) Ca2+
entry at indicated transfections. (C) Mean nuclear/cytosol fluorescence intensity ratio (±SEM, n > 11) of HEK293 cells expressing GFP-NFAT and the respectivechannel protein after stimulation and application of the same protocol as used in fura-2 experiments. Black bar (basal) represents mean nuclear/cytosolfluorescence intensity ratio in HEK293 cells transfected with GFP-NFAT only. (B and C) Asterisks indicate statistically significance of difference to TRPC3-WT–expressing cells. (D) Representative fluorescence images recorded in GFP-NFAT translocation experiments shown at Left. Positions of nuclei are indicated byarrowheads. Nucleus/cytosol fluorescence ratios of example images are indicated.
13876–13878 | PNAS | August 16, 2011 | vol. 108 | no. 33 www.pnas.org
A
B
C
D
Fig. 3. Ca2+ entry through TRPC3 is critical for activation of the calcineurin/NFAT pathway in HL-1 atrial myocytes. HL-1 cells were transfected with vectorcontrol (A), TRPC3-WT (B), or the indicated pore mutant (C and D). (Left) Representative traces of fura-2 imaging in cells at basal conditions (unstimulated +Ca2+ readdition) and stimulated with 100 nM endothelin (+ ET-1, arrow) in the absence or presence of 3 μM nifedipine (+ Nif). (Center Left) Mean fura-2 Δratio values (±SEM, n > 20). (Center Right) Mean nuclear/cytosolic NFAT-GFP fluorescence ratio (±SEM, n > 8) in unstimulated HL-1 cells (basal), HL-1 cellsstimulated with 100 nM endothelin (+ ET-1) in the absence and presence of 3 μM nifedipine (+ Nif) and application of the same protocol as used in fura-2experiments. Asterisks indicate statistically significant inhibition by nifedipine. (Right) Representative images of NFAT-localization before stimulation and Ca2+
readdition (control), after Ca2+ readdition (basal), and after stimulation by endothelin and subsequent Ca2+ readdition (+ ET-1) in the absence and presence of3 μM nifedipine (+ Nif). Positions of nuclei are indicated by arrowheads. Nucleus/cytosol fluorescence ratios of example images are indicated.
PNAS | August 16, 2011 | vol. 108 | no. 33 | 13877
CORR
ECTION
www.pnas.org/cgi/doi/10.1073/pnas.1111388108
A B C D
Fig. 5. Phosphorylation of threonine 573 of the TRPC3 channel protein is essential for the activation of the calcineurin/NFAT pathway in HL-1 atrial myocytes.(A) Representative traces of fura-2 Ca2+-imaging experiments in cells transfected with either TRPC3-WT or TRPC3-T573A and TRPC3-WT (DNA ratio 3:1, MWK/TRPC3-WT), stimulated by 100 nM endothelin (arrow) and in the absence or presence of 3 μM nifedipine (+ Nif) or 2 μM GF109203X (+ GFX), as indicated. (B)Mean Δ ratio values (±SEM, n > 30) of fura-2 Ca2+-imaging experiments. Asterisks indicate statistically significant nifedipine-induced inhibition. (C) Meannucleus/cytosol fluorescence intensity ratio (±SEM, n > 9) in HL-1 cells expressing GFP-NFAT and the respective (mutant) channel protein after stimulation andapplication of the same protocol as used in fura-2 experiments. (B and C) Asterisks indicate statistically significant difference to TRPC3-WT–expressing cells inthe absence of inhibitors (white bar). (D) Representative fluorescence images recorded in GFP-NFAT translocation experiments. Individual nucleus/cytosolfluorescence ratios are given, and positions of nuclei are indicated by arrowheads.
13878 | www.pnas.org
Supporting InformationPoteser et al. 10.1073/pnas.1106183108SI Materials and MethodsSequence Alignment. Sequence alignments between TRPC3 andthe template protein were carried out by applying a hierarchicalapproach using the alignment programs T-Coffee, M-Coffee (1),and Muscle (2). Sequencing of TRPC, KcsA, and Kv1.2 channelswere collected with Blast (3, 4) by using the query sequences ofhuman TRPC3 (UniProtKB ID: Q13507), KcsA (UniProtKBID: P0A334), and the sequence of the Kv1.2–2.1 chimera asdeposited in the Protein Data Bank database (PDB ID code:2R9R). Initial alignments for each subset were performed withMuscle by using gapopen = −3. Only the pore-forming regions(TM5 to TM6) were retained. The ≈30 most diverse sequenceswere extracted from the alignment by using T-Coffee. In thesecond step, the three (TRPC, KcsA, and Kv1.2) alignmentswere merged by a consensus alignment using M-Coffee applyingt-coffee probcons (5), muscle, kalign (6), and clustalW (7) scores.The merged alignment was realigned with Muscle, the numberof sequences was again reduced to 40 sequences, and a finalalignment was created by using T-Coffee applying the blosum40matrix (8), using a gapopen penalty of 300, and applying t-coffeeprobcons, muscle and kalign scores. Variation of parameters andprocedure resulted in three possible alignments.
Alignment Evaluation.Alignment evaluation was carried out at thesequence level and at the 3D structural level. At the sequence level,clustalW scores, secondary structure prediction, alignment of ar-omatic residues in the pore helix, and hydrophobic-hydrophilicpatter were used. The secondary structure prediction was carriedout by using Jpred3 (9, 10). The predictions were compared withthe 3D model created and evaluated for compatibility.3D homology models were built as described below. Com-
patibility of resulting models with protein structure stability(charged groups should be water exposed, helix-helix packing andposition of helix breaking residues) and similarity of hydrophobic,polar and charged residues between the model and the templatewere evaluated. One alignment showed much higher likeliness ofbeing correct.
Homology Model. The homology model of the TRPC3 poreforming region (TM5 to TM6) was generated by using the Kv1.2–2.1 structure with the PDB ID code 2R9R as a template andapplying the final sequence alignment. Model creation was donewith the program modeler (11, 12) applying the multichainprotocol that allows for maintaining the same conformation inevery protomer of the TRPC3 tetramer.
DNAandMutagenesis.TheQuikChangeIISit-DirectedMutagenesisKit (Stratagene) was used formutagenesis. Template was a plasmidconstruct with wild-type human TRPC3 cloned into pEYFP-C1,creating an N-terminally YFP-tagged fusion protein. The plasmidswere electrotransformed into the XL-1 e.coli strain for amplificationand correct nucleotide exchange was verified by sequencing.For some experiments, an untagged TRPC3-WT cloned into
the pcDNA3 vector was coexpressed with the mutants. For ob-servation of NFAT nuclear translocation and for immunopreci-pitations, an N-terminally HA-tagged TRPC3 construct was used.The empty peYFP-C1 vector served as control.
Electrophysiology. Patch pipettes were pulled from borosilicateglass (Clark Electromedical Instruments; 3–5 MΩ). Currentswere recorded at room temperature by using a List EPC7 patchclamp amplifier (HEKA Instruments). Signals were low-pass
filtered at 1 kHz and digitized with 5 kHz. Voltage-clamp pro-tocols (voltage ramps from –100 to +80 mV, holding potential0 mV) were controlled by pClamp software (Axon Instruments).
NFAT-Translocation. Measurements were performed at roomtemperature and started in a Ca2+-free buffer containing 140 mMNaCl, 2 mM MgCl2, 10 mM Glucose, and 10 mM Hepes at pH7.4. After 5 min, incubation cells were challenged with 100 μMcarbachol (HEK293) or 100 nM endothelin (HL-1) and 2 mMCa2+
was readded in a low sodium buffer: 100 mM NaCl, 40 mMNMDG, 2 mM Ca2+, 3 mM MgCl2, 10 mM glucose, and 10 mMHepes at pH 7.4 and incubated for 15 min.
Measurement of Intracellular Ca2+ Signaling. Cells were loaded with2 μM fura 2-AM (Molecular Probes) for 45 min in Optimem me-dium (Invitrogen) and washed. Cells were continuously perfused atroom temperature with calcium-free buffer and challenged withcarbachol (HEK293) or endothelin (HL-1). Agonists as well asinhibitors remained present continuously after administration.For calcium readdition, 2 mM extracellular CaCl2 was added. Ex-citation light was supplied via a Polychrome II polychromator(TILL Photonics) and emission was detected by a Sensicam CCD-camera (PCOComputer Optics). Ca2+-sensitive fluorescence ratios(340 nm/380 nm excitation; 510 nm emission) were recorded andanalyzed by using Axon Imaging Workbench (Axon Instruments).
Cell Culture and Transfection. For transfection, HEK293 cells or HL-1atrial myocytes were seeded at 105 cells per well into 30-mm dishes.After ≈18 h, adherent cells were transfected either using Transfast(Promega; for HEK293) or FuGENE (Roche; for HL-1) trans-fection reagent according to the manufacturer’s instructions. DNA(4–5 μg) was used for single transfections (YFP-TRPC3-WT/-mu-tant, YFP-vector) per dish. For the measurement of NFAT-trans-location, double transfection of 2 μg of GFP-NFAT+ 2 μg of YFP-TRPC3-WT/-mutant or triple transfection of 1 μg of GFP-NFATand 4 μg of YFP-TRPC3-T573A (Mwk) + untagged TRPC3-WT(in 3:1) was used. Approximately 18 h after the transfection, cellswere trypsinized and reseeded 1:2 on polylysine (for HEK293) orfibronectin (for HL-1) coated on 12 mm or 6 × 6 mm coverslips andincubated for 18 h before experiments.
Immunoprecipation. HEK293 cells were grown in 100-mm Petridishes and, if required, transfected. Eighteen to twenty-four hoursafter transfection or for wild-type cells at confluency, cells wereincubated as for the NFAT translocation experiments. At the end,cells were scraped off the dishes, resuspended in 5 mL of PBS, andcentrifuged for 5 min [188 × g, Sorvall (Asheville, NC), RT 7,RTH-250, 4 °C]. The pellet was lysed with 500 μL of MammalianCell Lysis Buffer (QIAGEN), containing 5 μL of protease in-hibitor solution. The lysate was shaken overhead for 30 min at4 °C and centrifuged with high speed for 5 min. Proteins from celllysates (500 mg) were incubated with 50 μL of washed protein-Aor protein-G beads (Merck) and PBS for a final volume of 500 μLand gently rotated for 40–50 min at 4 °C to remove nonspecificbound proteins. Precleared supernatants were incubated over-night at 4 °C under rotation with 3 μg of antibody against HA andcalcineurin, respectively. On the following day, 60 μL of washedprotein-A or protein-G beads were added and gently rotated for2 h at room temperature. The beads were washed three timeswith ice-cold PBS containing 1% Triton and heated to 95 °C afterresuspension in 50 μL of 2× Lämmli buffer. The immuno-complexes were separated by SDS/PAGE and transferred to ni-trocellulose membranes (Hybond ECL nitrocellulose, Amersham
Poteser et al. www.pnas.org/cgi/content/short/1106183108 1 of 7
Biosciences), followed by blocking for 1 h at RT with 5% nonfatdry milk in PBST (0.1% Tween in PBS) and TBST (0.2% Tween20 in TBS), respectively. Nitrocellulose membranes were incubatedovernight at 4 °C with antibodies against HA (1:800 in TBST;Roche), phospho-threonine (1:1,000 in TBST, Cell Signaling orcalcineurin (1:2,000 in TBST; BD Transduction Laboratories).After four washes (10 min each), the secondary antibodies, anti-rabbit IgG (Sigma Aldrich), anti-mouse IgM (Sigma Aldrich) or
anti-mouse IgG (BD Transduction Laboratories) cross-linkedwith horseradish peroxidase (1:5,000) were applied for 1 h atroom temperature and the washing was repeated. Membraneswere detected by the Chemi Glow West ChemiluminescenceSubstrate Sample Kit (Alpha Innotech) and developed by usinga Herolab RH-5.2 Darkroom Hood with an E.A.S.Y 1.3 HCcamera (Herolab).
1. Wallace I-M, et al. (2006) M-Coffee: Combining multiple sequence alignment methodswith T-Coffee. Nucleic Acids Res 34:1692–1699.
2. Edgar R-C (2004) MUSCLE: Multiple sequence alignment with high accuracy and highthroughput. Nucleic Acids Res 32:1792–1797.
3. Altschul S-F, Gish W, Miller W, Myers E-W, Lipman D-J (1990) Basic local alignmentsearch tool. J Mol Biol 215:403–410.
4. Altschul S-F, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of proteindatabase search programs. Nucleic Acids Res 25:3389–3402.
5. Do C-B, Mahabhashyam M-S-P, Brudno M (2005) ProbCons: Probabilistic consistency-based multiple sequence alignment. Genome Res 15:330–340.
6. Lassmann T, Sonnhammer E-L-L (2005) Kalign - an accurate and fast multiple sequencealignment algorithm. BMC Bioinformatics 6:298.
7. Chenna R, Sugawara H, Koike T (2003) Multiple sequence alignment with the Clustalseries of programs. Nucleic Acids Res 31:3497–3500.
8. Henikoff S, Henikoff J-G (1993) Performance evaluation of amino-acid substitutionmatrices. Protein-Structure Function and Genetics 17:49–61.
9. Cuff J-A, Clamp M-E, Siddiqui A-S, Finlay M, Barton G-J (1998) Jpred: A ConsensusSecondary Structure Prediction Server. Bioinformatics 14:892–893.
10. Cole C, Barber J-D, Barton G-J (2008) The Jpred 3 secondary structure predictionserver. Nucleic Acids Res. 36(Web Server issue):W197–W201.
11. Marti-RenomM-A, et al. (2000) Comparative protein structure modeling of genes andgenomes. Annu Rev Biophys Biomol Struct 29:291–325.
12. Sali A, Blundell T-L (1993) Comparative protein modelling by satisfaction of spatialrestraints. J Mol Biol 234:779–815.
Fig. S1. Computational homology model of the TRPC3 pore region, based on the structure of KcsA and Kv1.2. (A) Side view on TRPC3 TM5 + TM6 of twoopposite subunits (Left) and view from the extracellular side (Right) showing the pore structure of the homotetrameric channel. Charged glutamate residuesare marked in red, green, and yellow. (B) Sequence alignment of the putative pore region of KcsA and TRPC3. Glutamate and aspartate residues of the TRPC3pore region and the corresponding amino acid residues of KcsA are indicated as well as the calculated accessibility prediction (B = buried) and the accessibilityprediction probability value (0–9) for the corresponding residue. Charged amino acids E616, E630, and D639 appear accessible for ion binding.
Poteser et al. www.pnas.org/cgi/content/short/1106183108 2 of 7
Fig. S3. Currents through TRPC3-E630Q do not display changes in reversal potential upon reduction in extracellular Ca2+. (A) Current recordings of un-stimulated (control, 1) or stimulated (+ CCh, 100 μM) TRPC3-E630Q expressing HEK293 cells in Na+/Ca2+ solution (2) and Na+ only (3). (B) Mean reversal potential(± SEM, n > 6) of carbachol-induced currents in TRPC3-E630Q expressing HEK293 cells in Na+/Ca2+ solution and Na+ only.
Fig. S2. Stimulation by carbachol does not induce currents in TRPC3-E630K transfected HEK293 cells. (A) Representative current recordings of HEK293 cellstransfected with TRPC3-E630K (E630K) unstimulated (basal, black) and stimulated with 100 μM carbachol (red). (B) Mean current densities (± SEM, n > 6) ofHEK293 cells transfected with TRPC3-WT unstimulated (basal), HEK293 cells transfected with either TRPC3-WT or E630K and stimulated with 100 μM carbachol(+ CCh). Asterisk indicates statistical significant difference to stimulated TRPC3-WT expressing cells.
Poteser et al. www.pnas.org/cgi/content/short/1106183108 3 of 7
Fig. S4. Receptor stimulated Ca2+ entry into TRPC3-WT transfected HEK293 cells (A) is insensitive to 3 μM nifedipine (Nif, arrow) but inhibited by 3 μM Pyr3and moderately reduced by acute administration (arrow) of 5 μM KB-R7943 (KBR), whereas receptor-stimulated Ca2+ entry in atrial myocytes (HL-1; B) is highlysensitive to inhibtion 3 μM nifedipine (Nif, arrow) as well as 3 μM Pyr3, but not to acute administration of 5 μM KBR (arrow). (Left) Representative traces offura-2 Ca2+-imaging experiments. Arrows indicate the time points of addition of either 100 nM endothelin or the inhibitory drugs. (Right) Inhibition (in percent ±SEM, n > 30) of readdition-induced Ca2+ plateau by 3 μM Pyr3, 3 μM Nif, or 5 μM KBR. Arrowheads denote values close to zero.
Poteser et al. www.pnas.org/cgi/content/short/1106183108 4 of 7
Fig. S5. TRPC3 channels are still active at the time of Ca2+ readdition in fura-2 imaging experiments. (A) Representative time course of currents at 70 mVand −70 mV in TRPC3-WT expressing HEK293 cells stimulated with 100 μM carbachol in the absence of extracellular Ca2+. (B) Mean current densities (70 mV,−70 mV, ± SEM) of TRPC3wt expressing HEK293 cells, recorded during before stimulation (1) and 100 s after stimulation with carbachol (time point of Ca2+
entry initiation in fura-2 imaging experiments; 2).
Fig. S6. The pyrazole compound Pyr3, but not KB-R7943, inhibits NFAT translocation in TRPC3-WT expressing HEK293 cells. (Left) Mean nuclear/cytosolfluorescence intensity ratio (± SEM, n > 11) of HEK293 cells expressing GFP-NFAT and TRPC3-WT before (basal) and after stimulation with 100 μM carbachol(CCh) and application of the same protocol as used in fura-2 experiments in the absence or presence of 5 μM KB-R7943 (KBR) or 3 μM Pyr3. Asterisks indicatestatistically significant difference to basal conditions. (Right) Representative images of NFAT translocation at basal conditions (basal), after stimulation with100 μM carbachol (+CCh), and after stimulation by carbachol after incubation by 5 μM KB-R7943 (KBR) or 3 μM Pyr3. Individual nuclear/cytosol fluorescenceratios are indicated. Positions of nuclei are indicated by arrowheads.
