Mutational Analysis of Cysteine Residues of the InsectOdorant Co-receptor (Orco) from Drosophila melanogasterReveals Differential Effects on Agonist- and Odorant-tuningReceptor-dependent Activation*□S

Received for publication, August 8, 2014, and in revised form, September 29, 2014 Published, JBC Papers in Press, September 30, 2014, DOI 10.1074/jbc.M114.603993

Rebecca M. Turner‡, Stephen L. Derryberry§, Brijesh N. Kumar‡, Thomas Brittain‡, Laurence J. Zwiebel§,Richard D. Newcomb‡¶, and David L. Christie‡1

From the ‡School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand, the §Departmentof Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232, and ¶Plant & Food Research, Private Bag 92169,Auckland 1142, New Zealand

Background: Orco is a highly conserved co-receptor required for insects to respond to odorants.Results: Mutation of two cysteine residues in the third intracellular loop of Orco increases direct agonist, but reduces odorant-tuning receptor-dependent channel activation.Conclusion: Intracellular loop 3 is important for activation of the Orco channel.Significance: The research identifies a region of Orco that may regulate odorant sensing by insects.

Insect odorant receptors are heteromeric odorant-gated cat-ion channels comprising a conventional odorant-sensitive tun-ing receptor (ORx) and a highly conserved co-receptor known asOrco. Orco is found only in insects, and very little is knownabout its structure and the mechanism leading to channel acti-vation. In the absence of an ORx, Orco forms homomeric chan-nels that can be activated by a synthetic agonist, VUAA1. Dro-sophila melanogaster Orco (DmelOrco) contains eight cysteineamino acid residues, six of which are highly conserved. In thisstudy, we replaced individual cysteine residues with serine oralanine and expressed Orco mutants in Flp-In 293 T-Rex cells.Changes in intracellular Ca2� levels were used to determineresponses to VUAA1. Replacement of two cysteines (Cys-429and Cys-449) in a predicted intracellular loop (ICL3), individu-ally or together, gave variants that all showed similar increasesin the rate of response and sensitivity to VUAA1 compared withwild-type DmelOrco. Kinetic modeling indicated that theresponse of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of theCys mutants was confirmed by whole-cell voltage clamp electro-physiology. In contrast to the results from direct agonist activa-tion of Orco, the two cysteine replacement mutants whenco-expressed with a tuning receptor (DmelOR22a) showed an�10-fold decrease in potency for activation by 2-methylhexanoate. Our work has shown that intracellular loop 3 isimportant for Orco channel activation. Importantly, this studyalso suggests differences in the structural requirements for theactivation of homomeric and heteromeric Orco channelcomplexes.

Many of the behaviors important for insects depend on olfac-tion. Odorant receptors are one of the main chemosensoryreceptor families in insects and are responsible for detecting awide variety of volatile molecules in the environment (1). Thesereceptors are located on the dendrites of odorant receptor neu-rons, where they interact with ligands, resulting in depolariza-tion and signals that reach the corresponding glomerulus in theantennal lobe.

Insect ORs2 are novel seven-transmembrane domain pro-teins with an inverted topology compared with G protein-cou-pled receptors (2– 4). They have been shown to function asodorant-gated, non-selective cation channels (5, 6). Thesechannels may also be regulated by metabotropic pathways (1, 5,7–9). Insect ORs comprise heteromeric complexes containingboth a conventional, odorant-sensing, or tuning receptor(ORx), and a co-receptor, now known as Orco (2, 10). The stoi-chiometry of OR complexes is unknown. The conventional ORsare highly divergent and provide selectivity to a broad range ofodorant compounds (11). Their expression is restricted to spe-cific odorant receptor neurons (11–13). In contrast, Orco isbroadly expressed in odorant receptor neurons and is essentialfor the response of ORs to odorants (14). Orco is highly con-served across insects, and in vivo, it is required for conventionalOR trafficking and the localization of ORs to ciliated dendritesof odorant receptor neurons (2, 15). Because Orco is requiredfor every insect OR, chemicals that affect Orco function maydisrupt insect behavior and provide a means of pest control.When heterologously expressed, Orco is capable of formingfunctional channels in the absence of a conventional receptor(5, 16). Screening of cell lines expressing these homomericchannels led to the discovery of VUAA1 as a novel allostericagonist of insect ORs (16). Further exploration of the structure-* This work was supported by Grant PAF-09-01 from the Marsden Fund

administered by the Royal Society of New Zealand (to R. D. N.).□S This article contains supplemental Fig. 1.1 To whom correspondence should be addressed: School of Biological Sci-

ences, University of Auckland, Private Bag 92019, Auckland, New Zealand.Tel.: 64-937-37599; E-mail: d.christie@auckland.ac.nz.

