Modulation of TRPV1 function by phosphorylation and site-
directed mutagenesis: from structure to biophysics
Die Modulation der TRPV1 Funktion durch Phosphorylierung und ortsspezifische
Mutagenese: von der Struktur zur Biophysik
Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften
(Dr. rer. nat.)
Der Fakultt fr Biologie und Vorklinischen Medizin
der Universitt Regensburg
vorgelegt von
Thomas Jendryke
aus
Hagen
Im Jahr
2016
Das Promotionsgesuch wurde eingereicht am:
18.03.2016
Die Arbeit wurde angeleitet von:
Prof. Dr. C. H. Wetzel
Unterschrift:
I
Table of contents
1 Zusammenfassung .............................................................................................................. 1
2 Summary ............................................................................................................................. 2
3 Introduction ........................................................................................................................ 3
3.1 Nociception and pain ............................................................................................... 3
3.2 Transient receptor potential (TRP) ion channels ..................................................... 4
3.3 TRPV ion channel subfamily ..................................................................................... 5
3.4 Structural and functional features of the TRPV1 receptor ...................................... 6
3.5 Activation and inhibition of TRPV1 .......................................................................... 9
3.6 Regulation of TRPV1 receptors .............................................................................. 11
3.7 TRPV1 and Cyclin-dependent kinase 5 ................................................................... 13
4 Aims ................................................................................................................................... 18
5 Methods ............................................................................................................................ 19
5.1 Molecular biological methods ................................................................................ 19
5.1.1 Chemocompetent E.coli bacteria ....................................................................... 19
5.1.2 Transformation of vectors into chemocompetent bacteria .............................. 19
5.1.3 Isolation of plasmid DNA .................................................................................... 20
5.1.4 Restriction digestion of plasmid DNA ................................................................. 20
5.1.5 Agarose gel electrophoresis ............................................................................... 20
5.1.6 Gel extraction ..................................................................................................... 21
5.1.7 Ligation of digested DNA fragments .................................................................. 21
5.1.8 Polymerase chain reaction (PCR)........................................................................ 21
5.1.9 Cloning of Cdk5-mCherry, p35-CFP, and TRPV1-GFP fusion proteins ................ 22
5.1.10 TRPV1 mutagenesis via Overlap extension PCR ................................................. 24
5.1.11 Site-directed TRPV1 mutagenesis via pGEM-T vector cloning ........................... 25
5.2 Cell culture ............................................................................................................. 28
5.2.1 Cultivation of CHO and HEK293 cells .................................................................. 28
5.2.2 Transient transfection of CHO and HEK293 cells ............................................... 28
5.3 Electrophysiological experiments .......................................................................... 30
5.3.1 The patch-clamp technique ................................................................................ 30
II
5.3.2 Experimental design ........................................................................................... 31
5.3.3 Electrode preparation ........................................................................................ 31
5.3.4 Application system ............................................................................................. 32
5.3.5 Preparation of TRPV1 stimuli ............................................................................. 32
5.3.6 Voltage-ramp and voltage-step protocols .......................................................... 32
5.3.7 Analysis of voltage-ramp and voltage-step protocols ........................................ 33
5.3.8 Cell-attached single-channel recordings ............................................................ 34
5.3.9 Analysis of single-channel recordings ................................................................ 34
5.4 Fura-2 Ca2+ imaging ................................................................................................ 35
5.4.1 Experimental design ........................................................................................... 35
5.4.2 Recording settings .............................................................................................. 36
5.4.3 Ca2+ imaging data analysis .................................................................................. 36
5.5 TIRF microscopy ..................................................................................................... 36
5.6 Statistical analysis................................................................................................... 37
5.7 Equations ................................................................................................................ 37
6 Results ............................................................................................................................... 38
6.1 TRPV1 desensitization is modulated by co-expression of Cdk5 and p35 .............. 38
6.2 TRPV1, Cdk5 and p35 co-expression does not affect the capsaicin sensitivity ..... 41
6.3 TRPV1 T406 is located close to the TRP domain .................................................... 42
6.4 TRPV1 T406 mutagenesis modulates receptor function ....................................... 44
6.5 Use-dependent activation of TRPV1T406D ............................................................... 47
6.6 Membrane trafficking of TRPV1 and TRPV1T406 mutants ....................................... 51
6.7 T406D mutagenesis affects the voltage-dependence of TRPV1 ............................ 53
6.8 TRPV1T406 mutation alters the capsaicin sensitivity ............................................... 57
6.9 TRPV1 T407 mutagenesis affects the proton activation ........................................ 62
6.10 Heat activation of TRPV1T406D in Ca2+ imaging experiments .................................. 68
6.11 TRPV1 single-channel characteristics are altered in T406D mutants .................... 70
6.12 What is the molecular mechanism that alters the ion channel gaiting? ............... 74
6.13 Desensitization of transition pathway mutants ..................................................... 76
6.14 TP-mutant capsaicin concentration/response-relationship .................................. 78
6.15 Voltage-dependence of transition pathway mutants ............................................ 81
6.16 Low pH activation of TRPV1 transition pathway mutants ..................................... 83
III
7 Discussion ......................................................................................................................... 87
7.1 TRPV1 phosphorylation and nociception ............................................................... 87
7.2 The molecular mechanism of TRPV1 phosphorylation .......................................... 88
7.3 TRPV1T406 mutagenesis........................................................................................... 89
7.4 TRPV1T406D voltage-dependence ............................................................................ 91
7.5 The TRPV1 ion channel sensitivity to capsaicin ..................................................... 92
7.6 Proton and heat induced activation of TRPV1T406 mutants ................................... 93
7.7 Single channel properties of TRPV1T406D receptors................................................ 94
7.8 The high resolution structure of TRPV1 ................................................................. 95
7.9 The TRPV1 transition pathway of Cdk5-mediated phosphorylation ..................... 96
7.10 The Ca2+-dependent desensitization of TRPV1 mutants ........................................ 97
8 Conclusion ......................................................................................................................... 99
9 Materials ......................................................................................................................... 100
9.1 Consumables ........................................................................................................ 100
9.2 Chemicals ......................