Poteser et al. www.pnas.org/cgi/content/short/1106183108 5 of 7
Fig. S7. Thapsigargin promotes NFAT translocation in TRPC3-WT expressing HEK293 cells, but not in HL-1 cells. (A) Mean nuclear/cytosolic NFAT-GFP fluorescenceratio (± SEM, n > 11) in unstimulated cells (basal), in cells challenged by 100 μM carbachol (CCh) or 1 μM thapsigargin (TG). (A Right) Representative images ofNFAT localization before Ca2+ readdition (control), after Ca2+ readdition (basal), after stimulation by 100 μM CCh or 1 μM TG and subsequent Ca2+ readdition.Nuclear/cytosol fluorescence ratios and positions of nuclei (arrowheads) are indicated. (B) Mean nuclear/cytosolic NFAT-GFP fluorescence intensity ratio (± SEM, n >8), in unstimulated HL-1 cells (basal), HL-1 cells challenged by 1 μM TG, and TG stimulated in the presence of 3 μM nifedipine (Nif). (B Right) Representative imagesof NFAT translocation before stimulation and Ca2+ readdition (control), after Ca2+ readdition (basal), after stimulation by 1 μM TG and subsequent Ca2+ readdition(+ TG) as well as after stimulation and Ca2+ readdition in the presence of 3 μMnifedipine (+ TG + Nif). Individual nuclear/cytosol fluorescence ratios and positions ofnuclei are indicated (arrows). Standard Ca2+ readdition protocols were used. Asterisks indicate statistical significance vs. basal.
Poteser et al. www.pnas.org/cgi/content/short/1106183108 6 of 7
Fig. S8. Heteromeric channels consisting of the mutant TRPC3-T573A (MWK) and TRRPC3-WT (MWK:WT = 3:1) show enhanced current densities and per-sistent activity upon stimulation in HEK293 cells. (A) Representative time-course of currents at 70 mV and −70 mV recorded from HEK293 cells expressingTRPC3-T573A (MWK, circles) or TRRPC3-WT (squares) and stimulated with 100 μM carbachol (arrow). (B) Mean current densities (± SEM, n > 8) of TRPC3-WT andTRPC3-T573A (MWK) expressing HEK293 cells at basal, unstimulated conditions (B) and peak current after stimulation with 100 μM carbachol (P). (B Right)Representative images of HEK293 cells expressing TRPC3-WT or TRPC3-T573A (MWK)/TRPC3-WT (3:1). (C) Current recordings of unstimulated (black) andstimulated (red, carbachol 100 μM) TRPC3-T573A/TRPC3-WT (3:1, MWK; Left) and TRPC3-E630Q (E630Q; Right) expressing HEK293 cells in Na+-free, Ca2+
containing solution (2 mM).
Poteser et al. www.pnas.org/cgi/content/short/1106183108 7 of 7
38
Novel pyrazole compounds for pharmacological discrimination
between receptor-operated and store-operated Ca2+ entry pathways.
Br J Pharmacol (2012) - submitted 31.01.2012
H Schleifer1, B Doleschal2, M Lichtenegger2, R Oppenrieder2, I Derler3, I Frischauf3, T N
Glasnov4, C O Kappe4, C Romanin3 and K Groschner1,2
1
Institute of Biophysics, Medical University of Graz, 8010 Graz, Austria
2 Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, 8010
Graz, Austria
3 Institute for Biophysics, University of Linz, 4040 Linz, Austria and
4 Christian Doppler Laboratory for Microwave Chemistry, Institute of Chemistry, University of Graz, 8010 Graz,
Austria
For Peer Review
Novel pyrazole compounds for pharmacological discrimination between receptor-operated and store-
operated Ca2+ entry pathways
Journal: British Journal of Pharmacology
Manuscript ID: 2012-BJP-0113-RP
Manuscript Type: Research Paper
Date Submitted by the Author:
31-Jan-2012
Complete List of Authors: Schleifer, Hannes; Medical University of Graz, Institute of Biophysics
Doleschal, Bernhard; University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology Lichtenegger, Michaela; University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology Oppenrieder, Regina; University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology Derler, Isabella; University of Linz, Institute of Biophysics Frischauf, Irene; University of Linz, Institute of Biophysics Glasnov, Toma; University of Graz, Christian Doppler Laboratory for Microwave Chemistry, Institute of Chemistry
Kappe, Christian; University of Graz, Christian Doppler Laboratory for Microwave Chemistry, Institute of Chemistry Romanin, Christoph; University of Linz, Institute of Biophysics Groschner, Klaus; Medical University of Graz, Institute of Biophysics; University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology
Major area of pharmacology:
Endocrine pharmacology
Cross-cutting area: Immunopharmacology, Electrophysiology
Additional area(s): Ion channels, TRP, ROCs
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Novel pyrazole compounds for pharmacological discrimination between
receptor-operated and store-operated Ca2+
entry pathways
H Schleifer1, B Doleschal2, M Lichtenegger2, R Oppenrieder2, I Derler3, I Frischauf3, T N
Glasnov4, C O Kappe4, C Romanin3 and K Groschner1,2*
Institutions
1Institute of Biophysics, Medical University of Graz, Harrachgasse 21/IV, 8010 Graz, Austria
2Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology,
University of Graz, Universitätsplatz 2, 8010 Graz, Austria
3Institute for Biophysics, University of Linz, Gruberstraße 40-42, 4040 Linz, Austria
4Christian Doppler Laboratory for Microwave Chemistry, Institute of Chemistry, University
of Graz, Heinrichstraße 28, 8010 Graz, Austria
Corresponding Author:
Dr. Klaus Groschner
Email: [email protected]
Running Header:
Modulation of cellular Ca2+ handling by pyrazoles
Page 3 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Summary:
Background and purpose
Pyrazole derivatives have recently been suggested as selective blockers of TRPC channels but
their ability to distinguish between TRPC and Orai pore complexes is ill-defined. This study
was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai
selectivity and to delineate consequences of selective suppression of these pathways for mast
cell activation.
Experimental approach
Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca2+ entry
by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were
performed in HEK293 cells overexpressing TRPC3, in RBL-2H3 mast cells, which express
classical store-operated Ca2+ entry mediated by Orai channels and HEK293 cells
overexpressing Orai1 along with Stim1. The consequences of inhibitory effects on Ca2+
signalling in RBL-2H3 cells were investigated at the level of both degranulation and NFAT
activation
Key Results
Pyr3, a previously suggested selective inhibitor of TRPC3 channels, inhibited Orai1- and
TRPC3-mediated Ca2+ entry and currents as well as mast cell activation with similar potency.
By contrast, Pyr6 exhibited an about >30-fold higher potency to inhibit Orai1-mediated Ca2+
entry as compared to TRPC3-mediated Ca2+ entry and potently suppressed mast cell
activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated
responses (>50-fold) and the selective block of TRPC3 channels by Pyr10 barely affected
mast cell activation.
Conclusions and Implications
Page 4 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-
mediated Ca2+ entry and may serve as useful tools for the analysis of cellular functions of the
underlying Ca2+ channels.
Keywords:
pyrazole Ca2+ channel blockers, transient receptor potential (TRPC), Orai, store-operated Ca2+
entry (SOCE), receptor operated Ca2+ entry (ROCE), NFAT signalling, mast cell
degranulation
Page 5 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Introduction
Changes in cytosolic Ca2+ concentration control a broad range of cell- and tissue specific
processes reaching from B-cell activation, mast cell degranulation or cardiac pathologies to
cell proliferation and gene expression. Ca2+ entry via plasma membrane channels can be
mediated by a diverse array of extra- and intracellular stimuli (Berridge et al., 2000).
Characterization of the mechanisms that govern Ca2+ channel function has resulted in a
commonly accepted distinction between “receptor operated Ca2+ entry” (ROCE) pathways
that take place in response to receptor agonist/ligand-induced phospholipase C-mediated
phosphoinositol-4,5-bisphosphate (PIP2) hydrolysis formation (Hofmann et al., 1999;
Lemonnier et al., 2008) and “store operated Ca2+ entry (SOCE), which is activated as a
consequence of depletion of endoplasmic reticulum (ER) Ca2+ stores. Until the discovery of
stromal interaction molecule 1 (STIM1) and Orai1 as key components of the later process
(Prakriya et al., 2006; Zhang et al., 2005) the family of canonical transient receptor potential
channels (TRPC) (Abramowitz et al., 2009; Nilius et al., 2007; Pedersen et al., 2005) has
been considered the prime candidates for both Ca2+ entry pathways. Due to inherent overlap
and crosstalk of the two mechanisms as well as the paucity of model systems that
unequivocally lack one of these components a clear-cut distinction between Orai1-meditated
SOCE and TRPC-channel-mediated ROCE appears difficult. Moreover, physical interactions
between these two channel proteins in either a direct or indirect way has been proposed
(Cheng et al., 2011; Jardin et al., 2008; Liao et al., 2008; Liao et al., 2007; Woodard et al.,
2010; Yuan et al., 2009) and both Ca2+ influx pathways are tightly linked to downstream gene
transcription via nuclear factor of activated T-cells (NFAT) (Gwack et al., 2007; Sinkins et
al., 2004).
Therefore, specific and potent pharmacological tools are highly desirable for further analysis
of the contribution of either protein species or Ca2+ channel complexes to Ca2+ signalling and
Page 6 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
downstream cellular events. Inorganic blockers like Gd3+ or La3+ had been used extensively
for this purpose considering a specific interaction with TRPC channel pores (Trebak et al.,
2002). Similarly, organic compounds such as SKF-96365 (Harteneck et al., 2011), 2-APB
(DeHaven et al., 2008) and SK66 (Ng et al., 2008) have also been used due to their blocking
effects on TRPC and store-operated Ca2+ conductance with half-maximal concentrations in
the low to intermediate micromolar range. All these pharmacological tools share common
drawbacks in terms of lacking specificity for blocking a certain Ca2+ entry channel. The
blockers not only lack selectivity for subtypes of TRPC channels (Harteneck et al., 2011), but
exert in addition complex effects on Ca2+ entry and currents mediated by the STIM1/Orai1
SOCE pathway (DeHaven et al., 2008).
3,5-bis(trifluoromethyl)pyrazole derivatives (BTPs), especially BTP2/Pyr2, in contrast have
been proposed as small molecule inhibitors with selectivity for SOCE. Historically, this tool
evolved from a class of immunosuppressants aside from Cyclosporine A or so called –“limus
drugs” such as Sirolimus and Tacrolimus (FK506) (Chen et al., 2002; Djuric et al., 2000;
Ishikawa et al., 2003). Although primarily affecting NFAT activation and as a consequence
cytokine production in immune cells, experiments showed that BTPs are capable of blocking
store depletion-activated Ca2+ entry into a wide variety of cells at nanomolar to low
micromolar concentrations with appreciable selectivity over voltage-gated Ca2+ entry. For a
general review on BTPs as SOCE blockers see (Sweeney et al., 2009).
Recently, Pyr3 a pyrazole derivate has been proposed as highly subtype-specific inhibitor of
TRPC3 channel activity (Kiyonaka et al., 2009). Specificity over other TRPC family
members and other TRP subtypes in transfected HEK293 cells has been clearly demonstrated
and the tricholoroarylic amide bond-linked side group was identified as relevant for this
property. Moreover, electron drawing side groups in C3 position of the pyrazole ring of BTP
and Pyrs were proposed as key structural determinants of the inhibitory effect (Law et al.,
2011). Studies demonstrating BTP2/Pyr2 as an inhibitor of receptor-operated TRPC
Page 7 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
functions, thus lacking selectivity for SOCE (He et al., 2005) and, in turn, Pyr3 as potent
inhibitor of SOCE (Kim et al., 2011; Salmon et al., 2010) raised doubts about suitability of
these compounds for pharmacological dissection of these Ca2+ entry mechanism and
supported the hypothesis of a substantial overlap of these pathways and/or contribution of
TRPC3 to SOCE. Applying a recently published synthesis strategy to generate pyrazole
derivatives (Obermayer et al., 2011), we characterized the selectivity of four pyrazole
compounds, including a new structure, designated as Pyr10, in cell systems that express high
levels of well characterized receptor-operated or store-operated Ca2+ channels. We employed
HEK293 cells overexpressing TRPC3 as a ROCE model and native RBL-2H3 mast cells as an
established Orai-mediated SOCE model (Di Capite et al., 2009).
Our results demonstrate the ability of two pyrazole derivatives to discriminate between the
classical Orai-mediated, highly Ca2+ selective signalling pathway and the phospholipase C-
dependent Ca2+ entry-mediated by TRPC channels, specifically by TRPC3. We present Pyr6
and Pyr10 as valuable tools to dissect these signalling pathways.
Methods
DNA, cell culture and transfection
RBL-2H3 and HEK293 cells were cultivated in DMEM medium (Invitrogen) supplemented
with 10% FBS. HEK293 cells seeded out in adequate density were transiently transfected by
lipofection using FuGENE® (Roche) according to manufacturer’s protocol with an n-
terminally YFP-tagged TRPC3 cDNA clone to be used as ROCE model or with a CFP-tagged
STIM1 and YFP-tagged Orai1 clone to reconstitute the CRAC pore in HEK293 cells (Muik et
al., 2008). For NFAT translocation experiments RBL-2H3 cells were electroporated with a
GFP-tagged NFAT construct (at 300 V and 275 µF) with 20 µg of DNA 12-18 h before and
Page 8 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
seeded out on glass coverslips. For detailed procedures and DNA constructs see (Poteser et
al., 2011). All subsequent measurements were performed at room temperature.
Reagents & synthesis of pyrazole compounds
If not mentioned otherwise chemicals were purchased from Sigma Aldrich. Pyrazole
compounds were synthesized according to a strategy as published recently (Glasnov et al.,
2009; Obermayer et al., 2011). Molecular properties were calculated using Molinspiration
Property Calculation Service (www.molinspiration.com).
Electrophysiology
Patch pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, resistance
3-5 MΩ). Currents were recorded at room temperature using a List EPC7 patch clamp
amplifier (HEKA Instruments). Signals were low-pass filtered at 3 and 10 kHz and digitized
with 5 kHz. For HEK293 cells voltage-clamp protocols (voltage ramps from -130 to +80 mV,
holding potential 0 mV) were controlled by pClamp software (Axon Instruments).
Extracellular solution (ECS) contained (in mM) 140 NaCl, 2 CaCl2, 2 MgCl2, 10 Glucose, pH
adjusted to 7.4 with NaOH. The pipette solution (ICS) contained (in mM) 120 cesium
methanesulphonate, 20 CsCl, 15 HEPES, 5 MgCl2, 3 EGTA, pH adjusted to 7.3 with CsOH.
To activate the TRPC3 current cells were challenged with 100 µM carbachol. For RBL-2H3
cells CRAC measurement, standard protocols and buffers were modified from (Derler et al.,
2009). In brief voltage ramps from -90 to +90 mV over 1 second (holding potential +30 mV)
were applied controlled by pClamp software. ECS contained (in mM) 130 NaCl, 5 CsCl, 1
MgCl2, 10 HEPES, 10 Glucose, 20 CaCl2 at pH 7.4. ICS was comprised of 3.5 MgCl2, 145
cesium methanesulphonate, 8 NaCl, 10 HEPES, 20 EGTA at pH 7.2. Experiments in HEK-
293 cells expressing STIM1 and Orai1 to reconstitute the CRAC pore were done as in (Muik
et al., 2008). ECS contained (in mM) 145 NaCl, 5 CsCl, 1 MgCl2, 10 HEPES, 10 Glucose, 10
Page 9 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
CaCl2 at pH 7.4. ICS was comprised of 3.5 MgCl2, 145 cesium methanesulphonate, 8 NaCl,
10 HEPES, 20 EGTA at pH 7.2. For measuring the sodium currents in divalent-free
conditions protocols and buffers from (Bergsmann et al., 2011) were used. If not mentioned
otherwise for the experiments cells were preincubated for 3 min and measured in presence of
either 3 µM Pyr2, Pyr3, Pyr6 or Pyr10.
Measurement of intercellular Ca2+
Cells were loaded with 2 µM Fura-2-AM (Molecular Probes) for 45 min in Optimem®
medium (Invitrogen) and washed. Cells were continuously perfused with Ca2+ free ECS buffer
for any cell type as above and either challenged by depletion of intracellular Ca2+ stores with
1 µM thapsigargin (RBL-2H3) for 5 min or by acute application of 100 µM carbachol
(HEK293). Pyrazole compounds were supplied in corresponding concentration in the buffer at
least 5 min before the start of the measurement. Agonists as well as inhibitors remained
present continuously. For Ca2+ readdition 2 mM extracellular CaCl2 was added. Excitation
light was supplied via a Polychrome II polychromator (TILL Photonics) and emission was
detected by a Sensicam CCDcamera (PCO Computer Optics). Ca2+-sensitive fluorescence
ratios (340 nm/380 nm excitation; 510 nm emission) were recorded and analysed by using
Axon Imaging Workbench (Axon Instruments).
NFAT translocation
For NFAT imaging experiments, coverslips with transfected RBL-2H3 cells were transferred
into nominally Ca2+-free RBL-2H3 buffer described above and incubated with thapsigargin (1
µM) for 5 min to deplete the internal Ca2+ stores. NFAT translocation was triggered by adding
2 mM extracellular CaCl2. Pyrazole compounds were present in all buffers at 10 µM. Basal
NFAT localization was assessed from cells before store depletion.
Page 10 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
GFP-NFAT translocation was monitored (488 nm laser excitation) with standard fluorescence
microscopy (Zeiss Axiovert equipped with Coolsnap HQ). Nuclear/cytosol fluorescence
intensity ratios of cells were calculated with ImageJ software.