2 The abbreviations used are: OR, odorant receptor; ORx, odorant-sensing ortuning receptor; ICL, intracellular loop; Dmel, Drosophila melanogaster,DmelOrco, Drosophila melanogaster Orco; TM, transmembrane.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 46, pp. 31837–31845, November 14, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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activity relationships of VUAA1 identified several more potentOrco allosteric agonists (17–19). A number of compoundsstructurally related to VUAA1 that are inactive as agonists actas competitive inhibitors of VUAA1 (17, 18, 20). Both hetero-meric and homomeric Orco complexes can be activated byVUAA1 (16). Heteromeric and homomeric OR complexes arealso blocked by the general cation channel blocker rutheniumred (21, 22) and by amiloride derivatives (23, 24) .

Currently, there is little knowledge about the structure of theOrco channel, how Orco interacts with an ORx, and the mech-anisms involved in channel activation and permeability. Thepredicted topology of Orco consisting of seven transmembranedomains with the N terminus facing the cytoplasm and C ter-minus on the extracellular surface is supported by a range ofexperimental techniques (2, 4). Bioluminescence and Förster/fluorescence resonance energy transfer experiments have beenused to show interactions between Orco and an ORx (25, 26).The predicted intracellular loop 3 (ICL3) regions of bothDmelOR43a and DmelOrco were shown to interact in a yeasttwo-hybrid assay, consistent with the observation that theC-terminal region of OR43a is sufficient for Orco dependenttransport to olfactory cilia (2). Although Orco can form chan-nels on its own (5, 16) and Orco does not contribute to theodorant binding site (21, 27), studies indicate the presence ofodorant-specific ORs may influence ion permeability andinhibitor sensitivity of heteromeric OR complexes (21, 22, 28).Taken together, these studies support the idea that both theodorant-specific tuning OR and Orco contribute to the channelpore. Alternatively, it is possible that the interaction of specificORs with Orco may indirectly affect the properties of the chan-nel pore. There is some information on the contribution ofindividual amino acid positions of Orco to OR function. Mod-ification of a TVVGYLG sequence in TM6 of DmelOrcoreduced K� permeability (5) and a Y464A mutation in TM7 ofthe Bombyx mori Orco in combination with B. mori OR-1results in a small increase in K� selectivity (28). Furthermore,we recently showed that a conserved aspartic acid residue inTM7 is important for the activation of both homomeric chan-nels by VUAA1 and heteromeric channels by odorants (29).

DmelOrco contains a number of cysteine residues in pre-dicted ICLs (see Fig. 1). We hypothesized that these may con-tribute to structural features important for function. Here, wehave carried out a mutagenesis study and find that replacementof two cysteines in ICL3 has differential effects on agonist- andodorant-tuning receptor-dependent activation.

EXPERIMENTAL PROCEDURES

Expression Plasmids for DmelOrco and DmelOR22a—Themodification of DmelOrco to include an N-terminal Mycepitope and its cloning into the pcDNA5/FRT/TO vector hasbeen described previously (29). The Drosophila melanogasterOR22a cDNA was obtained from Dr. Coral Warr (Monash Uni-versity, Melbourne, Australia). This was cloned into pIB/V5-His (Invitrogen) using KpnI and SacII sites and subsequentlytransferred into pcDNA3.1� (Invitrogen). Lastly, a FLAGepitope (DYKDDDK) was inserted after the initiator methio-nine, and the sequence around the initiation code was altered toa mammalian Kozak consensus sequence by PCR.

Site-directed Mutagenesis and Preparation of Flp-In 293T-Rex Cell Lines—The pcDNA5/FRT/TO-DmelOrco templatewas mutated to encode the C87S, C216A, C221S, C228S,C409S, C429S, C446S, and C449S Orco variants by GenScriptUSA, Inc. The C429S/C449S double mutant was preparedusing C429S as a template and a method adapted from theQuikChange site-directed mutagenesis kit (Stratagene) andRef. 30. Two complementary oligonucleotides (29 –33 bp)encoding the C449S mutation were obtained from IntegratedDNA Technologies. The cycling reaction used Pfu Turbo DNAPolymerase (Agilent Technologies) and the presence of 1X PCREnhancer solution (Invitrogen). The PCR products weretreated with DpnI (Invitrogen) to remove the template DNAand used to transform competent Escherichia coli DH5� cellsprepared as described in Ref. 30. The presence of the desiredmutation and absence of introduced mutations were confirmedby sequencing the N-terminal Myc DmelOrco insert. KpnI andNotI sites were used to transfer the insert into fresh vector.Plasmids encoding the cysteine replacement mutants weretransfected into Flp-In 293 T-Rex cells that were grown andselected for hygromycin resistance as described previously (29).

Ca2� Imaging—Flp-In 293 T-Rex cells encoding WTDmelOrco and the Cys replacement mutants were plated(50,000 cells/well) in 96-well clear bottom, black-walled plates(BD Biocoat, catalog no. 356640). After 1 day, cells were treatedwith 0.1 �g/ml doxycycline for 24 h to induce Orco expression.The medium was then removed, and the cells were loaded (30min at 37 °C, followed by 1 h at room temperature) with Fluo-4NW (Invitrogen) prepared as suggested by the manufacturer inHank’s buffer containing Ca2� and Mg2�. To investigate odor-ant activation, WT DmelOrco and Orco Cys replacementmutants were plated in six-well plates (400,000 cells/well), leftfor 24 h, and transfected with DmelOR22a (2 �g/well) usingFuGENE 6 (Promega, 6 �l/well) for 12 h. Cells (80,000 cells/wellwere transferred to 96 well assay plates prior to being inducedwith 0.3 �g/ml of doxycycline for 12–13 h prior to the assay.The cells were loaded with Fluo-4 AM (Molecular Probes) andwashed prior to the assay as described previously (29). Ca2�

fluorescence was measured in an Envision multilabel platereader (PerkinElmer Life Science). The following settings wereused: excitation filter, FITC 485 nm; emission filter, 520 nm;bottom-fitted dichroic mirror, FITC 505; bottom excitation,bottom sensor; measurement distance: 6.5 mm. Fluorescencereadings were taken every 0.4 s, except for the analysis of thetime course kinetics when readings were taken every 0.1 s. TheOrco allosteric agonist, VUAA1 (N-(4-ethylphenyl)-2-((4-eth-yl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)thio)acetamide), pur-chased from Interbioscreen, Ltd. (ID STOC3S-70586), or odor-ant methyl hexanoate (Sigma, CAS 106-70-7) were freshlydiluted from dimethyl sulfoxide stocks into Hank’s buffer to sixtimes the desired test concentration and injected (20 �l into100 �l) automatically after 8 s. The final concentration ofdimethyl sulfoxide did not exceed 0.25%, and all experimentsincluded controls with buffer and dimethyl sulfoxide alone.

Cell Surface Biotinylation and Western Blotting—Flp-In 293T-Rex cell lines expressing DmelOrco and its variants wereplated (700,000 cells/well in six-well plates, grown for 24 h, andinduced with 0.1 �g/ml doxycycline for 24 h. Proteins at the cell

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surface were labeled with a membrane impermeable biotinyla-tion reagent. The procedure for biotinylation was based on thatused previously (29). Briefly, the cells were washed with PBSand labeled with EZ-Link-Sulfo-NHS-SS-Biotin (ThermoSci-ence, 1.5 mg/ml; 0.6 ml/well) for 20 min on ice. The labeling wasrepeated once with fresh reagent. The reagent was removed,and the cells were washed and lysed in 350 �l of 20 mM Tris, 137mM NaCl, 1 mM EDTA, pH 7.6 buffer, containing 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhib-itors (CompleteTM Mini protease inhibitor mixture, RocheMolecular Biochemicals). The lysate was obtained by centrifu-gation, and the protein concentration was determined (Bio-RadDC Protein Assay). Lysates were adjusted if necessary by dilu-tion with lysis buffer. Biotinylated proteins were purified fromlysate samples (typically 286 �l, 1.4 mg/ml protein) by the addi-tion (50 �l of a 50% suspension) of Neutravidin beads (Ther-moScience). Following incubation overnight at 4 °C, the beadswere recovered by centrifugation and washed to remove non-specifically bound proteins. The beads were resuspended withan equal volume of a high-SDS reducing buffer (125 mM Tris,8% SDS, 20% glycerol, and 10% �-mercaptoethanol) and incu-bated for 1 h at room temperature. Aliquots (17.5 �l) of thelysate and biotinylated samples were run on 10% SDS-poly-acrylamide gels and subjected to Western blotting (31). Theblot was probed with mouse anti-Myc antibodies (Santa CruzBiotechnology, sc-40) followed by goat anti-mouse horseradishperoxidase conjugate (Bio-Rad, 170-6516). Chemilumines-cence was detected using a Fuji LAS-1000 digital imaging sys-tem. A WesternBright ECL detection kit (Advansta) was usedfor samples from total cell lysates, whereas the more sensitiveWesternBright Sirius kit (Advansta) was used for biotinylatedsamples. Exposure times were between 20 – 60 s for both detec-tion systems. Bands were quantified using MultiGauge software(version 2.2, Fujifilm). Band intensity for each lane was calcu-lated from the number of black pixels in a box placed around theband minus the black pixels in a box of the same size placedaround a background region from the same blot. The expres-sion of Orco Cys-replacement mutants is expressed relative tosamples of WT Orco run in every experiment.