Degranulation assay
Degranulation of RBL-2H3 cells was measured by determining the level of secreted β-
hexosaminidase similar to (Law et al., 2011). Cells were seeded out in 24-well plates and
grown to confluency. All subsequent incubations were done at 37°C. After washing with 2
mM CaCl2 containing RBL-2H3 buffer described above, cells were incubated with 10µM
pyrazole compounds for 15 min. Ionomycin was added to a final concentration of 0,4 µM per
well and incubation prolonged for further 30 min. 30 µl aliquots of each well’s supernatant
were transferred to 96 well plates containing 50 µl of 1,3 mg·ml-1 p-nitrophenyl-n-acetyl-β-d-
glucosaminide in 100 mM Na-citrate buffer (pH 4,5) as substrate. Aliquots of untreated and
triton incubated (final concentration 2 % triton, used in 1:10 dilution for substrate assay) cells
were used as basal and maximal granula content references. After 45 min the enzymatic
reaction was stopped with 50 µl 0,4 M glycine buffer (at pH 10.7). Absorbance was measured
at 405 nm in a plate reader.
Data evaluation and statistics
Data is presented as mean values +/- S.E.M. and was tested for statistical significance using
one-way ANOVA in Sigma Plot®. * indicates p < 0.05, ** p < 0.01 and *** p < 0.001.
Page 11 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Results
Nomenclature and structure of pyrazole compounds
Pyrazole compounds were designated according to the previous literature and for novel
structures following order of synthesis in our laboratory. Correct chemical nomenclature is
Pyr2/BTP2 - N-(4-(3,5-Bis(trifluoromethyl)-1H-pyrazole-1-yl)phenyl)-4-methyl-1,2,3-
thiadiazole-5-carboxamide, Pyr3 - Ethyl 1-(4-(2,3,3-Trichloroacrylamido)phenyl)-5-
(trifluoromethyl)-1H-pyrazole-4-carboxylate, Pyr6 - N-(4-(3,5-bis(trifluoromethyl)-1H-
pyrazol-1-yl)phenyl)-3-fluoroisonicotinamide, Pyr10 - N-(4-(3,5-Bis(trifluoromethyl)-1H-
pyrazole-1-yl)phenyl)-4-methylbenzenesulfonamide (Obermayer et al., 2011). As illustrated
in figure 1 Pyr2, Pyr6 and Pyr10 share the common backbone of BTPs with a trifluoromethyl-
group on position C3 and C5 of the pyrazole ring, whereas Pyr3 lacks this at position C3 and
is substituted with a carboxylate group on position C4 of the pyrazole ring. Estimating the
octanol-water-coefficient (logP) of the four compounds regarding the hydro-/lipophilic
behaviour revealed two groups Pyr2, Pyr3 and Pyr6 (values 3.87; 3.89 and 3.84) versus a
higher value of 5.14 for Pyr10. Calculation of the total molecular polar surface area (tPSA) as
second indicator for membrane permeability yielded 59.81 Å (Pyr6), 63.99 Å (Pyr10), 72.71
Å (Pyr2) and 73.23 Å (Pyr3), and did not suggest substantial differences in membrane
permeability of these compounds.
Potency and selectivity of pyrazole compounds in ROCE and SOCE model systems
The group of Mori reported a high selectivity of Pyr3 for TRPC3 channels as compared to
channels formed by other TRPC species (Kiyonaka et al., 2009). The potency and selectivity
of this compound for classical SOCE, which was repeatedly suggested to overlap with TRPC
signalling, has so far not been delineated. By contrast, another pyrazole, Pyr2 (BTP2) is
commonly accepted as a pharmacological tool to inhibit classical SOCE pathways mediated
by Orai1 (Zitt et al., 2004). Here we set out to compare the TRPC/Orai1 selectivity of Pyr2
Page 12 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
and Pyr3 along with Pyr6, and a newly synthesized structure designated as Pyr10 (Figure 1).
The inhibitory potential of these four compounds on ROCE and SOCE was tested by
measuring Ca2+ entry into stimulated cells using Fura-2 and classical Ca2+ re-addition
protocols. As a ROCE model, HEK293 cells overexpressing YFP-tagged TRPC3, as a typical
lipid/second messenger-controlled TRPC channel were used. TRPC3 channels were activated
by stimulating endogenous muscarinic receptors with 100 µM carbachol (Mundell et al.,
2000; Thyagarajan et al., 2001). As a SOCE model native RBL-2H3 cells were employed,
which display the prototypical STIM1/Orai1-mediated SOCE based on the classical CRAC
conductance (Calloway et al., 2009; Hoth et al., 1992). In this system SOCE was activated by
passively depleting the intracellular stores with thapsigargin before Ca2+ readdition.
Table 1 and figure 2 show the calculated IC50 values obtained from fitted dose-response
curves. Notably, Pyr3 lacked selectivity for ROCE (TRPC3) in this test and displayed a
similar potency for SOCE inhibition in native RBL-2H3 cells like Pyr2, which, in line with
earlier reports, was found 7-fold (0.85 orders of magnitude [OM]) more potent in the SOCE
than in the ROCE model (Sweeney et al., 2009). Direct inhibition of the STIM1/Orai1-
mediated CRAC currents was confirmed by reconstitution of the CRAC pore in HEK293
cells. Inhibition of CRAC elicited by passive store depletion using EGTA (10 mM) in the
pipette solution, was observed upon acute administration in a, rapid and dose-dependent
manner. This effect was not readily reversed upon washout and was evident also in divalent-
free condition (supplemental figure 1) representing monovalent permeation through CRAC
channels (DeHaven et al., 2007).
The most striking selectivity was obtained with Pyr6 and Pyr10. In line with earlier reports
suggesting Pyr6 as a rather selective SOCE inhibitor (Sweeney et al., 2009; Yonetoku et al.,
2008), Pyr6, displayed 37-fold (1.58 OM) higher potency for RBL SOCE than for TRPC3
ROCE, with an IC50 comparable to Pyr2 or Pyr3. Interestingly, the sulphonamide-substituted
Page 13 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
compound Pyr10 by contrast, showed substantial selectivity for TRPC3 ROCE exhibiting
marginal inhibitory action on SOCE.
Pyr6 and Pyr10 – tools to distinguish between TRPC3 ROCE and STIM1/Orai1 SOCE
As Pyr6 and Pyr10 affected cellular Ca2+ handling in a divergent manner, we investigated
their effects on TRPC and CRAC channels more directly by electrophysiology. In line with
the Fura-2 imaging results, preincubation of cells with Pyr2 and Pyr3 at 3 µM cells
completely eliminated the Ca2+ entry-mediating conductances in both cell types (figure 3 and
table 2). On the contrary, Pyr6 and Pyr10 interfered with these conductances in a selective
manner. While completely inhibiting CRAC currents, Pyr6 at 3 µM diminished TRPC3
ROCE to only 52 %. Pyr10 (3 µM) eliminated TRPC3 ROCE currents but suppressed SOCE
in RBL-2H3 cells to only 60 % of control. These results suggest Pyr6 and Pyr10 as potential
tools for selectively affecting TRPC3 or Orai1 channels and demonstrate a clear lack of
selectivity for Pyr3.
Physiological consequences of Pyr-mediated inhibition of SOCE in RBL-2H3 cells
Due to the key role of Ca2+ acting as important second messenger for transduction of plasma
membrane signals to cellular functions including gene transcription, the effects of the four
compounds on NFAT translocation and mast cell degranulation were examined. As shown in
figure 4A and 4B SOCE inhibition by 10 µM Pyr2, Pyr3 or Pyr6 clearly inhibited NFAT
translocation, whereas the selective ROCE inhibitor Pyr10 failed to suppress NFAT activation
significantly.
RBL-2H3 degranulation initiated with ionomycin was inhibited by all pyrazol compounds,
with Pyr10 being the weakest inhibitor. At 10 µM, the potent SOCE inhibitors Pyr2, Pyr3 and
Pyr6 reduced degranulation to basal levels, while Pyr10 (10 µM) prevented degranulation to
only 65% of control. Therefore, Orai1-mediated SOCE without significant contribution of
Page 14 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
TRPC permeation pathways is clearly demonstrated as a master regulator of degranulation
and Ca2+-dependent transcriptional control in RBL-2H3 cells.
Discussion and Conclusion
Pharmacological dissection of SOCE and ROCE pathways
In view of the currently incomplete understanding of the molecular structures involved in
agonist/receptor-operated control of Ca2+ entry into many native tissues, it is highly desirable
to identify potent inhibitors for specific Ca2+ channel pore structures that are controlled via
receptor-phospholipase C-dependent mechanisms. A principal problem is the typical
simultaneous activation of pathways activated by Ca2+ store depletion and by second
messenger mechanisms such as generation or depletion of lipid mediators. These mechanisms
not only overlap upon stimulation of PLC but may also both involve TRPC channel proteins
as essential components (Putney, 2004). Nonetheless, pharmacological dissection of these
mechanisms appears possible, based on different pore complexes of receptor/second
messenger-operated and store-operated channels. Prototypical molecules mediating these Ca2+
entry mechanism are on the one hand TRPC3, which, upon overexpression, forms
diacylglycerol-regulated non selective cation channels and on the other hand Orai1, which
forms a highly Ca2+ selective channel activated by interaction of the ER Ca2+ sensor STIM1 in
response to a reduction of ER Ca2+ levels. Here we tested several potential blockers of TRPC
and Orai channels for selectivity including a recently synthesized, novel pyrazol compound
termed Pyr10. We report the ability of two pyrazole compounds Pyr6 and Pyr10 to
discriminate between receptor-operated TRPC3 and native STIM1/Orai1 channels. At low
micromolar concentrations, the novel structure Pyr10 completely eliminated TRPC3 currents
as well as Ca2+ entry while exerting modest effects on Orai-mediated responses. Pyr6
Page 15 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
displayed inverse selectivity, resulting in weak inhibition of TRPC3 currents at concentrations
that eliminated Orai-mediated currents. Interestingly, the recently proposed TRPC3-selective
blocker Pyr3 (Kiyonaka et al., 2009) as well as Pyr2, a previously suggested preferential
inhibitor of store-operated Ca2+ entry and inhibitor of the classical CRAC current in immune
cells, was barely able to distinguish between TRPC3 and Orai1 (He et al., 2005; Kiyonaka et
al., 2009; Zitt et al., 2004). Our current findings confirm the principle activity of pyrazole
derivatives as inhibitors of CRAC currents and, thus, of Orai channels. For Pyr3 we found
that this compound rapidly suppresses both native CRAC currents as well as heterologously
reconstituted CRAC currents upon acute administration (supplementary Figure 1A and 1B).
The rapid, acute inhibitory effect of Pyr3 may be interpreted as an interaction of the pyrazole
with an extracellular target site at the Orai channel complex. Consistent with inhibition of
Orai channel activity, Pyr2, Pyr3 or Pyr6 substantially inhibited typical Orai downstream
signalling events in RBL mast cells (NFAT activation and degranulation) activated by passive
store depletion. Unequivocally, Pyr2 and Pyr3 inhibit also TRPC-mediated Ca2+ entry (He et
al., 2005; Kiyonaka et al., 2009) and investigations performed in human neutrophils (Salmon
et al., 2010), pancreatic and salivary gland acinar cells (Kim et al., 2011) demonstrated
inhibition of SOCE by Pyr3, which was interpreted as a contribution of TRPC proteins in
SOCE phenomena (Salmon et al., 2011). However, insufficient selectivity of Pyr3 in terms of
discrimination between TRPC and Orai channel pores as demonstrated here, weakens this
conclusion.
The observed selectivity of Pyr6 and Pyr10 suggest that these compounds may be useful to
identify and analyse TRPC- and Orai-mediated conductances in native tissues. Our results
obtained in RBL-2H3 mast cells and STIM1/Orai1-expressing HEK293 cells were in line
with the concept that store-operated Ca2+ entry in these two cell systems occurs via the same
channels, which are characterized by sensitivity to Pyr6 being clearly higher than to Pyr10.
By contrast TRPC3 homomeric pore structures are highly sensitive to Pyr10 but weakly
Page 16 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
sensitive to Pyr6. Thus, we suggest a pyrazole sensitivity of Pyr6 > Pyr10 as a characteristics
of Orai1-mediated Ca2+ entry. As RBL-2H3 cells express TRPC genes including TRPC3 (Ma
et al., 2008), our results may be taken as an indication that TRPC3 does not contribute to
store-operated Ca2+ entry in mast cells. Nonetheless, we cannot exclude contribution of a
TRPC3 containing channel complex in local Ca2+ signalling events that are not detectable as
global cellular Ca2+ changes but could be pivotal for certain downstream signalling processes
as recently reported for cardiac TRPC3 channels (Poteser et al., 2011). Our finding of NFAT
translocation being highly sensitivity to Pyr6 but not to Pyr10 is in line with the concept that
in RBL-2H3 cells, NFAT is exclusively activated via Orai channels (Gwack et al., 2007). It is
of note that pyrazole structures have initially been recognized as effective inhibitors of NFAT
signalling via ill-defined mechanism downstream of Ca2+ signalling (Djuric et al., 2000;
Trevillyan et al., 2001). Here we report that the inhibitory effects of Pyr6 and Pyr10 correlate
well with their efficacy as CRAC/Orai1 inhibitors. The observation that Pyr6 was more potent
than Pyr10 as suppressant of mast cell degranulation corroborates the view that Orai channels
represent the main source of Ca2+ for exocytosis in RBL-2H3 cells. Consistent with previous
reports, Pyr6 was found highly effective as inhibitor of immune cell transcriptional activation
and cytokine production (Birsan et al., 2004; Ishikawa et al., 2003; Law et al., 2011;
Shirakawa et al., 2010), underscoring the potential value of this chemical structure for the
development of potent immune modulators (Chen et al., 2002; Zitt et al., 2004).
Structural basis of selective modulation of Ca2+
signalling by pyrazoles
The structural basis of Ca2+ entry block by pyrazoles has recently been analysed. The C3
position in the pyrazole ring was recognized as a critical determinant of the inhibitory effects
on mast cell degranulation, most likely corresponding to inhibition of SOCE. Block of SOCE
apparently requires substitution with bulky, electron-drawing groups in C3. However, a
similar substitution in C5, as present in Pyr3, by itself was found insufficient to provide
Page 17 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
SOCE blocking activity. (Law et al., 2011) Our finding of a high SOCE blocking potency of
Pyr3 indicates that introduction of an ethyl-carboxylate in C4 of Pyr3 restores inhibitory
potency although appropriate C3 substitution is lacking. Interestingly, substitution in C4 was
suggested of importance for the Pyr3 blocking potency regarding TRPC family members
(Kiyonaka et al., 2009). Moreover, this later study also proposed the amid-bond linked side-
group as pivotal for TRPC channel subtype selectivity, with the tricholoraryl-substitution in
Pyr3 as the TRPC3 selectivity encompassing structural element. This amid-bond linked side
group is, according to our results, also a potential structural determinant for the CRAC/SOCE
inhibitory action, as this structural feature is lacking in the weak SOCE inhibitor Pyr10.
Notably, Pyr10 contains a sulphonamide-linked side-group at the BTP backbone. One might
speculate about substantial changes within the molecule generated by different interactions
between the backbone and the side-group resulting in structures that discriminate between the
two channel types. As electronegativity and polarity of the amine-linked side groups in Pyr3
and Pyr10 are similar, it is tempting to speculate that these structures are essential for
inhibition of ROCE with high potency.
In this study we obtained evidence for a possible value of pyrazoles as selective modulators of
cellular Ca2+ handling, widening the view on both their therapeutic potential as
immunosuppressants as well as their utility for experimental dissection of Ca2+ signalling
pathways. In aggregate, we introduce a novel pharmacological approach to distinguish
between second messenger-gated TRPC-mediated and store-operated, Orai-mediated Ca2+
entry using selective pyrazole compounds including Pyr10 as a novel TRPC3-selective
inhibitor. The identified ability of certain pyrazole structures to discriminate between
receptor- and store operated Ca2+ signalling pathway is expected to pave the way towards
both better experimental analysis and understanding of Ca2+ entry mechanisms in native
tissues and for the development of novel therapeutic strategies.
Page 18 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Acknowledgements
This work was funded by FWF (Austrian research fund) projects P21925-B19 (to K.
Groschner), P22565-B18 (to C. Romanin) and the DK+ Metabolic and Cardiovascular Disease
grant W2126-B18. We like to thank Renate Schmidt and Ines Neubacher for their technical
assistance, Sonia Stürmer for support with preliminary experiments and Dr. Kehlenbach for
kindly providing the GFP-NFAT construct.
References
Abramowitz J, Birnbaumer L (2009). Physiology and pathophysiology of canonical transient
receptor potential channels. FASEB J 23(2): 297-328.
Bergsmann J, Derler I, Muik M, Frischauf I, Fahrner M, Pollheimer P, et al. (2011).
Molecular determinants within N terminus of Orai3 protein that control channel activation
and gating. J Biol Chem 286(36): 31565-31575.
Berridge MJ, Lipp P, Bootman MD (2000). The versatility and universality of calcium
signalling. Nat Rev Mol Cell Biol 1(1): 11-21.
Birsan T, Dambrin C, Marsh KC, Jacobsen W, Djuric SW, Mollison KW, et al. (2004).
Preliminary in vivo pharmacokinetic and pharmacodynamic evaluation of a novel calcineurin-
independent inhibitor of NFAT. Transpl Int 17(3): 145-150.
Page 19 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Calloway N, Vig M, Kinet JP, Holowka D, Baird B (2009). Molecular clustering of STIM1
with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+
stores and on electrostatic interactions. Mol Biol Cell 20(1): 389-399.
Chen Y, Smith ML, Chiou GX, Ballaron S, Sheets MP, Gubbins E, et al. (2002). TH1 and
TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles, a novel class of
immunomodulators. Cell Immunol 220(2): 134-142.
Cheng KT, Liu X, Ong HL, Swaim W, Ambudkar IS (2011). Local Ca(2)+ entry via Orai1
regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca(2)+ signals
required for specific cell functions. PLoS Biol 9(3): e1001025.
DeHaven WI, Smyth JT, Boyles RR, Bird GS, Putney JW, Jr. (2008). Complex actions of 2-
aminoethyldiphenyl borate on store-operated calcium entry. J Biol Chem 283(28): 19265-
19273.
DeHaven WI, Smyth JT, Boyles RR, Putney JW, Jr. (2007). Calcium inhibition and calcium
potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels. J Biol
Chem 282(24): 17548-17556.
Derler I, Fahrner M, Carugo O, Muik M, Bergsmann J, Schindl R, et al. (2009). Increased
hydrophobicity at the N terminus/membrane interface impairs gating of the severe combined
immunodeficiency-related ORAI1 mutant. J Biol Chem 284(23): 15903-15915.
Di Capite J, Parekh AB (2009). CRAC channels and Ca2+ signaling in mast cells. Immunol
Rev 231(1): 45-58.
Page 20 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Djuric SW, BaMaung NY, Basha A, Liu H, Luly JR, Madar DJ, et al. (2000). 3,5-
Bis(trifluoromethyl)pyrazoles: a novel class of NFAT transcription factor regulator. J Med
Chem 43(16): 2975-2981.
Glasnov TN, Groschner K, Kappe CO (2009). High-speed microwave-assisted synthesis of
the trifluoromethylpyrazol-derived canonical transient receptor potential (TRPC) channel
inhibitor Pyr3. ChemMedChem 4(11): 1816-1818.
Gwack Y, Feske S, Srikanth S, Hogan PG, Rao A (2007). Signalling to transcription: store-
operated Ca2+ entry and NFAT activation in lymphocytes. Cell Calcium 42(2): 145-156.
Harteneck C, Gollasch M (2011). Pharmacological modulation of diacylglycerol-sensitive
TRPC3/6/7 channels. Curr Pharm Biotechnol 12(1): 35-41.
He LP, Hewavitharana T, Soboloff J, Spassova MA, Gill DL (2005). A functional link
between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole
derivative, BTP2. J Biol Chem 280(12): 10997-11006.
Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G (1999). Direct
activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397(6716): 259-
263.
Hoth M, Penner R (1992). Depletion of intracellular calcium stores activates a calcium current
in mast cells. Nature 355(6358): 353-356.
Page 21 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Ishikawa J, Ohga K, Yoshino T, Takezawa R, Ichikawa A, Kubota H, et al. (2003). A
pyrazole derivative, YM-58483, potently inhibits store-operated sustained Ca2+ influx and
IL-2 production in T lymphocytes. J Immunol 170(9): 4441-4449.
Jardin I, Lopez JJ, Salido GM, Rosado JA (2008). Orai1 mediates the interaction between
STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+
channels. J Biol Chem 283(37): 25296-25304.
Kim MS, Lee KP, Yang D, Shin DM, Abramowitz J, Kiyonaka S, et al. (2011). Genetic and
pharmacologic inhibition of the Ca2+ influx channel TRPC3 protects secretory epithelia from
Ca2+-dependent toxicity. Gastroenterology 140(7): 2107-2115, 2115 e2101-2104.
Kiyonaka S, Kato K, Nishida M, Mio K, Numaga T, Sawaguchi Y, et al. (2009). Selective
and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole
compound. Proc Natl Acad Sci U S A 106(13): 5400-5405.
Law M, Morales JL, Mottram LF, Iyer A, Peterson BR, August A (2011). Structural
requirements for the inhibition of calcium mobilization and mast cell activation by the
pyrazole derivative BTP2. Int J Biochem Cell Biol 43(8): 1228-1239.
Lemonnier L, Trebak M, Putney JW, Jr. (2008). Complex regulation of the TRPC3, 6 and 7
channel subfamily by diacylglycerol and phosphatidylinositol-4,5-bisphosphate. Cell Calcium
43(5): 506-514.
Liao Y, Erxleben C, Abramowitz J, Flockerzi V, Zhu MX, Armstrong DL, et al. (2008).
Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated
Page 22 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
heteromeric Orai/TRPC model for SOCE/Icrac channels. Proc Natl Acad Sci U S A 105(8):
2895-2900.
Liao Y, Erxleben C, Yildirim E, Abramowitz J, Armstrong DL, Birnbaumer L (2007). Orai
proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl
Acad Sci U S A 104(11): 4682-4687.
Ma HT, Peng Z, Hiragun T, Iwaki S, Gilfillan AM, Beaven MA (2008). Canonical transient
receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal
influx of Ca2+, and degranulation in a rat mast cell line. J Immunol 180(4): 2233-2239.
Muik M, Frischauf I, Derler I, Fahrner M, Bergsmann J, Eder P, et al. (2008). Dynamic
coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel
activation. J Biol Chem 283(12): 8014-8022.
Mundell SJ, Benovic JL (2000). Selective regulation of endogenous G protein-coupled
receptors by arrestins in HEK293 cells. J Biol Chem 275(17): 12900-12908.
Ng SW, di Capite J, Singaravelu K, Parekh AB (2008). Sustained activation of the tyrosine
kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated
Ca2+ channels. J Biol Chem 283(46): 31348-31355.
Nilius B, Owsianik G, Voets T, Peters JA (2007). Transient receptor potential cation channels
in disease. Physiol Rev 87(1): 165-217.
Page 23 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Obermayer D, Glasnov TN, Kappe CO (2011). Microwave-assisted and continuous flow
multistep synthesis of 4-(pyrazol-1-yl)carboxanilides. J Org Chem 76(16): 6657-6669.
Pedersen SF, Owsianik G, Nilius B (2005). TRP channels: an overview. Cell Calcium 38(3-
4): 233-252.
Poteser M, Schleifer H, Lichtenegger M, Schernthaner M, Stockner T, Kappe CO, et al.
(2011). PKC-dependent coupling of calcium permeation through transient receptor potential
canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes. Proc Natl Acad Sci U S A
108(26): 10556-10561.
Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A, Hogan PG (2006). Orai1 is an essential
pore subunit of the CRAC channel. Nature 443(7108): 230-233.
Putney JW, Jr. (2004). The enigmatic TRPCs: multifunctional cation channels. Trends Cell
Biol 14(6): 282-286.
Salmon MD, Ahluwalia J (2010). Discrimination between receptor- and store-operated
Ca(2+) influx in human neutrophils. Cell Immunol 265(1): 1-5.
Salmon MD, Ahluwalia J (2011). Pharmacology of receptor operated calcium entry in human
neutrophils. Int Immunopharmacol 11(2): 145-148.
Shirakawa H, Sakimoto S, Nakao K, Sugishita A, Konno M, Iida S, et al. (2010). Transient
receptor potential canonical 3 (TRPC3) mediates thrombin-induced astrocyte activation and
upregulates its own expression in cortical astrocytes. J Neurosci 30(39): 13116-13129.
Page 24 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Sinkins WG, Goel M, Estacion M, Schilling WP (2004). Association of immunophilins with
mammalian TRPC channels. J Biol Chem 279(33): 34521-34529.
Sweeney ZK, Minatti A, Button DC, Patrick S (2009). Small-molecule inhibitors of store-
operated calcium entry. ChemMedChem 4(5): 706-718.
Thyagarajan B, Poteser M, Romanin C, Kahr H, Zhu MX, Groschner K (2001). Expression of
Trp3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+
handling: evidence for a role of Trp3 as a subunit of capacitative Ca2+ entry channels. J Biol
Chem 276(51): 48149-48158.
Trebak M, Bird GS, McKay RR, Putney JW, Jr. (2002). Comparison of human TRPC3
channels in receptor-activated and store-operated modes. Differential sensitivity to channel
blockers suggests fundamental differences in channel composition. J Biol Chem 277(24):
21617-21623.
Trevillyan JM, Chiou XG, Chen YW, Ballaron SJ, Sheets MP, Smith ML, et al. (2001).
Potent inhibition of NFAT activation and T cell cytokine production by novel low molecular
weight pyrazole compounds. J Biol Chem 276(51): 48118-48126.
Woodard GE, Lopez JJ, Jardin I, Salido GM, Rosado JA (2010). TRPC3 regulates agonist-
stimulated Ca2+ mobilization by mediating the interaction between type I inositol 1,4,5-
trisphosphate receptor, RACK1, and Orai1. J Biol Chem 285(11): 8045-8053.
Page 25 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Yonetoku Y, Kubota H, Miyazaki Y, Okamoto Y, Funatsu M, Yoshimura-Ishikawa N, et al.
(2008). Novel potent and selective Ca2+ release-activated Ca2+ (CRAC) channel inhibitors.
Part 3: synthesis and CRAC channel inhibitory activity of 4'-[(trifluoromethyl)pyrazol-1-
yl]carboxanilides. Bioorg Med Chem 16(21): 9457-9466.
Yuan JP, Kim MS, Zeng W, Shin DM, Huang G, Worley PF, et al. (2009). TRPC channels as
STIM1-regulated SOCs. Channels (Austin) 3(4): 221-225.
Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, et al. (2005). STIM1 is a
Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma
membrane. Nature 437(7060): 902-905.
Zitt C, Strauss B, Schwarz EC, Spaeth N, Rast G, Hatzelmann A, et al. (2004). Potent
inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the
pyrazole derivative BTP2. J Biol Chem 279(13): 12427-12437.
Page 26 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Figure legends
Figure 1: Chemical structures of tested pyrazole compounds
Molecular structures suggested of importance for Ca2+ channel blocking activity are
highlighted
Figure 2: Concentration dependence of Ca2+
entry inhibition by pyrazoles in TRPC3
overexpressing HEK293 cells and in native RBL-2H3 mast cells representing model
systems of receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+
entry (SOCE).
Fura-2 Ca2+ imaging experiments for YFP-TRPC3 transfected cells (ROCE model, blue
symbols and line) or native RBL-2H3 (SOCE model, red symbols and line) with fitted dose-
response curve. Inhibition is presented as percentage of peak Ca2+ entry level in cells without
incubation with pyrazole compound (minimum n ≥ 29 cells in 3 experiments for HEK293
YFP-TRPC3 cells and n ≥ 72 cells in 3 experiments for RBL-2H3 and each concentration).
HEK293 YFP-TRPC3 cells were challenged with carbachol (100 µM) to stimulate ROCE.
Native RBL-2H3 cells were incubated with thapsigargin (1 µM) before the experiment to
elucidate SOCE by depleting intracellular Ca2+ stores.
Figure 3: Divergent selectivity of Pyr6 and Pyr10 in blocking TRPC3- and
STIM1/Orai1-mediated membrane currents. (A) Top panel: Time course of currents
measured at -90 mv (n ≥ 7 experiments for each condition) after incubation with pyrazoles for
5 min and stimulation of HEK-293 cells transiently expressing TRPC3 with carbachol (100
µM). Lower panel: Representative I-V relations of carbachol-stimulated currents in cells pre-
treated with pyrazole compound versus control. (B) Top panel: Time course of CRAC
currents in native RBL-2H3 cells after incubation with pyrazoles and store depletion with
EGTA in the patch pipette (n ≥ 6). Lower panel: Representative I-V relation of EGTA-
induced currents in store depleted RBL-2H3 cells, pre-treated with pyrazole compound,
Page 27 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
versus control. Mean values ± S.E.M. are given. In all experiments pyrazoles were
administrated at 3 µM concentrations. Symbols/colours: filled black square/black trace -
untreated control, open orange square/orange trace - Pyr2, filled green triangle/green trace -
Pyr3, filled red circle/red trace - Pyr6, filled blue circles/blue trace - Pyr10
Figure 4: Pyrazol effects on NFAT activation and degranulation in RBL-2H3 cells.
(A) Mean values ± S.E.M. of NFAT nuclear to cytosolic ratio (n ≥ 18 cells for each condition)
Values were determined after depletion of intracellular Ca2+ stores with thapsigargin and
readdition of extracellular Ca2+ for control (thapsigargin only). Pyrazole treated (10 µM) cells
as indicated are compared to basal condition (dashed line). (B) Representative images of
NFAT localization and DIC microscopy images for basal, control and pyrazole-treated cells.
Arrows indicate positions of nuclei in fluorescence images. (C) Mean values ± S.E.M. of
native RBL-2H3 degranulation measured by a β-hexosaminidase-assay (n = 3 experiments
from different passages) for control (ionomycin only) and pyrazole treated (10 µM) cells
compared to basal degranulation (dashed line). Asterisks indicate statistical significant
difference.
Statement of conflict of interest
The authors declare no conflict of interest.
Page 28 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Tables
Pyrazole IC50 (ROCE) [µM] IC50 (SOCE) [µM]
Pyr2 4.21 0.59
Pyr3 0.55 0.41
Pyr6 18.46 0.49
Pyr10 0.98 n.a.
Table 1: IC50 of Ca2+
influx inhibition by pyrazoles in carbachol stimulated YFP-TRPC3
transfected HEK293 cells for ROCE or thapsigargin depleted native RBL-2H3 cells for
SOCE. n.a. = not applicable
Page 29 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Pyrazole IROCE
[pA·pF-1]
statistical
significance
ISOCE
[pA·pF-1]
statistical
significance
Pyr2 -1.54 ± 0.53 * -1,13 ± 0,16 ***
Pyr3 -2.27 ± 0.96 * -1.45 ± 0.19 ***
Pyr6 -9.50 ± 2.66 n.a. -1.31 ± 0.15 ***
Pyr10 -1.01 ± 0.57 * -4.49 ± 0.75 n.a.
no pyrazole -18.50 ± 3.47 * -7.50 ± 0.64 ***
Table 2: Peak ROCE and SOCE currents in the absence and presence of 3 µM
pyrazoles. Currents were measured at at maximal carbachol activation of ROCE in YFP-
TRPC3 transfected HEK-293 cells or at -85 mV 300 seconds after perforating the cell for
fully developed SOCE in native RBL-2H3 cells. Statistical significance was calculated
compared to Pyr6 incubated cells for ROCE and compared to Pyr10 for SOCE. Values are
mean values of net-currents (measured current minus basal current) ± SEM. n.a. = not
applicable
Page 30 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
946x700mm (600 x 600 DPI)
Page 31 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
134x112mm (300 x 300 DPI)
Page 32 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
1916x1454mm (600 x 600 DPI)
Page 33 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
155x161mm (300 x 300 DPI)
Page 34 of 34
British Pharmacological Society
British Journal of Pharmacology
For Peer Review
Supplementary data
Supplementary figure 1: Pyr3 acutely blocks a reconsituted CRAC pore in a dose-
dependent, non-reversible manner as well affecting sodium influx through it. (A) Time
course of measured currents (n = 4 cells) of acute Pyr3 inhibition of CRAC in a HEK293 cell
system expressing STIM1 and Orai1 to reconstitute the CRAC pore. (B) Time course of acute
Pyr3 inhibition (3µM) in native RBL-2H3 cells (n = 6 cells). (C) Time course of currents (n =
8 cells) with acute Pyr3 inhibition of a reconstituted CRAC pore. Pyrazole compound was
applied for only a limited time to examine reversibility of inhibitory effect. (D) Time course
of Pyr3-effect (10 µM) on divalent-free sodium current through CRAC-pore after activation
of current in Ca2+
containing buffer (n = 4 cells). All values are mean values ± S.E.M.
Page 35 of 34
British Pharmacological Society
British Journal of Pharmacology
73
TRPC3 links Orai1-mediated Ca2+ entry to CaN/NFAT activation in RBL
mast cells
PLoS one (2012) - in preparation
Hannes Schleifer1,2+, Bernhard Doleschal2+, Michael Poteser1,2, Michaela Lichtenegger2, Irene
Frischauf3, Christoph Romanin3, and Klaus Groschner1,2
+
authors contributed equally
1 Institute of Biophysics, Medical University of Graz, 8010 Graz, Austria
2 Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, 8010
Graz, Austria
3 Institute for Biophysics, University of Linz, 4040 Linz, Austria
TRPC3 links Orai1-mediated Ca2+
entry to calcineurin/NFAT activation in
RBL mast cells
Short title: TRPC3/Orai1 signalling partnership in RBL mast cells
Keywords: TRPC3, Orai1, PKC, NFAT signalling, Ca2+
signalling
Authors and Affiliations
Hannes Schleifer1,2+
, Bernhard Doleschal2+
, Michael Poteser1,2
, Michaela Lichtenegger2, Irene
Frischauf3, Christoph Romanin
3 and Klaus Groschner
1,2*
1Institute of Biophysics, Medical University Graz, 8010 Graz, Austria
2Institute of Pharmaceutical Sciences - Pharmacology and Toxicology, University of Graz,
8010 Graz, Austria
3Insitute of Biophysics, University of Linz, 4040 Linz Austria
+ authors contributed equally
* address for correspondence:
Klaus Groschner
Institute of Biophysics
Medical University Graz
Harrachgasse 21/IV
8010 Graz, Austria
phone: +43 316 380-4137
Fax: +43 316 380-9660
Email: [email protected]
Abstract
Orai and STIM proteins have been identified as the central constituents of store-operated Ca2+
entry into mast cells and are thereby essential in the control of degranulation and gene
transcription. As mast cells express also TRPC channels, which may as well contribute to
Ca2+
entry in response to lipid (PLC) signalling and have been found to interact physically
and functionally with FKBP/CaN complexes, we tested for a potential crosstalk between
Orai1 and TRPC3 signalling in terms of Ca2+
entry and CaN-mediated NFAT activation.
Although TRPC3 expression in mast cells showed a distinctively changed I/V relation, the
channel activity did not affect the amplitude of Orai1-mediated currents. Orai1 activity in
contrast seemed to be a determinant of TRPC3 function in mast cells, promoting TRPC3
presentation in the PM. For downstream signalling via the CaN/NFATpathway different
TRPC3 mutants with impaired protein-protein interaction or lacking regulatory
phosphorylation sites revealed a PKC-dependent scaffold function of the channel.