Whole-cell Patch Clamp Electrophysiology—Stably trans-fected cells were induced using 0.1 �g/ml doxycycline for 12 to24 h before recording. Whole-cell recording was carried out asdescribed previously (16). All cells were voltage clamped at �60mV and Clampfit 10 (pClamp 10, Axon Instruments) wasemployed to calculate the rise time, which was defined asthe time during which the magnitude of the inward currentremained within 10 and 90% of the maximal inward current.

Data Analysis—Curve fitting, EC50 determination, and statis-tical analysis was performed using Prism software (GraphPad). Thelog values of relative protein expression data were used for anal-ysis of variance and t tests to determine statistical differences.The changes in Ca2� fluorescence in response to Orco activa-tion was defined as the fluorescence minus the background fluo-rescence, all divided by the background fluorescence (deter-mined from the average of the first 20 readings prior to theaddition of agonist). Log values of the relative activity of Cys-replacement Orco mutants compared with WT Orco deter-mined in the same experiment were used for statistical analysis.

To analyze the time course of the Ca2� response followingactivation of WT Orco and the Cys replacement mutants withVUAA1, data were collected at 0.1-s intervals. Digitized timecourse data for the increase of intracellular calcium fluores-cence signals were fitted to the minimum model using non-linear least squares criteria. The fitting was performed usingTableCurve 2D software (Jandel Scientific). Best fits typicallydisplayed r2 values better than 0.998. The analysis yielded bestfit parameters for the apparent rates of the two phases of reac-tion. The apparent rates for the first phase of the reaction (k1)were found to be concentration-dependent. Second order rateconstants were determined from the slope of linear plotsobtained by least squares fitting of the rate versus concentrationdata.

RESULTS

Cysteine residues are present at positions 87, 216, 221, 228,409, 429, 446, and 449 of DmelOrco (Fig. 1). Six of the eightcysteines are present in equivalent positions in Orco sequencesrepresenting species from six insect orders (supplemental Fig.1). The conserved Cys residues are located in two predictedICLs: Cys-221 and Cys-228 are in ICL2 and Cys-409, Cys-429,Cys-446, and Cys-449 are in ICL3 (Fig. 1). Of the two non-conserved cysteine residues, Ser or Thr is found in several spe-cies in place of Cys-87, whereas Ala, Leu, Met, or Val canreplace Cys-216. We carried out a mutagenesis study by replac-ing individual Cys residues with Ser, except for Cys-216, whichwas replaced with Ala, an amino acid present at this position inOrco from some species. Each Orco variant was expressedin Flp-In 293 T-Rex cells, and activity was determined fromchanges in intracellular Ca2� levels in response to the syntheticallosteric agonist, VUAA1. The time course for Ca2� influxidentified Cys mutants with maximum activity levels higher,lower, and similar to WT Orco (Fig. 2A). Fig. 2B shows a moredetailed quantitative comparison where the data for individualmutants has been normalized as a ratio relative to WT Orcoactivity determined in the same experiment. Mutants are com-pared both in relation to their maximum response levels andalso the responses obtained after 6 s, a time that indicates theinitial rates of activity (Fig. 2A). The activity level for the C87S,C216A, and C221S mutants is very similar to WT Orco. Twomutants, C228S and C446S, had significantly reduced activity,whereas C87S showed a small but significant reduction in activ-ity. C409S, C429S, and C449S all showed significantly higherlevels of activity particularly when measured at 6 s (Fig. 2B). Theactivities of the C228S and C446S mutants were also comparedwith each other. Their activities, determined after 6 s, weresignificantly different (analysis of variance with a Turkey’s posttest, p � 0.001), although differences between the two mutantscould not be confirmed for the maximum levels of activity. Sim-ilar analyses for the two mutants C429S and C449S confirmedthat their activities, although significantly different from Orco,were not significantly different from each other. The activity ofthe C429S/C449S double mutant was significantly increasedcompared with WT Orco (Fig. 2B) and indistinguishable fromeither of the contributing single mutants.

Some of the differences observed in the activity of Cysreplacement mutants compared with WT Orco may be due to

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variations in cell surface expression. To address this, Westernblot experiments were carried out to compare the expression ofOrco mutants with WT Orco in both total cell lysates and in apurified “biotinylated” fraction obtained after labeling of intactcells with sulfo-NHS-SS-biotin. Here, Orco was detected withantibodies against the Myc epitope, and data from individualrepresentative experiments are shown in Fig. 3, A and B. TheC409S mutant appeared to be present at higher levels than WTOrco in both total and cell surface fractions. A quantitativecomparison of the relative expression of mutants confirmedthat C409S Orco was expressed at significantly higher levels(�3-fold) than WT Orco (Fig. 3C). A small but significantincrease in total and surface expression was also observed forC446S Orco. Otherwise, the expression levels of the Cysreplacement mutants were similar to WT Orco.

The properties of mutants (C409S, C429S, C449S, and theC429S/C449S double mutant) showing increased levels ofVUAA1-stimulated activity in the Ca2� flux assay (Fig. 2B)were studied in more detail. Fig. 4A shows representative con-centration response curves for the Cys replacement variants,and WT Orco and indicates that the curves of variants contain-ing the C429S and C449S mutations individually and in combi-

nation are “left-shifted” compared with WT Orco. The EC50values determined from multiple experiments were �2-foldlower than WT Orco (Fig. 4B), indicating the enhanced sensi-tivity of these variants to activation by VUAA1. The EC50 valueof C409S was similar to WT Orco but gave a higher Emax valuein accordance with the increased expression level this mutant.

Although the concentration response curves indicate thatthe C429S, C449S, and the C429S/C449S mutants haveenhanced sensitivity to activation by VUAA1, the experimentsin Fig. 2 suggested that they may also respond faster than WTOrco. However, the time course for the change in intracellularCa2� in response to VUAA1 is complex (Fig. 2A). There aremultiple components, most obviously an increase followed by adecrease in intracellular Ca2� (most clearly seen for themutants showing the highest Ca2� response). To further clarifythis point, we more closely analyzed the response of WT Orcoduring the phase where intracellular Ca2� is increasing, reveal-ing that the initial Orco response exhibits a sigmoid time course(Fig. 5). We further asked whether a simple two-step reactionmechanism A � B3 C3D could explain the observed kinet-ics. As can be seen, a mathematical simulation based on thisequation generates a curve that provides an excellent fit to the

FIGURE 1. Schematic diagram of the topology of Orco from Drosophila melanogaster (DmelOrco). The TM domains were predicted using TMHMM (33, 34),and the topology diagram was generated with TOPO2 (S. J. Johns, TOPO2, Transmembrane protein display software). The amino acid residues of the Mycepitope present at the N terminus and used to detect Orco by Western blotting are shown as white letters on gray circles. The location of Cys residues targetedin the mutagenesis study are shown as white letters on black circles and numbered according to their positions in WT DmelOrco.

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observed kinetics (Fig. 5). Data collected from the response ofWT Orco and the Cys replacement mutants at differentVUAA1 concentrations were used to calculate the second orderrate constant (k1) for the first reaction (A � B3C) and the rateconstant (k2) of the second step (C3 D). This modeling sug-gests that each of the Cys replacement mutants facilitate theactivation response in two respects: a concentration-dependentinteraction of Orco with the agonist VUAA1 and also a non-VUAA1 dependent step required for entry of Ca2� into the cell.The rate constants also suggest that the overall response of theCys replacement mutants to VUAA1 is faster than for WTOrco.

Measurements of changes in intracellular [Ca2�] are fast andsensitive and have been used extensively to characterize thedose-response characteristics of allosteric Orco agonists and

antagonists (16, 18, 19) as well as Orco mutations that affect theactivation of both homomeric channels by VUAA1 and hetero-meric channels by odorants (29). Although these assays do notdirectly measure voltage changes, the response to VUAA1 doeshowever depend on fluxing of extracellular Ca2� (data notshown), so increases in Ca2� fluorescence do reflect cationchannel activity. Nevertheless, we did elect to provide addi-tional data of these new Orco mutants through recording ofionic currents in patch clamped single cells (Fig. 6). Asexpected, VUAA1 elicited concentration-dependent increasesin the inward current of WT and mutant Orco cell lines (Fig.6A). The concentration response curves indicated that the two

FIGURE 2. Effect of replacement of the endogenous cysteine residues ofDrosophila melanogaster Orco (DmelOrco) on VUAA1-stimulated Ca2�

influx activity. A, the time course for Ca2� influx stimulated by the additionof 100 �M VUAA1 to Flp-In 293 T-Rex cells expressing WT Dmel Orco and eightindividual cysteine replacement mutants. The curves are representative ofthe responses obtained for WT and cysteine replacement Orco variants. Thedata represent the means � S.D. of 6 replicates (solid and dashed lines). Thevertical dashed line indicates the time point (6 s) used to compare relativeactivities prior the response of any of the Orco variants reaching a maximumeffect on intracellular Ca2�. B, comparison of the relative response of individ-ual mutants. The data are normalized as a ratio of the activity of each mutantrelative to WT Orco determined in the same experiment. The values shownare the means � S.E. of five to six separate experiments each with five to sixreplicates. The normalized responses are compared in two ways: first after 6 s,a time at which all the mutants are showing increases in intracellular Ca2� andprior to any of them reaching a maximum level and second, comparison ofthe maximum response levels reached by each mutant. The activities for eachof the mutants were compared (individual t tests, using the log of the ratiodata) against WT DmelOrco: *, 0.01 � p � 0.05; **, 0.001 � p � 0.01; and ***,p � 0.001.