Therefore, we report here for the first time on an essential participation of TRPC3 in
STIM1/Orai1-mediated SOCE phenomena in RBL-2H3 mast cells.
Introduction
Ca2+
entry into cells and changes in cytosolic Ca2+
levels are important mediators for a vast
number of cellular processes [1]. In immune cells, especially RBL-2H3 mast cells, cytosolic
Ca2+
signals control the expression of interleukins and cytokines, degranulation and
proliferation [2]. It is well accepted that this Ca2+
entry is initiated after depletion of
intracellular stores like the ER in a variety of cells and the underlying Ca2+
entry mechanism
has been first described in immune cells as an inwardly rectifying, Ca2+
-selective current
[3,4,5].
For more than two decades the key proteins involved this process were sought to be TRPC
channels proteins. This “canonical” family of transient receptor potential channels, a family of
proteins identified by homology to a pore protein in the light-signal-transducing process of
drosophila, comprises of 6 members expressed in humans (plus one pseudogene expressed in
rodents) and is expressed in a range of tissues [6,7,8]. TRPC channels have been
demonstrated to contribute to cellular Ca2+
entry linked to lipid signalling initiated by Gq-
coupled receptors and PLC-dependent PIP2 hydrolysis into DAG and IP3. With the
identification of Orai1 as a store-operated Ca2+
channel protein and STIM1 as Ca2+
sensor
which cooperate to enable SOCE in mast cells a novel model for SOCE activation has been
established [9,10].
In basal conditions STIM1 is the ER-residing Ca2+
sensor, which upon store-depletion
transmits the reduced filling state of these Ca2+
stores to PM-localized, tetramer Orai1
channels. These channels are activated by direct protein-protein interaction with STIM1,
which beforehand clustered and translocated to PM-near ER [10,11,12].
Recent findings indicate a complex functional and maybe even physical interaction of STIM1
and Orai1 with TRPC channels. The group of S. Muallem showed that STIM1 interaction
with TRPC1 and TRPC3 is abrogated by mutations of only two charged aminoacids (aa 684
& 685 in STIM1 and 697 & 698 in TRPC3) [13]. Interaction of TRPC1, STIM1 and Orai1 in
various SOCE situations has been observed as well in salivary gland cells [14,15], leading to a
model of increased membrane representation of TRPCs second to Orai1-mediated Ca2+
entry
[16]. Furthermore, a SOCE and ROCE activation and protein-protein interaction model
involving Orai1 just as regulatory subunit with solely TRPC responsible for Ca2+
permeation
was proposed [17,18]. There an even more differentiated model of TRPC3 function in
receptor- or store-operated mode dependent on its localization in or out of lipid-rafts was
suggested too [19]. In HEK-293 cells formation of a STIM1/Orai1/TRPC3 complex with
RACK1 as mediator was found [20]. In aggregate this points to involvement of TRPCs in
STIM/Orai-mediated SOCE.
Here we explored the contribution of TRPC3 - a prototypical member of the TRPC-family
and STIM/Orai1 to SOCE in RBL-2H3 mast cells at the level of membrane currents as well as
on the level of Ca2+
-dependent gene transcription after NFAT activation. Our results
demonstrate that TRPC3 expression exerts only minor impact on Orai1-mediated SOCE. By
contrast, we observed a clear SOCE-dependence of TRPC3 channel function in RBL mast
cells and report hereon an essential scaffold function of TRPC3 in Orai1-mediated
Ca2+
/transcription coupling.
Results
STIM1/Orai1-mediated (SOCE) currents in RBL-2H3 cells
A standard store-depletion protocol (20 mM EGTA in the patch pipette) initiated a current in
RBL-2H3 mast cells, exhibiting the characteristics of the well described ICRAC (Fig. 1) [21].
This current was eliminated by overexpression of a dominant-negatively impermeable pore
mutant of Orai1 - Orai1E106Q [22] - substantiating the concept of this ICRAC conductance being
entirely based on STIM1/Orai1 channel complexes.
TRPC3-induced changes in RBL-2H3 SOCE current properties
Overexpression of wildtype TRPC3 generated a distinct change in the store-depletion-induced
conductance of RBL-2H3 cells. Reversal potential of the current shifted to neutral potential
and the I/V relation resembled that of the TRPC3 conductance typically observed in classical
expression systems such as HEK293. This was specifically evident by an outward rectifying
component of the store depletion-induced current (Fig. 2A middle panel) [23,24,25].
Evaluation of current densities indicated that the conductance observed in TRPC3
overexpressing cells was not a simple combination of Orai1-mediated ICRAC and a TRPC3
conductance. Comparing the net inward currents between vector transfected cells and TRPC3
expressing cells revealed a small but significantly reduced conductance at negative potentials.
This suggests a negative functional impact of TRPC3 on Orai1 activity (Fig 2A left & middle
panel).
Experiments expressing a dominant negative TRPC3 pore mutant of TRPC3 (Fig. 2A right
panel) [26] and use of Pyr3, a recently described TRPC3 blocker [27] demonstrated that the
observed conductance is based on permeation through the TRPC3 pore (Fig 2B upper left for
time course & lower panels for representative I/V). To exclude effects of Ca2+
buffering or
microdomain arrangement of TRPC3 and Orai1 [28,29], stores were also depleted with
BAPTA revealing the same characteristics as with EGTA (Fig. 2B upper left for time course
& upper right panel for representative I/V).
Orai1 function is essential for activation and plasma membrane presentation of TRPC3
Considering a potential role of Ca2+
entry through Orai channels in the observed activation of
TRPC3 in response to store depletion, we co-expressed TRPC3 and Orai1E106Q to knock-down
the Orai1 conductance. The results show complete elimination of SOCE currents (Fig. 3).
This is in-line with a previously proposed model of a dynamic functional interaction between
the two proteins potentially involving a Ca2+
dependent promotion of TRPC3 recruitment into
the PM [16,30].
The group of I. Ambudkar observed a similar crosstalk based on Orai1-mediated Ca2+
entry as
a key trigger for TRPC1 membrane presentation in human salivary gland cells. Ca2+
entering
via the CRAC pore appears to initiate fusion of TRPC1 containing vesicles with the PM
thereby increasing presentation of TRPC complexes in the cellular surface [16].
Consequently, we explored a potential change of TRPC3 PM presentation upon Orai1
activation using TIRF microscopy and co-expression of YFP-TRPC3 and CFP-Orai1 or CFP-
Orai1E106Q. Activation of SOCE by thapsigargin administration in Ca2+
-free solution and
subsequent Ca2+
re-addition showed increased YFP-fluorescence in the TIRF plain in cells
expressing wildtype Orai1 but not in cells expressing the dominant negative Orai1 mutant.
This result clearly suggested increased TRPC3 presentation produced by Orai1-dependent
Ca2+
permeation (Fig. 4A & 4B). Nonetheless, we did not detect any direct physical
interaction by FRET analysis. Moreover, fluorescence microscopy on cells expressing CFP-
tagged Orai1 and YFP-tagged TRPC3 revealed that the two channel types lacked a general
co-localization with an overlap of fluorescence only in restricted areas of the PM.
Orai-induced activation of CaN/NFAT signalling is dependent on TRPC3 and PKC-
phosphorylation
Our results identified a principle functional crosstalk between Orai and TRPC3 in terms of
membrane conductance. Consequently we set out to investigate the potential interaction of
these different Ca2+
entry pathways in terms of downstream signalling events. NFAT plays a
prominent role in linking Ca2+
entry to gene transcription [31]. Consistent with our
observation of neither unchanged nor even diminished ICRAC upon TRPC3 activity, nor
TRPC3wt neither TRPC3E630K affected NFAT translocation (Fig. 5A & 5B). Surprisingly,
TRPC3P704Q, a mutant lacking the binding site for FKB12 [32], showed complete suppression
of NFAT translocation. FKBP12 is an immunophilin connecting TRPCs to downstream
effectors like CaM/CaN and NFAT [32]. This interaction can be blocked by inhibition of
PKC-dependent phosphorylation of TRPC3 at aa 712 [33] or via application of the PKC
inhibitor GF109203X. Both conditions reduced or even suppressed NFAT translocation. As
illustrated in Fig. 5C both TRPC3 mutants still exhibit expected either reduced (TRPC3P704Q)
or increased (TRPC3S712A) permeability, proving their channel function. This indicates a
PKC-phosphorylation-dependent scaffold function of TRPC3.
Further investigation of this scaffold function was performed by co-expressing the channel
with CaN, the primary Ca2+
sensor and upstream signalling molecule required for NFAT
activation. We analysed the cellular localization of CaN and TRPC3 during cell activation.
These experiments revealed a distinct localization of the effector after store-depletion and
Ca2+
re-addition, when co-expressed with TRPC3wt (Fig. 6) but not when co-expressed with
TRPC3P704Q (data not shown). This CaN localization effect therefore matches with the NFAT
translocation.
Discussion
TRPC3 expression determines SOCE characteristics in RBL-2H3 mast cells
SOCE in RBL-2H3 cells is considered as mainly mediated by STIM1/Orai1 complexes and is
electrophysiologically characterized by the properties originally described as ICRAC. In terms
of cation influx our results indicate that TRPC3 expression either lacks effects on or may
slightly reduce native ICRAC. The observation was independent of the properties of the used
Ca2+
chelator, as the use of EGTA and BAPTA [28] generated similar results. This suggests
only little impact of TRPC3 activity on Orai1 function. Our results obtained with dominant-
negative constructs comprising impermeable pores (TRPC3E630K and Orai1E106Q) or
application of the blocking agent Pyr3 on TRPC3-expressing cells strongly supported the
concept of activation of two permeation pathways by store depletion, involving activation of
two distinct channels. This is inconsistent with a model of L. Birnbaumer, where Orai1 is
considered as a regulatory subunit of a either ROCE- or SOCE-active TRPC3 channel [17].
Thus a SOCE-operated mode of TRPC3 in RBL-2H3 mast cells with the expression-level of
the protein being important may be concluded, in-line with previously published models
[34,35,36,37]. This is borne out by the following observations: i) The TRPC3-like currents
were only seen in cells over-expressing TPRC3, which is endogenously expressed at rather
low levels in mast cells [6]; ii) The conductance was clearly sensitive to Pyr3; and iii) The
store-operated conductance was eliminated by expression of dominant negative TRPC3.
Analysis of the vice-versa-situation, considering a potential Orai1-dependency of TRPC3
function, provided evidence that Orai1 activation is indeed important for TRPC3 activity. The
dominant negative Orai1E106Q mutant completely suppressed native CRAC as well as “store-
operated” TRPC3-like currents in RBL-2H3 mast cells. This may be interpreted either by
TRPC3 activity requiring Orai as a pore forming subunit, Ca2+
permeation through Orai1
being essential due to an impact on metabolism of upstream mediators (e.g. PLC-dependency)
or changes in TRPC3 PM recruitment or localization.
Orai1-mediated Ca2+
entry promotes membrane presentation of TRPC3
Although the lack of a general overlap between localization of Orai1 and TRPC3 in
unstimulated mast cells argues against a stable direct protein-protein interaction, the
beforehand mentioned results may still be explained by a dynamic Orai1-dependent
recruitment of TRPC3 into distinct microdomains, similar to a model proposed by I.
Ambudkar [16]. Indeed, TIRF microscopy experiments suggested that TRPC3 presentation in
the PM increases with Orai1 activity. This effect was missing in cells expressing the
dominant-negative Orai1E106Q pointing towards an Orai1-mediated Ca2+
entry as being a
central first step in a cascade of TRPC3 channel activation processes associated with store
depletion.
Ca2+
/transcription coupling involves TRPC3 as scaffold and depends on PKC-activity
NFAT translocation to the nucleus to elicit gene transcription is mediated by Ca2+
-dependent
activation of CaM and CaN and is a key event of immune cell activation [31]. In-line with our
electrophysiological data showing that inward currents are barely changed by TRPC3
expression, we consistently observed no change in NFAT activation after expression of
TRPC3wt or TRPC3E630K. The important role of Orai1 in native SOCE and NFAT regulation
was further substantiated by expression of Orai1E106Q (data not shown). Surprisingly,
overexpression of TRPC3P704Q, a mutant lacking immunophilin-binding capability, eliminated
NFAT translocation. These immunophilins link TRPCs to the NFAT-effector proteins CaM
and CaN. This interaction can be interrupted in a phosphorylation-dependent manner as
shown in various cell types [26,32]. Phosphorylation-dependence is indicated further by the
diminished or suppressed NFAT translocation with TRPC3S712A, lacking a negative regulatory
PKC-site [33], or after direct PKC-inhibition with GF109203X. According to a previous
report [32] both conditions mimic the TRPC3P704Q binding defect. The expected phenotype of
increased channel activity of the TRPCS712A-mutant is observable in patch clamp experiments
and the TRPC3P704Q mutant was as well still permeable. These results clearly point to PKC-
phosphorylation-dependent scaffold function of TRPC3 in RBL-2H3 mast cells.
This scaffold function of TRPC3 was further strengthened by results showing a changed
distinct cellular CaN localization upon co-expression with TRPC3wt or TRPC3P704Q, where the
later combination did not show a distinct CaN presentation during Ca2+
re-addition after store-
depletion.
Endowed by various studies implicating a direct or indirect function of TRPCs and
STIM1/Orai1 in SOCE phenomena [16,19,38,39], we present results for an Orai1/TRPC3
interaction on the level of biophysical properties and the downstream effector of
Ca2+
/transcription coupling. Although upon store depletion a TRPC3-mediated outward
conductance was clearly observed in RBL-2H3 mast cells, TRPC3 Ca2+
influx appears to be
without impact on Orai1 function. Orai1 activity by contrast seems to be an absolute
prerequisite of TRPC3 activation in response to store depletion of RBL-2H3 mast cells, as
Orai1 activity leads to increased TRPC3 PM presentation. Our experiments with TRPC3
constructs lacking FKBP12 and CaN interaction [26,32] strongly suggest a crucial PKC-
dependent scaffold function of TRPC3 in CaM/CaN/NFAT activation.
In aggregate, we report hereon the essential participation of TRPC3 in STIM1/Orai1-mediated
activation of RBL-2H3 mast cells.
Materials and Methods
Cell culture and DNA
RBL-2H3 cells were cultivated in DMEM medium (Invitrogen) supplemented with 10% FBS.
For electrophysiology, fluorescence microscopy and NFAT translocation experiments they
were seeded on glass-coverslips or glass-bottom petri dishes. Transfections were done 12-18h
before the experiments by electroporation (at 300 V and 275 µF) with (if not stated otherwise)
20 µg DNA of each construct. Used constructs were n-terminally YFP- or CFP-tagged cDNA
clones of human TRPC3, Orai1, corresponding mutants or CaN (expression vectors were
peYFP-C1 or peCFP-C1). For NFAT translocation experiments an n-terminally GFP-tagged
NFAT construct was employed. For details on DNA and mutagenesis see [26]. All subsequent
measurements were performed at room temperature.
Electrophysiology
Patch pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, resistance
3-5 MΩ). Currents were recorded at room temperature using a List EPC7 patch clamp
amplifier (HEKA Instruments). Signals were low-pass filtered at 3 and 10 kHz and digitized
with 5 kHz. For RBL-2H3 cells CRAC measurement, standard protocols and buffers were
modified from [40]. In brief voltage ramps from -90 to +90 mV over 1 second (holding
potential +40 mV) were applied controlled by pClamp software. ECS contained (in mM) 115
NaCl, 5 CsCl, 1 MgCl2, 10 HEPES, 10 Glucose, 20 CaCl2 at pH 7.4. ICS was comprised of
3.5 MgCl2, 145 caesium methanesulphonate, 8 NaCl, 10 HEPES, 20 EGTA at pH 7.2.
Fluorescence microscopy
Epifluorescene and TIRF images were recorded with a Zeiss Axiovert 200M microscope
(Zeiss) equipped with a Coolsnap HQ CCD-camera (Visitron) at respective excitation
wavelengths. Cells were transfected accordingly and seeded out on glass coverslips or glass
bottom petri dishes (for TIRF imaging) one day before the experiments. Used buffers and
perfusion conditions were the same as for electrophysiology.
NFAT translocation
For NFAT imaging experiments, coverslips with single- or double-transfected RBL-2H3 cells
were transferred into nominally Ca2+
-free RBL-2H3 buffer described above and incubated
with thapsigargin (1 µM) for 5 min to deplete the internal Ca2+
stores. NFAT translocation
was triggered by adding 2 mM extracellular CaCl2. Any inhibitors were present in all buffers
at indicated concentrations. Basal NFAT localization was assessed from cells before store
depletion.
GFP-NFAT translocation was monitored (488 nm laser excitation) with standard fluorescence
microscopy (Zeiss Axiovert equipped with a Coolsnap HQ camera). Nuclear/cytosol
fluorescence intensity ratios of cells were calculated with ImageJ software.
Data evaluation and statistics
Data is presented as mean values +/- S.E.M. and was tested for statistical significance using
student’s t-test or one way ANOVA. * indicates p < 0.05, ** p < 0.01 and *** p < 0.001.
Acknowledgments
We like to thank Dr. Kehlenbach for kindly providing the GFP-NFAT construct. This work
was funded by FWF projects P21925-B19 (to K Groschner), P21118-B09 (to C. Romanin)
and the DK+ Metabolic and Cardiovascular Disease grant W2126-B18.
Abbreviations
aa - aminoacid, CRAC - Ca2+
release activated current, CaM - calmodulin, CaN – calcineurin,
DAG - diacylglycerol, IP3 - inositol-triphosphate, I/V - current to voltage (relation), FRET -
fluorescence/Foerster resonance energy transfer, NFAT - nuclear factor of activated T-cells,
PLC - phospholipase C, PIP2 - phosphoinostitol-bisphosphate, PM - plasma membrane,
ROCE – receptor-operated Ca2+
entry, SOCE – store-operated Ca2+
entry, STIM1 - stromal
interaction molecule 1, TIRF(M) - total internal reflection fluorescence (microscopy), TRP(C)
- transient receptor potential (canonical), wt - wildtype
References
1. Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol
Cell Biol 1: 11-21.