FIGURE 3. Comparison of the expression levels of Cys replacementmutants of Orco with WT Orco from D. melanogaster. Equal amounts ofprotein from whole cell lysates (A) and biotinylated fractions (B) of Flp-In 293T-Rex cells expressing cysteine replacement mutants of Orco and WT Orcowere run on SDS-polyacrylamide gels and detected by Western blotting withMyc antibodies. The immunoblots shown for lysate and biotinylated fractionsare representative of the many experiments carried out to analyze the indi-vidual mutants. Experiments for the individual mutants were replicated fiveto nine times in different experiments. Extracts from WT Orco were run andprocessed identically for each experiment. C, expression levels of individualCys replacement mutants are shown relative to WT Orco. Orco expression,normalized at 100%, is shown by the dashed line. The ratio of the expressionlevels of Cys replacement mutants were expressed as a ratio relative to WTOrco (normalized to 1.0). The values represent the mean � S.E. of five to ninedeterminations. These values were log-transformed to determine statisticaldifferences in expression levels compared with WT Orco (two-tailed t tests, *,0.01 � p � 0.05; and ***, p � 0.001).

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Cys replacement mutants were more sensitive to VUAA1 thanWT Orco (Fig. 6B), although only the EC50 values for C449Sand the C429S/C449S double mutant were significantly differ-

ent. To compare the relative rates of response, we defined “risetime” as the time during which the magnitude of the inwardcurrent remained within 10 and 90% of the maximal inwardcurrent. A comparative analysis of the rise times suggested thatthe Cys replacement mutants responded faster than WT Orco,although the values were only significantly different from WTOrco for the C429S/C449S mutant (Fig. 6).

We wanted to determine whether the increased activationresponses of C429S, C449S, and C429S/C449S Orco to VUAA1(Figs. 4 – 6) extended to odorant activation of Orco when co-expressed with a conventional ORx. Cells stably expressing WTOrco or the Cys replacement mutants were transiently trans-fected with DmelOR22a and the change in intracellular Ca2�

determined in response to the addition of the OR22a agonist,methyl hexanoate (Fig. 7). The concentration response curvesfor the Cys replacement mutants were all “shifted to the right”compared with Orco. This change reflects an �10-folddecrease in the potency of the Cys replacement mutants tomethyl hexanoate compared with WT Orco. Thus, it appearsthat the replacement of cysteine residues at these positionsimpairs the activation of Orco by odorants in the presence of anORx.

DISCUSSION

Although the molecular mechanisms by which direct ago-nists and odorants through interactions with specific odorantreceptors activate the Orco channel remain largely unknown,the studies described here show the involvement of the ICL3region in the activation of Orco. Interestingly, our functionalanalyses of cysteine replacement mutants suggest perturbationof ICL3 differentially affects direct Orco agonist- versus odor-ant-tuning receptor-dependent activation of the channel.

The conservation of six of the eight cysteines in DmelOrco inequivalent positions in Orco orthologues across a wide range ofinsect taxa provided a strong rationale to investigate their func-tional importance. We chose initially to characterize cysteinereplacement mutants in the absence of a tuning receptor. Thisapproach avoids complications from the possible effects ofmutations on the interactions of Orco with the tuning receptoror from the presence of mixtures of both homomeric and het-eromeric Orco complexes. Cys replacement Orco mutantswere stably expressed in Flp-In 293 T-Rex cells lines to avoidpotential difficulties from differences in transfection efficiency.These tetracycline-inducible lines are suitable for studies ofOrco function by both microplate assays of Ca2� influx anddirect current recording of patch clamped cells (16, 29). Ideally,the surface expression of all mutants would be compared withWT Orco through direct binding assays with specific agonistsor antagonists. In the absence of specific reagents for Orco, weused an N-terminal Myc epitope to compare the expression ofmutants relative to WT Orco by cell-surface biotinylation andWestern blotting. C409S Orco was the only mutant found to beexpressed at substantially higher levels than WT Orco (Fig. 3).Importantly, the increased expression affected only the Emaxvalue and not the EC50 value of this mutant for VUAA1.

The individual C429S and C449S and double C429S/C449SOrco mutants all showed increases in response to VUAA1accompanied by decreases in the their EC50 values compared

FIGURE 4. Sensitivity of WT and the Cys replacement mutants of D. melano-gaster Orco to VUAA1. A, concentration response analysis of WT Orco andC409S, C429S, C449S, and C429S/C449S Orco to VUAA1. The data for the concen-tration response curves were obtained from a single experiment (mean � S.D. offour to six replicates). B, log EC50 values for the response of WT Orco and C409S,C429S, C449S, and C429S/C449S Orco to VUAA1. The values represent themean � S.E. from three to seven experiments. The values for C429S, C449S, andC429S/C449S Orco were significantly different from WT Orco. ***, p � 0.001; n.s.,not significant. The mean EC50 values for WT, and C409S, C429S, C449S, andC429S/C449S Orco are 40.9, 54.2, 23.7, 21.6, and 24.5 �M VUAA1, respectively.

FIGURE 5. Analysis of the kinetics of the time course for the increase in intra-cellular Ca2� following activation by VUAA1. A, the fluorescence for intracel-lular Ca2� following the addition of 100 �M VUAA1 to Flp-In 293 T-Rex cellsexpressing WT Orco from Drosophila melanogaster was determined at 0.1-s inter-vals. To simplify presentation, only every third data point (black circles) is shown.The complete set of data was fitted to the minimum model (A � B3 C3 D)using non-linear least squares criteria. The red line shows the simulated curvesbased on this model. The analysis yielded best fit parameters for the apparentrates of the two phases of reaction. The apparent rate for the first phase of thereaction (k1) was found to be concentration dependent. Second order rate con-stants were determined from the slope of linear plots obtained by least squaresfitting of the rate versus concentration data. B, similar analyses for the data fromexperiments with C429S, C449S, and C429S/C449S Orco were carried out. The k1and k2 values for the Cys replacement mutants are compared with WT Orco; S.E.indicates S.E. of the fits used to estimate the rate constants.

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with WT Orco (Figs. 2 and 4). The increased sensitivity of thecysteine replacement mutants to VUAA1, shown by assays ofCa2� influx, was supported by direct current analysis of WTand mutant Orco cell lines (Fig. 6B), although the effects inthese whole cell patch clamp studies were not as dramatic. Thedifferences between the EC50 values of the WT and Cys replace-ment mutants were greater in the Ca2� flux assays than for thepatch clamp experiments. There are several inherent differ-ences between whole cell patch clamp studies and Ca2� fluxassays that likely contribute to this partial disconnect. The mostobvious difference is that data from the Ca2� assays is obtainedfrom the collective response of large numbers of cells to a singleconcentration of VUAA1, whereas in the whole cell patchexperiments, direct voltage recordings are taken from individ-ual cells exposed to increasing doses of VUAA1 separated bywashing. In this light, we cannot rule out that this paradigmdoes not result in some degree of Orco channel desensitization.It is also apparent that in some cases the inward current has notreached a steady state that may also likely affect the EC50 values.That said, it is especially salient that the increased sensitivity toVUUA1, most notably for the C449S and the C429S/C449S

double mutant, is also seen in the whole cell current recordingexperiments.

There are many possible reasons for the increases in sensi-tivity to VUAA1 shown by the mutants. Replacement of a cys-teine with a serine is commonly used in mutagenesis experi-ments, but nevertheless, differences between the properties ofthe two amino acid side chains may affect the structure of theICL3 region. Such an alteration may improve the direct inter-actions with VUAA1 resulting in increased sensitivity to acti-vation by this agonist. Some possibilities include improvedaccess for VUAA1 to its binding site or by making it easier forVUAA1 binding to initiate the conformational changesrequired for activation of the channel.

We wanted to probe the mechanism for the observedincrease in the efficacy of VUAA1 to activate the C229S, C449S,and the C229S/C449S variants in more detail. First, we investi-gated whether a simple two-step reaction (A � B 3 C 3 D)could be used to model the kinetics for the increase in intracel-lular Ca2� following VUUA1 activation. This is very similar tothe del Castillo-Katz mechanism for the transduction mecha-nism for the endplate nicotinic acetylcholine receptor, as

FIGURE 6. Comparison of the response of WT and the Cys replacement mutants of D. melanogaster Orco to VUAA1 determined by assay of currents involtage clamped cells. A, representative whole-cell recordings from DmelOrco lines stimulated with ascending concentrations of VUAA1. Stimulus barsindicate log(M) concentrations of VUAA1. B, the concentration response curves for WT Orco, C429S, C449S, and C429S/C449S Orco to VUAA1 (mean values �S.E.). The mean EC50 values determined by analysis of the curves for WT and C429S, C449S, and C429S/C449S mutants were 50.89, 49.02, 45.90, and 41.97 �M

VUAA1, respectively. Their 95% confidence intervals indicate that C449S and C429S/C449S are significantly more potent than the WT Orco. C, the calculated risetimes (mean � S.E.) for the WT and mutant DmelOrco cell lines when stimulated at their respective EC50 concentrations of VUAA1 as determined in B. Rise timewas defined as the time during which the induced inward current magnitude remained within 10 and 90% of the maximum response. C429S, C449S, andC429S/C449S appear to have smaller rise times (are kinetically faster) when compared with the WT; however, only the double mutant is significantly faster (p �0.05, Dunnett’s, post test).