2. Ching TT, Hsu AL, Johnson AJ, Chen CS (2001) Phosphoinositide 3-kinase facilitates antigen-stimulated
Ca(2+) influx in RBL-2H3 mast cells via a phosphatidylinositol 3,4,5-trisphosphate-sensitive Ca(2+)
entry mechanism. J Biol Chem 276: 14814-14820.
3. Zweifach A, Lewis RS (1996) Calcium-dependent potentiation of store-operated calcium channels in T
lymphocytes. J Gen Physiol 107: 597-610.
4. Zweifach A, Lewis RS (1995) Slow calcium-dependent inactivation of depletion-activated calcium current.
Store-dependent and -independent mechanisms. J Biol Chem 270: 14445-14451.
5. Putney JW, Jr. (1986) A model for receptor-regulated calcium entry. Cell Calcium 7: 1-12.
6. Ma HT, Peng Z, Hiragun T, Iwaki S, Gilfillan AM, et al. (2008) Canonical transient receptor potential 5
channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and
degranulation in a rat mast cell line. J Immunol 180: 2233-2239.
7. Nilius B, Owsianik G (2011) The transient receptor potential family of ion channels. Genome Biol 12: 218.
8. Abramowitz J, Birnbaumer L (2009) Physiology and pathophysiology of canonical transient receptor potential
channels. FASEB J 23: 297-328.
9. Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A, et al. (2006) Orai1 is an essential pore subunit of the
CRAC channel. Nature 443: 230-233.
10. Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, et al. (2005) STIM1 is a Ca2+ sensor that activates CRAC
channels and migrates from the Ca2+ store to the plasma membrane. Nature 437: 902-905.
11. Luik RM, Wu MM, Buchanan J, Lewis RS (2006) The elementary unit of store-operated Ca2+ entry: local
activation of CRAC channels by STIM1 at ER-plasma membrane junctions. J Cell Biol 174: 815-825.
12. Luik RM, Wang B, Prakriya M, Wu MM, Lewis RS (2008) Oligomerization of STIM1 couples ER calcium
depletion to CRAC channel activation. Nature 454: 538-542.
13. Zeng W, Yuan JP, Kim MS, Choi YJ, Huang GN, et al. (2008) STIM1 gates TRPC channels, but not Orai1,
by electrostatic interaction. Mol Cell 32: 439-448.
14. Liu X, Cheng KT, Bandyopadhyay BC, Pani B, Dietrich A, et al. (2007) Attenuation of store-operated Ca2+
current impairs salivary gland fluid secretion in TRPC1(-/-) mice. Proc Natl Acad Sci U S A 104:
17542-17547.
15. Singh BB, Zheng C, Liu X, Lockwich T, Liao D, et al. (2001) Trp1-dependent enhancement of salivary
gland fluid secretion: role of store-operated calcium entry. FASEB J 15: 1652-1654.
16. Cheng KT, Liu X, Ong HL, Swaim W, Ambudkar IS (2011) Local Ca(2)+ entry via Orai1 regulates plasma
membrane recruitment of TRPC1 and controls cytosolic Ca(2)+ signals required for specific cell
functions. PLoS Biol 9: e1001025.
17. Liao Y, Erxleben C, Yildirim E, Abramowitz J, Armstrong DL, et al. (2007) Orai proteins interact with
TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci U S A 104: 4682-
4687.
18. Liao Y, Erxleben C, Abramowitz J, Flockerzi V, Zhu MX, et al. (2008) Functional interactions among Orai1,
TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels.
Proc Natl Acad Sci U S A 105: 2895-2900.
19. Liao Y, Plummer NW, George MD, Abramowitz J, Zhu MX, et al. (2009) A role for Orai in TRPC-mediated
Ca2+ entry suggests that a TRPC:Orai complex may mediate store and receptor operated Ca2+ entry.
Proc Natl Acad Sci U S A 106: 3202-3206.
20. Woodard GE, Lopez JJ, Jardin I, Salido GM, Rosado JA (2010) TRPC3 regulates agonist-stimulated Ca2+
mobilization by mediating the interaction between type I inositol 1,4,5-trisphosphate receptor, RACK1,
and Orai1. J Biol Chem 285: 8045-8053.
21. Hoth M, Penner R (1992) Depletion of intracellular calcium stores activates a calcium current in mast cells.
Nature 355: 353-356.
22. Vig M, Beck A, Billingsley JM, Lis A, Parvez S, et al. (2006) CRACM1 multimers form the ion-selective
pore of the CRAC channel. Curr Biol 16: 2073-2079.
23. Hardie RC, Minke B (1992) The trp gene is essential for a light-activated Ca2+ channel in Drosophila
photoreceptors. Neuron 8: 643-651.
24. Hurst RS, Zhu X, Boulay G, Birnbaumer L, Stefani E (1998) Ionic currents underlying HTRP3 mediated
agonist-dependent Ca2+ influx in stably transfected HEK293 cells. FEBS Lett 422: 333-338.
25. Kamouchi M, Philipp S, Flockerzi V, Wissenbach U, Mamin A, et al. (1999) Properties of heterologously
expressed hTRP3 channels in bovine pulmonary artery endothelial cells. J Physiol 518 Pt 2: 345-358.
26. Poteser M, Schleifer H, Lichtenegger M, Schernthaner M, Stockner T, et al. (2011) PKC-dependent coupling
of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling
in HL-1 myocytes. Proc Natl Acad Sci U S A 108: 10556-10561.
27. Kiyonaka S, Kato K, Nishida M, Mio K, Numaga T, et al. (2009) Selective and direct inhibition of TRPC3
channels underlies biological activities of a pyrazole compound. Proc Natl Acad Sci U S A 106: 5400-
5405.
28. Naraghi M, Neher E (1997) Linearized buffered Ca2+ diffusion in microdomains and its implications for
calculation of [Ca2+] at the mouth of a calcium channel. J Neurosci 17: 6961-6973.
29. Ait-Haddou R, Kurachi Y, Nomura T (2010) On calcium-buffer dynamics within the excess buffer regime. J
Theor Biol 264: 55-65.
30. Cheng KT, Ong HL, Liu X, Ambudkar IS (2011) Contribution of TRPC1 and Orai1 to Ca(2+) entry
activated by store depletion. Adv Exp Med Biol 704: 435-449.
31. Kar P, Nelson C, Parekh AB (2011) Selective activation of the transcription factor NFAT1 by calcium
microdomains near Ca2+ release-activated Ca2+ (CRAC) channels. J Biol Chem 286: 14795-14803.
32. Sinkins WG, Goel M, Estacion M, Schilling WP (2004) Association of immunophilins with mammalian
TRPC channels. J Biol Chem 279: 34521-34529.
33. Trebak M, Hempel N, Wedel BJ, Smyth JT, Bird GS, et al. (2005) Negative regulation of TRPC3 channels
by protein kinase C-mediated phosphorylation of serine 712. Mol Pharmacol 67: 558-563.
34. Putney JW, Jr. (2004) The enigmatic TRPCs: multifunctional cation channels. Trends Cell Biol 14: 282-286.
35. Vazquez G, Lievremont JP, St JBG, Putney JW, Jr. (2001) Human Trp3 forms both inositol trisphosphate
receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B
lymphocytes. Proc Natl Acad Sci U S A 98: 11777-11782.
36. Yildirim E, Kawasaki BT, Birnbaumer L (2005) Molecular cloning of TRPC3a, an N-terminally extended,
store-operated variant of the human C3 transient receptor potential channel. Proc Natl Acad Sci U S A
102: 3307-3311.
37. Vazquez G, Wedel BJ, Trebak M, St John Bird G, Putney JW, Jr. (2003) Expression level of the canonical
transient receptor potential 3 (TRPC3) channel determines its mechanism of activation. J Biol Chem
278: 21649-21654.
38. Ong HL, Cheng KT, Liu X, Bandyopadhyay BC, Paria BC, et al. (2007) Dynamic assembly of TRPC1-
STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in
store-operated and calcium release-activated calcium channel components. J Biol Chem 282: 9105-
9116.
39. Ambudkar IS, Ong HL, Liu X, Bandyopadhyay BC, Cheng KT (2007) TRPC1: the link between functionally
distinct store-operated calcium channels. Cell Calcium 42: 213-223.
40. Derler I, Fahrner M, Carugo O, Muik M, Bergsmann J, et al. (2009) Increased hydrophobicity at the N
terminus/membrane interface impairs gating of the severe combined immunodeficiency-related ORAI1
mutant. J Biol Chem 284: 15903-15915.
Figures
Figure 1: Native RBL-2H3 SOCE is characterized as ICRAC. Application of a store-
depletion protocol in patch clamp experiments with RBL-2H3 mast cells elicits typical ICRAC.
(left panel) Time course of current densities of vector (open symbols) and Orai1E106Q (filled,
red symbols) transfected RBL-2H3 cells depleted with EGTA (mean values ± S.E.M. of n > 5
cells each). Representative I/V relation of measured currents for control-transfected (middle
panel) and OraiE106Q (right panel) (black trace - immediately after break-in, red trace -
maximal current-amplitude).
Figure 2: TRPC3 induces only minor changes in RBL-2H3 SOCE currents. (A) (left
panel) time course of current densities of cells overexpressing TRPC3wt (red symbols) or
TRPC3E630K (black symbols) (mean values ± S.E.M. of n > 5 cells each). Representative I/V
relation of cells expressing TRPC3wt (middle panel) or TRPC3E630K. (B) (upper left) Time
course of cells expressing TRPC3wt with stores depleted by BAPTA (black symbols) or after
pre-application of 10 µM Pyr3 (red symbols) (mean values ± S.E.M. of n > 5 cells each).
Corresponding representative I/V relation (BAPTA - upper right, Pyr3 - lower left). (lower
right) Representative time course of acute Pyr3 administration on TRCP3wt-expressing RBL-
2H3 cells. (for all I/V relations: black trace - immediately after break-in, red trace - maximal
current-amplitude)
Figure 3: TRPC3 conductance is prevented by suppression of Orai1-activity. Time
course (mean values ± S.E.M. of n > 5 cells each) and representative I/V relations of RBL-
2H3 cells expressing TRPC3wt (middle panel) or TRPC3wt jointly with Orai1E106Q (right
panel) after store depletion with EGTA. (black trace - immediately after break-in, red trace -
maximal current-amplitude)
Figure 4: Orai1 activity facilitates TRPC3 PM recruitment in RBL-2H3 cells.
(A) Representative epifluorescence (A) and TIRF (B) images of CFP-Orai1 (red), YFP-
TRPC3 (green) expression and calculated overlay. Cells were unstimulated.
(C) (left) Bleaching-corrected time course of YFP-TRPC3wt TIRF intensity in RBL-2H3 cells
co-expressing YFP-TRPC3wt with Orai1wt or Orai1E106Q (mean values ± S.E.M. of n = 4 each)
during application of thapsigargin (arrow, 1 µM) and subsequent Ca2+
re-addition (2 mM).
Large error bars at the onset of the experiment are due to perfusion induced disturbances in
the optical system. (right) Representative TIRF images of YFP-TRPC3 expression in cells co-
expressing YFP-TRPC3 and Orai1 (top) or Orai1E106Q (bottom) at the indicated time points.
Increased YFP-TRPC3 signal is indicated by the white arrow. Images at time point 2 are
fainter due to bleaching.
Figure 5: TRPC3 permeation is not relevant for NFAT activation, but immunophilin
binding and phosphorylation is. (A) Mean values ± S.E.M. of NFAT nucleus to cytosol
signal ratio (n ≥ 24 cells for each experiment) at basal condition (grey bars) and after
depletion of intracellular calcium stores by thapsigargin and re-addition of extracellular Ca2+
(2 mM) (black bars). GFP-NFAT single-transfected cells were used as control. (B)
Representative images of NFAT localization and DIC microscopy images for mentioned
conditions. (C) Time course (mean values ± S.E.M. of n > 5 cells each) and representative I/V
relations of RBL-2H3 cells expressing TRPC3S712A (middle panel) or TRPC3P704Q (right
panel). (black trace - immediately after break-in, red trace - maximal current-amplitude)
Figure 6: TRPC3-dependent localization of CaN. Representative TIRFM images of cells
co-expressing YFP-tagged CaN (green) and CFP-tagged TRPC3wt (red) with calculated
overlay before and after store depletion with thapsigargin and Ca2+
re-addition.
94
Discussion, conclusions & outlook
Insights into key determinants of TRPC3 structure/function
relationship
Our combined strategy using a novel homology model for TRPC3 along with mutagenesis of
predicted, functionally relevant aa in the putative pore region (Poteser et al., 2011)
confirmed a previous hypothesis of R. Hardie about the aa essential for Ca2+ permeation in
the channel (Liu et al., 2007a) (see Figure 7).
Neutralising the charge at AA 630 by exchanging glutamate to glutamine virtually abolished
divalent permeability. Including a positive charge into the putative permeation pathway by
exchanging E630 with lysine blocked ion permeation completely, most likely by occluding
this narrowest part of the pore for cation permeation. Notably, both mutants (TRPC3E630Q &
TPRCE630K) seem to affect even the endogenously expressed TRPCs in a dominant-negative
way. Unlike in K+ channels, where a GYGD motif determines (monovalent) selectivity
(Chapman et al., 2001; Tytgat, 1994), no such motif is present in the putative TRPC3 pore.
Current attempts of our group to explore the narrowest pore region from aa 628 to 634 by
so-called “cysteine scanning” mutagenesis will reveal further details about pore size and
hydrophilic/water-accessible surface in the putative pore region (Anderluh et al., 1999; Chen
et al., 1997; Hruz et al., 1999; Kurz et al., 1995; Voets et al., 2004). This may lead to a better
understanding of permeation, selectivity and gating of the TRPC3 channel.
Aside selectivity, channel activation and regulation, specifically in the context of subtype-
specific regulatory properties of TRPCs is an unsolved question. Differences in the AA
sequence of the two groups in the region just adjacent to TM5, forming the pore vestibule,
point to the charged AA 615, 616 and 619 as good candidates that might confer such
regulatory differences. Previous studies indicated that even single AA can confer binding
sites for lipid messengers leading to channel activation (Branstrom et al., 2007) or change
channel selectivity (Voets et al., 2004).
Nonetheless, experimental consequences of single AA mutations have to be considered in
the context of charge dispersion along the full stretch of the protein throughout the
membrane and the localisation of the individual subunits (see figure 8 for an illustration of
TRPC3 subunit arrangement). Effects might therefore be caused by changes of the insertion
depth into the PM or the distance of subunits to each other created by electrostatic forces
95
between charged AA. Variation in relationship between extracellular localised AA (615, 616,
619, 630 or 644) with charges buried deeper in the PM (561, 562 or 630) might generate
forces distorting the permeation pathway leading to observable changes. A prediction of
charge dissemination in the hydrophilic/water-accessible surface on the cytosolic face of the
channel’s permeation pathway (see figure 9) formed by TM 5 & 6 leave room for
speculations about regulatory function of ion binding there as well (Obukhov et al., 2005).
Figure 7: Structure and key AA of the putative TRPC3 pore.
(A) alignment with the bacterial KcsA channel. JPred prediction (Cole et al., 2008) of AA accessibility is
annotated (B = potentially buried) (B) 3D-rendering of the putative pore region of TRPC3 including TM 5 & 6 (C)
3 subunits of a putative TRPC3 pore with positions of charged aa highlighted (E630 – yellow, E616 & D639 –
red, K619 – blue) (D) detailed view of the permeation pathway and “pore helix” with aa E630 highlighted
yellow. (models are based on KcsA channel.)
96
Figure 8: Tertiary structure of TRPC3.
Illustration of TRPC3 subunit arrangement
(computed surface is shown)
Figure 9: TRPC3 mutant E630Q significantly changes electrostatic
potential of TRPC3 permeation pathway’s cytosol-facing end.
Computed iso-electric surface of TRPC3wt and TRPC3E630Q (negative charge is depicted red).
(model is based on NaVAB channel. The figure was compiled by T. Stockner,
Institute of Pharmacology, Medical University Vienna)
New pyrazole derivatives as TRPC3 and Orai1 blockers
For experimental and pharmacological use the development of potent and selective blocking
agents for TRPC3 and Orai1 is highly desirable.
Out of a range of pyrazole derivatives, generated in collaboration with the CDLMC5 and the
group of C.O. Kappe (Glasnov et al., 2009; Obermayer et al., 2011), our lab identified two
compounds designated as Pyr6 and Pyr10, which show high potency on either Orai1 or
5 Christian Doppler laboratory for microwave chemistry at the Insitute of Chemistry at Graz University
97
TRPC3. In the future, aside components derived from GSK-66 (Li et al., 2011), they might be
valuable as new lead substances for further dissecting the contribution of either Ca2+ entry
pathway to cellular effects or even treatment of pathological conditions.
Additionally, our results strongly suggest that Pyr3 is not as selective for TRPC3 as initially
proposed (Schleifer et al., 2012 - British Journal of Pharmacology - submitted). This issue was
as well already considered by other groups, observing a potent effect on thapsigargin
mediated SOCE in various cell types (Gibon et al., 2010; Kim et al., 2011; Salmon et al.,
2010). Nonetheless, it is important to note that the described blocking effects of Pyr3 might
as well be due to a significant contribution of TRPC3 activity to store-operated Ca2+ influx in
the studied cell lines.
Effects of changes in TRPC3 function on downstream cellular
signalling
The existence of cellular pathways, which link Ca2+ entry to gene transcription is well
established in many cell types and tissues. Two classical examples are the Ca2+-mediated
cellular activation, in terms of production of cytokines as well as proliferation in mast cells
(He et al., 2005) and pathological progression of hypertrophic remodelling of the heart
(Brenner et al., 2007; Bush et al., 2006; Nakayama et al., 2006). In both cases activation of
the CaM/CaN/NFAT-pathway is of particular importance.
We recently demonstrated a clear separation of TRPC3 Ca2+ permeation-dependent NFAT
activation from global cytosolic Ca2+ signalling after receptor-stimulation of HL1 murine
cardiac myocytes(Poteser et al., 2011). Unlike the situation in HEK-293 cells, where NFAT
translocation was impaired and maximal cytosolic Ca2+ entry was reduced with inhibition of
TRPC3 permeability using either TRPC3E630Q or TRPC3E630K, TRPC3E630Q uncoupled NFAT
activation in HL-1 cells from the normal Ca2+ influx. The difference between the two cell lines
is that in HL-1 cells, the observed Ca2+ entry was mediated through endogenously expressed
CaV1.2 channels, as proven by sensitivity to the classical Ca2+ antagonist nifedepin.
Furthermore, impaired PKC regulation, as for example present in the mwk-mutant
(TRPC3T573A) (Becker et al., 2009) or suppression of PKC-phosphorylation with the inhibitor
GFX, uncoupled the two processes as well. This suggests that two different Ca2+ pools
apparently exist in excitable cells. On the one hand, a global Ca2+ level affecting,
transcriptionally silent Ca2+ entry, where TRPC3 might have only pivotal function activating
98
the Ca2+ subsequent influx effected by changing the membrane potential. On the other
hand, a transcriptionally active, non-global Ca2+ affecting microdomain centred TRPC3-
dependent Ca2+ entry leading to downstream signalling, which relies on phosphorylation-
dependent maintenance of TRPC3/CaN/NFAT complexes.
A similar phosphorylation-dependent scaffold function of TRPC3 for CaN and NFAT was
found in SOCE contribution of TRPC3 in RBL-2H3 mast cells as well (Schleifer et al. - in
preparation). Here TRPC3P704Q suppressed NFAT translocation and modulated distinct
cellular CaN localisation. NFAT activation was as well inhibited when applying GFX or
overexpressing a phosphorylation-defective TRPC3 mutant (TRPC3S712A) (Trebak et al., 2005).
Despite this proposed scaffold function, it appeared that the Ca2+ entry activating NFAT
translocation was effectuated by Orai1 only, as TRPC3 permeation alone was insufficient to
change subcellular localisation of the transcription factor.
The obtained novel insights into properties of TRPC3 permeation and selectivity, as well in
its role as a molecular mediator of transcriptional control mechanisms in both ROCE and
SOCE situations, combined with the identification of new tools to selectively suppress TRPC-
mediated Ca2+ entry are expected to promote better understanding TRPC3-dependent
cellular signalling processes.
99
Abbreviations
2-APB 2-aminoethoxydiphenyl AA aminoacid ANK ankyrin repeats BTP 3,5-bis(trifluoromethyl)pyrazole Ca2+ calcium CAD CRAC activating domain CaM calmodulin CaN calcineurin CC coiled-coil CIRB CaM/IP3-receptor binding site CMD CRAC modulatory domain CRACM CRAC modulator DAG diacylglycerol ER endoplasmic reticulum FKBP general abbreviation for FK506/Sirolismus/Rapamycin binding proteins GFX GF109203X, a PKC-inhibitor HEK-293 human embryonic kidney (cells), a cell line HL-1 a murine, cardiac cell line derived from AT-1 atrial myocytes IP3 inositol-triphosphate L-type „long lasting type“ mwk “moonwalker” (mouse), TRPC3T573A mutation in human TRPC3 NFAT nuclear factor of activated T-cells OASF Orai1 activating small fragment RBL-2H3 rat basophil leukaemia (cells), a mast cell line ROCE receptor operated Ca2+ entry SAM sterile-α-motif SCID severe combined immune deficiency (syndrome) SOAR STIM-Orai activating region SOCE store operated Ca2+ entry STIM stromal interaction molecule T-type „transient type“ TIRF total internal reflection fluorescence TM transmembrane (region/domain) TRP transient receptor potential trp abbreviation for the original drosophila TRP-channel TRPA transient receptor potential ankyrin TRPC transient receptor potential canonical TRPM transient receptor potential melastatin TRPP transient receptor potential polycystic TRPML transient receptor potential mucolipin TRPN transient receptor potential „no mechanopotential“ TRPV transient receptor potential vanilloid TRPY transient receptor potential yeast PIP2 phosphoinositol-bisphosphate PK phosphokinase PKC phosphokinase C PKG phosphokinase G PLC phospholipase C
100
List of Figures
Figure 1: Phylogenetic tree of the 6 mammalian TRP-superfamily subfamilies ...................... 8
Figure 2: Overview of structural features of TRP channels ...................................................... 9
Figure 3: Scheme of typical TRPC channel activation ............................................................. 11
Figure 4: (A) 3D reconstruction of TRPC3 cryo-electron microscopy
(B) illustration of TRPC3 key features ...................................................................... 14
Figure 5: Depiction key functional domain organisation of human (A) STIM1
and (B) Orai1 ............................................................................................................ 16
Figure 6: Structures of Ca2+ channel inhibitors ....................................................................... 18
Figure 7: Structure and key AA of the putative TRPC3 pore .................................................. 95
Figure 8: Tertiary structure of TRPC3 ..................................................................................... 96
Figure 9: TRPC3 mutant E630Q significantly changes electrostatic potential of TRPC3
permeation pathway’s cytosol-facing end .............................................................. 96
References to database entries and software
For modelling and sequence alignment purposes the following protein or DNA (Watson et
al., 1953) database entries, identified by the unique accession number were used:
UniProt (www.uniprot.org) (Uniprot, 2012): P48955 - human TRPC1, Q9R224 - murine
TRPC2, Q13507 - human TRPC3, Q2M2U7 - human TRPC4, Q9UL62 - human TRPC5, Q9Y210 -
human TRPC6, Q9HCX4 - human TRPC7, P0A334 - KcsA
NCBI protein: GI 339961372 - NaVAB
Visual molecular dynamics (www.ks.uiuc.edu/research/vmd/) (Humphrey et al., 1996)
combined with PovRay (www.povray.org) renderer was used for imaging the protein models.
Alignments were viewed and modified with Jalview (www.jalview.org) (Waterhouse et al.,
2009).
101
References
Abramowitz J, Birnbaumer L (2009). Physiology and pathophysiology of canonical transient receptor potential channels. FASEB J 23(2): 297-328. Abramowitz J, Yildirim E, Birnbaumer L (2007). The TRPC Family of Ion Channels: Relation to the TRP Superfamily and Role in Receptor- and Store-Operated Calcium Entry TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades. edn. Taylor & Francis Group, LLC: Boca Raton FL. Ambudkar IS (2007). TRPC1: a core component of store-operated calcium channels. Biochem Soc Trans 35(Pt 1): 96-100. Anderluh G, Barlic A, Podlesek Z, Macek P, Pungercar J, Gubensek F, et al. (1999). Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes. Eur J Biochem 263(1): 128-136. Baba Y, Hayashi K, Fujii Y, Mizushima A, Watarai H, Wakamori M, et al. (2006). Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum. Proc Natl Acad Sci U S A 103(45): 16704-16709. Bandyopadhyay BC, Ong HL, Lockwich TP, Liu X, Paria BC, Singh BB, et al. (2008). TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1. J Biol Chem 283(47): 32821-32830. Becker EB, Fogel BL, Rajakulendran S, Dulneva A, Hanna MG, Perlman SL, et al. (2011). Candidate screening of the TRPC3 gene in cerebellar ataxia. Cerebellum 10(2): 296-299. Becker EB, Oliver PL, Glitsch MD, Banks GT, Achilli F, Hardy A, et al. (2009). A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice. Proc Natl Acad Sci U S A 106(16): 6706-6711. Berridge MJ, Lipp P, Bootman MD (2000). The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1(1): 11-21. Birnbaumer L (2009). The TRPC class of ion channels: a critical review of their roles in slow, sustained increases in intracellular Ca(2+) concentrations. Annu Rev Pharmacol Toxicol 49: 395-426. Boulay G, Zhu X, Peyton M, Jiang M, Hurst R, Stefani E, et al. (1997). Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J Biol Chem 272(47): 29672-29680. Brandman O, Liou J, Park WS, Meyer T (2007). STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. Cell 131(7): 1327-1339. Branstrom R, Leibiger IB, Leibiger B, Klement G, Nilsson J, Arhem P, et al. (2007). Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: indications for a long-chain acyl-CoA ester binding motif. Diabetologia 50(8): 1670-1677. Brenner JS, Dolmetsch RE (2007). TrpC3 regulates hypertrophy-associated gene expression without affecting myocyte beating or cell size. PLoS One 2(8): e802. Bush EW, Hood DB, Papst PJ, Chapo JA, Minobe W, Bristow MR, et al. (2006). Canonical transient receptor potential channels promote cardiomyocyte hypertrophy through activation of calcineurin signaling. J Biol Chem 281(44): 33487-33496. Calloway N, Vig M, Kinet JP, Holowka D, Baird B (2009). Molecular clustering of STIM1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+ stores and on electrostatic interactions. Mol Biol Cell 20(1): 389-399. Chapman ML, Krovetz HS, VanDongen AM (2001). GYGD pore motifs in neighbouring potassium channel subunits interact to determine ion selectivity. J Physiol 530(Pt 1): 21-33.
102
Chen S, Hartmann HA, Kirsch GE (1997). Cysteine mapping in the ion selectivity and toxin binding region of the cardiac Na+ channel pore. J Membr Biol 155(1): 11-25. Cheng KT, Liu X, Ong HL, Ambudkar IS (2008). Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels. J Biol Chem 283(19): 12935-12940. Cheng KT, Liu X, Ong HL, Swaim W, Ambudkar IS (2011). Local Ca(2)+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca(2)+ signals required for specific cell functions. PLoS Biol 9(3): e1001025. Clapham DE (2003). TRP channels as cellular sensors. Nature 426(6966): 517-524. Cole C, Barber JD, Barton GJ (2008). The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36(Web Server issue): W197-201. Corey DP (2003). New TRP channels in hearing and mechanosensation. Neuron 39(4): 585-588. Corey DP, Garcia-Anoveros J, Holt JR, Kwan KY, Lin SY, Vollrath MA, et al. (2004). TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells. Nature 432(7018): 723-730. Cosens DJ, Manning A (1969). Abnormal electroretinogram from a Drosophila mutant. Nature 224(5216): 285-287. DeHaven WI, Smyth JT, Boyles RR, Bird GS, Putney JW, Jr. (2008). Complex actions of 2-aminoethyldiphenyl borate on store-operated calcium entry. J Biol Chem 283(28): 19265-19273. Denis V, Cyert MS (2002). Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue. J Cell Biol 156(1): 29-34. Derler I, Fahrner M, Carugo O, Muik M, Bergsmann J, Schindl R, et al. (2009). Increased hydrophobicity at the N terminus/membrane interface impairs gating of the severe combined immunodeficiency-related ORAI1 mutant. J Biol Chem 284(23): 15903-15915. Dietrich A, Mederos YSM, Gollasch M, Gross V, Storch U, Dubrovska G, et al. (2005). Increased vascular smooth muscle contractility in TRPC6-/- mice. Mol Cell Biol 25(16): 6980-6989. Djuric SW, BaMaung NY, Basha A, Liu H, Luly JR, Madar DJ, et al. (2000). 3,5-Bis(trifluoromethyl)pyrazoles: a novel class of NFAT transcription factor regulator. J Med Chem 43(16): 2975-2981. Eder P, Molkentin JD (2011). TRPC channels as effectors of cardiac hypertrophy. Circ Res 108(2): 265-272. Eder P, Poteser M, Groschner K (2007a). TRPC3: a multifunctional, pore-forming signalling molecule. Handb Exp Pharmacol(179): 77-92. Eder P, Probst D, Rosker C, Poteser M, Wolinski H, Kohlwein SD, et al. (2007b). Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex. Cardiovasc Res 73(1): 111-119. Fahrner M, Muik M, Derler I, Schindl R, Fritsch R, Frischauf I, et al. (2009). Mechanistic view on domains mediating STIM1-Orai coupling. Immunol Rev 231(1): 99-112. Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, et al. (2006). A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441(7090): 179-185. Feske S, Prakriya M, Rao A, Lewis RS (2005). A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients. J Exp Med 202(5): 651-662. Freichel M, Vennekens R, Olausson J, Hoffmann M, Muller C, Stolz S, et al. (2004). Functional role of TRPC proteins in vivo: lessons from TRPC-deficient mouse models. Biochem Biophys Res Commun 322(4): 1352-1358. Freichel M, Vennekens R, Olausson J, Stolz S, Philipp SE, Weissgerber P, et al. (2005). Functional role of TRPC proteins in native systems: implications from knockout and knock-down studies. J Physiol 567(Pt 1): 59-66.
103
Garcia RL, Schilling WP (1997). Differential expression of mammalian TRP homologues across tissues and cell lines. Biochem Biophys Res Commun 239(1): 279-283. Gaudet R (2007). Structural Insights into the Function of TRP Channels TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades. edn. Taylor & Francis Group, LLC: Boca Raton FL. Gaudet R (2008). TRP channels entering the structural era. J Physiol 586(Pt 15): 3565-3575. Gibon J, Tu P, Bouron A (2010). Store-depletion and hyperforin activate distinct types of Ca(2+)-conducting channels in cortical neurons. Cell Calcium 47(6): 538-543. Glasnov TN, Groschner K, Kappe CO (2009). High-speed microwave-assisted synthesis of the trifluoromethylpyrazol-derived canonical transient receptor potential (TRPC) channel inhibitor Pyr3. ChemMedChem 4(11): 1816-1818. Graziani A, Poteser M, Heupel WM, Schleifer H, Krenn M, Drenckhahn D, et al. (2010). Cell-cell contact formation governs Ca2+ signaling by TRPC4 in the vascular endothelium: evidence for a regulatory TRPC4-beta-catenin interaction. J Biol Chem 285(6): 4213-4223. Greka A, Navarro B, Oancea E, Duggan A, Clapham DE (2003). TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nat Neurosci 6(8): 837-845. Gwack Y, Srikanth S, Feske S, Cruz-Guilloty F, Oh-hora M, Neems DS, et al. (2007). Biochemical and functional characterization of Orai proteins. J Biol Chem 282(22): 16232-16243. Hardie RC (2007). TRP channels and lipids: from Drosophila to mammalian physiology. J Physiol 578(Pt 1): 9-24. Hardie RC, Minke B (1993). Novel Ca2+ channels underlying transduction in Drosophila photoreceptors: implications for phosphoinositide-mediated Ca2+ mobilization. Trends Neurosci 16(9): 371-376. Hardie RC, Minke B (1992). The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron 8(4): 643-651. Harteneck C, Gollasch M (2011). Pharmacological modulation of diacylglycerol-sensitive TRPC3/6/7 channels. Curr Pharm Biotechnol 12(1): 35-41. He LP, Hewavitharana T, Soboloff J, Spassova MA, Gill DL (2005). A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2. J Biol Chem 280(12): 10997-11006. Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G (1999). Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397(6716): 259-263. Hruz PW, Mueckler MM (1999). Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter. J Biol Chem 274(51): 36176-36180. Hu Y, Schilling WP (1995). Receptor-mediated activation of recombinant Trpl expressed in Sf9 insect cells. Biochem J 305 ( Pt 2): 605-611. Huang GN, Zeng W, Kim JY, Yuan JP, Han L, Muallem S, et al. (2006). STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels. Nat Cell Biol 8(9): 1003-1010. Humphrey W, Dalke A, Schulten K (1996). VMD: visual molecular dynamics. J Mol Graph 14(1): 33-38, 27-38. Jammes F, Hu HC, Villiers F, Bouten R, Kwak JM (2011). Calcium-permeable channels in plant cells. FEBS J 278(22): 4262-4276. Kim MS, Lee KP, Yang D, Shin DM, Abramowitz J, Kiyonaka S, et al. (2011). Genetic and pharmacologic inhibition of the Ca2+ influx channel TRPC3 protects secretory epithelia from Ca2+-dependent toxicity. Gastroenterology 140(7): 2107-2115, 2115 e2101-2104.