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reviewed in Ref. 32. In this mechanism, the agonist binds to areceptor; the receptor in the “bound” state is still inactive butcan undergo a conformational change to the active (open) state.Mathematical modeling of this mechanism was found to fit theexperimental data very well (best fits, typically displayed r2 val-ues better than 0.998). The equation enabled the calculation ofthe two rate constants: k1 (A � B3 C); and k2 (C3 D) for arange of VUAA1 concentrations for both WT Orco and the Cysreplacement mutants (Fig. 5). Separate graphical analysis iden-tified k1 as a second order rate constant (dependent on theconcentration of VUAAl), whereas k2 was not concentration-dependent, consistent with it being associated with a step sub-sequent to VUAA1 binding. Interestingly, the k1 values for theCys replacement mutants were higher than WT Orco, whichwould be consistent with VUAA1 binding occurring more rap-idly. The k2 rate constant indicates that the C3 D step is alsofaster for the Cys replacement mutants. The faster responsekinetics for the cysteine replacement mutants was supported bythe direct measurement of inward currents in the mutant celllines (Fig. 6C). The rise time for the C429S/C449S doublemutant was significantly shorter than for WT Orco, confirmingthat the mutant does indeed respond faster. We have only con-sidered the “forward” reaction leading to opening of the chan-nel gate and appreciate that k1 and k2 will be affected by both theforward and reverse reactions, e.g. improved efficacy to VUAA1could result from mutations that facilitate binding or access ofVUAA1, or alternatively, decrease the “off” reaction forVUAA1 binding. Similarly, our studies have not consideredalternatives such as deactivation of the Orco channel.

ICL3 is predicted to act as a cytoplasmic link between TM6and TM7, regions known to contain amino acid residuesimportant for channel activity (5, 28, 29). Thus, this region is

well positioned to play a role in the activation of insect odorantreceptors. Previously, we have shown that substitution of Asp-466 in TM7 of DmelOrco with Glu (D466E) increased the sen-sitivity of DmelOrco activation to both VUAA1 and also toodorants in the presence of a specific odorant receptors (29).An intriguing feature of the current work is that the increasedsensitivity of the C229S and C449S mutants in homomericOrco complexes for VUAA1 was not matched by increased sen-sitivity of an Orco/odorant receptor complex to an odorant. Infact, reduced sensitivity to the odorant was observed. We sug-gest that the Orco mutations in the ICL3 region impact theability of OR22a, in the presence of methyl hexanoate, to inducethe conformational changes required for activation of the chan-nel. An interaction between the ICL3 region of Orco and theICL3 region of an ORx has been demonstrated in yeast two-hybrid studies, a finding also consistent with regions requiredfor Orco-dependent trafficking of OR complexes to olfactorycilia (2). We consider the similar Emax values determined for theCys replacement mutants and Orco make it unlikely that the10-fold increase in the EC50 values of the C229S, C449S, andC229S/C449S Orco variants for methyl hexanoate can beexplained by these mutations affecting the interaction of Orcowith DmelOR22a or the trafficking of heteromeric complexesto the cell membrane. Unfortunately, we were unable to con-firm that similar amounts of OR22a were expressed at the sur-face of cells expressing WT and mutant Orco. Despite the pres-ence of an N-terminal FLAG epitope, we could not detectOR22a in whole cell lysates or biotinylated fractions of tran-siently transfected cells by Western blotting.

The D466E mutation was suggested to induce a conforma-tional state that favors opening of the channel common to bothVUAA1 and odorant/ORx agonism (29). In separate experi-ments, we combined the C429S/C449S mutation with the pre-vious D466E gain-of-activation mutant. The concentrationresponse curve of the triple mutant transfected with OR22a tomethyl hexanoate (data not shown) indicated that the addi-tional mutation partially (�50%) reversed the decrease in sen-sitivity resulting from the C229S and C449S mutations. Thissuggests that the ICL3 and TM7 mutations target differentstages of the activation response as the addition of D466Eimproves but does not restore the loss of sensitivity to activa-tion of Orco by OR22a.

In summary, we have provided evidence that ICL3 of Orcoplays an important role in the activation of insect odorant chan-nels by both direct agonists and odorants through tuning ORs.We suggest that an alteration to the structure of the ICL3region is responsible for the decreased responsiveness seen toodorants. It would seem quite plausible for a structural changethat improves activation by VUAA1, a non-physiological allo-steric agonist, to interfere with activation by a tuning receptorin the presence of an odorant.

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Activation of Orco Involves Intracellular Loop 3

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Laurence J. Zwiebel, Richard D. Newcomb and David L. ChristieRebecca M. Turner, Stephen L. Derryberry, Brijesh N. Kumar, Thomas Brittain,

Odorant-tuning Receptor-dependent Activation Reveals Differential Effects on Agonist- andDrosophila melanogaster(Orco) from

Mutational Analysis of Cysteine Residues of the Insect Odorant Co-receptor

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