104
Kiyonaka S, Kato K, Nishida M, Mio K, Numaga T, Sawaguchi Y, et al. (2009). Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound. Proc Natl Acad Sci U S A 106(13): 5400-5405. Kiyonaka S, Kato K, Nishida M, Mori Y (2010). [Pharmacological properties of novel TRPC channel inhibitors]. Yakugaku Zasshi 130(3): 303-311. Kurz LL, Zuhlke RD, Zhang HJ, Joho RH (1995). Side-chain accessibilities in the pore of a K+ channel probed by sulfhydryl-specific reagents after cysteine-scanning mutagenesis. Biophys J 68(3): 900-905. Kwan HY, Huang Y, Yao X (2006). Protein kinase C can inhibit TRPC3 channels indirectly via stimulating protein kinase G. J Cell Physiol 207(2): 315-321. Law M, Morales JL, Mottram LF, Iyer A, Peterson BR, August A (2011). Structural requirements for the inhibition of calcium mobilization and mast cell activation by the pyrazole derivative BTP2. Int J Biochem Cell Biol 43(8): 1228-1239. Lemonnier L, Trebak M, Putney JW, Jr. (2008). Complex regulation of the TRPC3, 6 and 7 channel subfamily by diacylglycerol and phosphatidylinositol-4,5-bisphosphate. Cell Calcium 43(5): 506-514. Lewis RS (2001). Calcium signaling mechanisms in T lymphocytes. Annu Rev Immunol 19: 497-521. Lewis RS (2007). The molecular choreography of a store-operated calcium channel. Nature 446(7133): 284-287. Leypold BG, Yu CR, Leinders-Zufall T, Kim MM, Zufall F, Axel R (2002). Altered sexual and social behaviors in trp2 mutant mice. Proc Natl Acad Sci U S A 99(9): 6376-6381. Li J, McKeown L, Ojelabi O, Stacey M, Foster R, O'Regan D, et al. (2011). Nanomolar potency and selectivity of a Ca(2) release-activated Ca(2) channel inhibitor against store-operated Ca(2) entry and migration of vascular smooth muscle cells. Br J Pharmacol 164(2): 382-393. Li Z, Lu J, Xu P, Xie X, Chen L, Xu T (2007). Mapping the interacting domains of STIM1 and Orai1 in Ca2+ release-activated Ca2+ channel activation. J Biol Chem 282(40): 29448-29456. Liao Y, Erxleben C, Abramowitz J, Flockerzi V, Zhu MX, Armstrong DL, et al. (2008). Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels. Proc Natl Acad Sci U S A 105(8): 2895-2900. Liao Y, Erxleben C, Yildirim E, Abramowitz J, Armstrong DL, Birnbaumer L (2007). Orai proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci U S A 104(11): 4682-4687. Liao Y, Plummer NW, George MD, Abramowitz J, Zhu MX, Birnbaumer L (2009). A role for Orai in TRPC-mediated Ca2+ entry suggests that a TRPC:Orai complex may mediate store and receptor operated Ca2+ entry. Proc Natl Acad Sci U S A 106(9): 3202-3206. Lievremont JP, Bird GS, Putney JW, Jr. (2005). Mechanism of inhibition of TRPC cation channels by 2-aminoethoxydiphenylborane. Mol Pharmacol 68(3): 758-762. Lis A, Peinelt C, Beck A, Parvez S, Monteilh-Zoller M, Fleig A, et al. (2007). CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional properties. Curr Biol 17(9): 794-800. Liu CH, Wang T, Postma M, Obukhov AG, Montell C, Hardie RC (2007a). In vivo identification and manipulation of the Ca2+ selectivity filter in the Drosophila transient receptor potential channel. J Neurosci 27(3): 604-615. Liu X, Cheng KT, Bandyopadhyay BC, Pani B, Dietrich A, Paria BC, et al. (2007b). Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1(-/-) mice. Proc Natl Acad Sci U S A 104(44): 17542-17547. Luik RM, Wang B, Prakriya M, Wu MM, Lewis RS (2008). Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation. Nature 454(7203): 538-542. Machaca K (2011). Ca(2+) signaling, genes and the cell cycle. Cell Calcium 49(5): 323-330.
105
Marumo M, Suehiro A, Kakishita E, Groschner K, Wakabayashi I (2001). Extracellular pH affects platelet aggregation associated with modulation of store-operated Ca(2+) entry. Thromb Res 104(5): 353-360. Maruyama T, Kanaji T, Nakade S, Kanno T, Mikoshiba K (1997). 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem 122(3): 498-505. McNally BA, Somasundaram A, Yamashita M, Prakriya M (2012). Gated regulation of CRAC channel ion selectivity by STIM1. Nature. Merritt JE, Armstrong WP, Benham CD, Hallam TJ, Jacob R, Jaxa-Chamiec A, et al. (1990). SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J 271(2): 515-522. Minke B (2010). The history of the Drosophila TRP channel: the birth of a new channel superfamily. J Neurogenet 24(4): 216-233. Minke B, Wu C, Pak WL (1975). Induction of photoreceptor voltage noise in the dark in Drosophila mutant. Nature 258(5530): 84-87. Mio K, Ogura T, Hara Y, Mori Y, Sato C (2005). The non-selective cation-permeable channel TRPC3 is a tetrahedron with a cap on the large cytoplasmic end. Biochem Biophys Res Commun 333(3): 768-777. Mio K, Ogura T, Kiyonaka S, Hiroaki Y, Tanimura Y, Fujiyoshi Y, et al. (2007). The TRPC3 channel has a large internal chamber surrounded by signal sensing antennas. J Mol Biol 367(2): 373-383. Moiseenkova-Bell V, Wensel TG (2011). Functional and structural studies of TRP channels heterologously expressed in budding yeast. Adv Exp Med Biol 704: 25-40. Montell C (2001). Physiology, phylogeny, and functions of the TRP superfamily of cation channels. Sci STKE 2001(90): re1. Montell C, Rubin GM (1989). Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2(4): 1313-1323. Moran MM, McAlexander MA, Biro T, Szallasi A (2011). Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 10(8): 601-620. Muik M, Fahrner M, Derler I, Schindl R, Bergsmann J, Frischauf I, et al. (2009). A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels. J Biol Chem 284(13): 8421-8426. Muik M, Frischauf I, Derler I, Fahrner M, Bergsmann J, Eder P, et al. (2008). Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J Biol Chem 283(12): 8014-8022. Nakayama H, Wilkin BJ, Bodi I, Molkentin JD (2006). Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. FASEB J 20(10): 1660-1670. Ng SW, di Capite J, Singaravelu K, Parekh AB (2008). Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels. J Biol Chem 283(46): 31348-31355. Nilius B, Owsianik G (2011). The transient receptor potential family of ion channels. Genome Biol 12(3): 218. Nilius B, Owsianik G, Voets T, Peters JA (2007). Transient receptor potential cation channels in disease. Physiol Rev 87(1): 165-217. Nishida M, Kurose H (2008). Roles of TRP channels in the development of cardiac hypertrophy. Naunyn Schmiedebergs Arch Pharmacol 378(4): 395-406. Numaga T, Nishida M, Kiyonaka S, Kato K, Katano M, Mori E, et al. (2010). Ca2+ influx and protein scaffolding via TRPC3 sustain PKCbeta and ERK activation in B cells. J Cell Sci 123(Pt 6): 927-938. O'Neil RG, Heller S (2005). The mechanosensitive nature of TRPV channels. Pflugers Arch 451(1): 193-203.
106
Obermayer D, Glasnov TN, Kappe CO (2011). Microwave-assisted and continuous flow multistep synthesis of 4-(pyrazol-1-yl)carboxanilides. J Org Chem 76(16): 6657-6669. Obukhov AG, Nowycky MC (2005). A cytosolic residue mediates Mg2+ block and regulates inward current amplitude of a transient receptor potential channel. J Neurosci 25(5): 1234-1239. Okada T, Inoue R, Yamazaki K, Maeda A, Kurosaki T, Yamakuni T, et al. (1999). Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor. J Biol Chem 274(39): 27359-27370. Ong HL, Cheng KT, Liu X, Bandyopadhyay BC, Paria BC, Soboloff J, et al. (2007). Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components. J Biol Chem 282(12): 9105-9116. Owsianik G, Talavera K, Voets T, Nilius B (2006). Permeation and selectivity of TRP channels. Annu Rev Physiol 68: 685-717. Padinjat R, Andrews S (2004). TRP channels at a glance. J Cell Sci 117(Pt 24): 5707-5709. Pak WL, Grossfield J, Arnold KS (1970). Mutants of the visual pathway of Drosophila melanogaster. Nature 227(5257): 518-520. Pang B, Shin DH, Park KS, Huh YJ, Woo J, Zhang YH, et al. (2011). Differential pathways for calcium influx activated by concanavalin A and CD3 stimulation in Jurkat T cells. Pflugers Arch. Parekh AB (2010). Store-operated CRAC channels: function in health and disease. Nat Rev Drug Discov 9(5): 399-410. Parekh AB, Putney JW, Jr. (2005). Store-operated calcium channels. Physiol Rev 85(2): 757-810. Park CY, Hoover PJ, Mullins FM, Bachhawat P, Covington ED, Raunser S, et al. (2009). STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell 136(5): 876-890. Poteser M, Graziani A, Rosker C, Eder P, Derler I, Kahr H, et al. (2006). TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells. J Biol Chem 281(19): 13588-13595. Poteser M, Schleifer H, Lichtenegger M, Schernthaner M, Stockner T, Kappe CO, et al. (2011). PKC-dependent coupling of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes. Proc Natl Acad Sci U S A 108(26): 10556-10561. Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A, Hogan PG (2006). Orai1 is an essential pore subunit of the CRAC channel. Nature 443(7108): 230-233. Putney JW, Jr. (2004). The enigmatic TRPCs: multifunctional cation channels. Trends Cell Biol 14(6): 282-286. Putney JW, Jr. (1986). A model for receptor-regulated calcium entry. Cell Calcium 7(1): 1-12. Quintana A, Griesemer D, Schwarz EC, Hoth M (2005). Calcium-dependent activation of T-lymphocytes. Pflugers Arch 450(1): 1-12. Reiser J, Polu KR, Moller CC, Kenlan P, Altintas MM, Wei C, et al. (2005). TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function. Nat Genet 37(7): 739-744. Rosker C, Graziani A, Lukas M, Eder P, Zhu MX, Romanin C, et al. (2004). Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger. J Biol Chem 279(14): 13696-13704. Salido GM, Sage SO, Rosado JA (2009). TRPC channels and store-operated Ca(2+) entry. Biochim Biophys Acta 1793(2): 223-230. Salmon MD, Ahluwalia J (2010). Discrimination between receptor- and store-operated Ca(2+) influx in human neutrophils. Cell Immunol 265(1): 1-5.
107
Schindl R, Bergsmann J, Frischauf I, Derler I, Fahrner M, Muik M, et al. (2008). 2-aminoethoxydiphenyl borate alters selectivity of Orai3 channels by increasing their pore size. J Biol Chem 283(29): 20261-20267. Schindl R, Romanin C (2007). Assembly domains in TRP channels. Biochem Soc Trans 35(Pt 1): 84-85. Shi J, Takahashi S, Jin XH, Li YQ, Ito Y, Mori Y, et al. (2007). Myosin light chain kinase-independent inhibition by ML-9 of murine TRPC6 channels expressed in HEK293 cells. Br J Pharmacol 152(1): 122-131. Singh BB, Zheng C, Liu X, Lockwich T, Liao D, Zhu MX, et al. (2001). Trp1-dependent enhancement of salivary gland fluid secretion: role of store-operated calcium entry. FASEB J 15(9): 1652-1654. Sinkins WG, Goel M, Estacion M, Schilling WP (2004). Association of immunophilins with mammalian TRPC channels. J Biol Chem 279(33): 34521-34529. Smyth JT, Dehaven WI, Bird GS, Putney JW, Jr. (2008). Ca2+-store-dependent and -independent reversal of Stim1 localization and function. J Cell Sci 121(Pt 6): 762-772. Smyth JT, DeHaven WI, Bird GS, Putney JW, Jr. (2007). Role of the microtubule cytoskeleton in the function of the store-operated Ca2+ channel activator STIM1. J Cell Sci 120(Pt 21): 3762-3771. Steinckwich N, Frippiat JP, Stasia MJ, Erard M, Boxio R, Tankosic C, et al. (2007). Potent inhibition of store-operated Ca2+ influx and superoxide production in HL60 cells and polymorphonuclear neutrophils by the pyrazole derivative BTP2. J Leukoc Biol 81(4): 1054-1064. Stowers L, Holy TE, Meister M, Dulac C, Koentges G (2002). Loss of sex discrimination and male-male aggression in mice deficient for TRP2. Science 295(5559): 1493-1500. Su Z, Anishkin A, Kung C, Saimi Y (2011). The core domain as the force sensor of the yeast mechanosensitive TRP channel. J Gen Physiol 138(6): 627-640. Sweeney ZK, Minatti A, Button DC, Patrick S (2009). Small-molecule inhibitors of store-operated calcium entry. ChemMedChem 4(5): 706-718. Takahashi Y, Murakami M, Watanabe H, Hasegawa H, Ohba T, Munehisa Y, et al. (2007). Essential role of the N-terminus of murine Orai1 in store-operated Ca2+ entry. Biochem Biophys Res Commun 356(1): 45-52. Tiruppathi C, Minshall RD, Paria BC, Vogel SM, Malik AB (2002). Role of Ca2+ signaling in the regulation of endothelial permeability. Vascul Pharmacol 39(4-5): 173-185. Tominaga M (2007). The Role of TRP Channels in Thermosensation TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades. edn. Taylor & Francis Group, LLC: Boca Raton FL. Trebak M (2010). The puzzling role of TRPC3 channels in motor coordination. Pflugers Arch 459(3): 369-375. Trebak M, Bird GS, McKay RR, Putney JW, Jr. (2002). Comparison of human TRPC3 channels in receptor-activated and store-operated modes. Differential sensitivity to channel blockers suggests fundamental differences in channel composition. J Biol Chem 277(24): 21617-21623. Trebak M, Hempel N, Wedel BJ, Smyth JT, Bird GS, Putney JW, Jr. (2005). Negative regulation of TRPC3 channels by protein kinase C-mediated phosphorylation of serine 712. Mol Pharmacol 67(2): 558-563. Trebak M, St JBG, McKay RR, Birnbaumer L, Putney JW, Jr. (2003a). Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels. J Biol Chem 278(18): 16244-16252. Trebak M, Vazquez G, Bird GS, Putney JW, Jr. (2003b). The TRPC3/6/7 subfamily of cation channels. Cell Calcium 33(5-6): 451-461. Trevillyan JM, Chiou XG, Chen YW, Ballaron SJ, Sheets MP, Smith ML, et al. (2001). Potent inhibition of NFAT activation and T cell cytokine production by novel low molecular weight pyrazole compounds. J Biol Chem 276(51): 48118-48126. Tytgat J (1994). Mutations in the P-region of a mammalian potassium channel (RCK1): a comparison with the Shaker potassium channel. Biochem Biophys Res Commun 203(1): 513-518.
108
Uniprot (2012). Reorganizing the protein space at the Universal Protein Resource (UniProt). Nucleic Acids Res 40(Database issue): D71-75. Varnai P, Hunyady L, Balla T (2009). STIM and Orai: the long-awaited constituents of store-operated calcium entry. Trends Pharmacol Sci 30(3): 118-128. Vazquez G, Lievremont JP, St JBG, Putney JW, Jr. (2001). Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes. Proc Natl Acad Sci U S A 98(20): 11777-11782. Vazquez G, Wedel BJ, Trebak M, St John Bird G, Putney JW, Jr. (2003). Expression level of the canonical transient receptor potential 3 (TRPC3) channel determines its mechanism of activation. J Biol Chem 278(24): 21649-21654. Venkatachalam K, Zheng F, Gill DL (2003). Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C. J Biol Chem 278(31): 29031-29040. Vig M, Beck A, Billingsley JM, Lis A, Parvez S, Peinelt C, et al. (2006a). CRACM1 multimers form the ion-selective pore of the CRAC channel. Curr Biol 16(20): 2073-2079. Vig M, DeHaven WI, Bird GS, Billingsley JM, Wang H, Rao PE, et al. (2008). Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. Nat Immunol 9(1): 89-96. Vig M, Peinelt C, Beck A, Koomoa DL, Rabah D, Koblan-Huberson M, et al. (2006b). CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science 312(5777): 1220-1223. Voets T, Janssens A, Droogmans G, Nilius B (2004). Outer pore architecture of a Ca2+-selective TRP channel. J Biol Chem 279(15): 15223-15230. Watanabe H, Takahashi R, Zhang XX, Kakizawa H, Hayashi H, Ohno R (1996). Inhibition of agonist-induced Ca2+ entry in endothelial cells by myosin light-chain kinase inhibitor. Biochem Biophys Res Commun 225(3): 777-784. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009). Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 25(9): 1189-1191. Watson JD, Crick FH (1953). The structure of DNA. Cold Spring Harb Symp Quant Biol 18: 123-131. Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, et al. (2005). A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science 308(5729): 1801-1804. Wolstenholme AJ, Williamson SM, Reaves BJ (2011). TRP channels in parasites. Adv Exp Med Biol 704: 359-371. Woodard GE, Lopez JJ, Jardin I, Salido GM, Rosado JA (2010). TRPC3 regulates agonist-stimulated Ca2+ mobilization by mediating the interaction between type I inositol 1,4,5-trisphosphate receptor, RACK1, and Orai1. J Biol Chem 285(11): 8045-8053. Xu SZ, Zeng F, Boulay G, Grimm C, Harteneck C, Beech DJ (2005). Block of TRPC5 channels by 2-aminoethoxydiphenyl borate: a differential, extracellular and voltage-dependent effect. Br J Pharmacol 145(4): 405-414. Yildirim E, Kawasaki BT, Birnbaumer L (2005). Molecular cloning of TRPC3a, an N-terminally extended, store-operated variant of the human C3 transient receptor potential channel. Proc Natl Acad Sci U S A 102(9): 3307-3311. Yuan JP, Zeng W, Dorwart MR, Choi YJ, Worley PF, Muallem S (2009). SOAR and the polybasic STIM1 domains gate and regulate Orai channels. Nat Cell Biol 11(3): 337-343. Zeng W, Yuan JP, Kim MS, Choi YJ, Huang GN, Worley PF, et al. (2008). STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction. Mol Cell 32(3): 439-448. Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, et al. (2005). STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 437(7060): 902-905.
109
Zhu X, Chu PB, Peyton M, Birnbaumer L (1995). Molecular cloning of a widely expressed human homologue for the Drosophila trp gene. FEBS Lett 373(3): 193-198. Zhu X, Jiang M, Birnbaumer L (1998). Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry. J Biol Chem 273(1): 133-142. Zhu X, Jiang M, Peyton M, Boulay G, Hurst R, Stefani E, et al. (1996). trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell 85(5): 661-671. Zitt C, Halaszovich CR, Luckhoff A (2002). The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry. Prog Neurobiol 66(4): 243-264.
![Metal-mediated base pairs in parallel-stranded DNA · acids with metal-mediated base pairs exist in numerous fields [11]. More recently investigated areas include charge transfer](https://img.pdfslide.org/doc/110x75/5e17603195358446205a8469/metal-mediated-base-pairs-in-parallel-stranded-dna-acids-with-metal-mediated-base.jpg)
