Characterization of a 5-HT3–ELIC Chimera ... - ACS Publications

Mar 6, 2018 - Cys-loop receptors are major sites of action for many important therapeutically active compounds, but the sites of action of those that ...
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Characterization of a 5‑HT3−ELIC Chimera Revealing the Sites of Action of Modulators Kerry L. Price and Sarah C. R. Lummis* Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, U.K. ABSTRACT: Cys-loop receptors are major sites of action for many important therapeutically active compounds, but the sites of action of those that do not act at the orthosteric binding site or at the pore are mostly poorly understood. To help understand these, we here describe a chimeric receptor consisting of the extracellular domain of the 5-HT3A receptor and the transmembrane domain of a prokaryotic homologue, ELIC. Alterations of some residues at the coupling interface are required for function, but the resulting receptor expresses well and responds to 5-HT with a lower EC50 (0.34 μM) than that of the 5-HT3A receptor. Partial agonists and competitive antagonists of the 5-HT3A receptor activate and inhibit the chimera as expected. Examination of a range of receptor modulators, including ethanol, thymol, 5-hydroxyindole, and 5chloroindole, which can affect the 5-HT3A receptor and ELIC, suggest that these compounds act via the transmembrane domain, except for 5-hydroxyindole, which can compete with 5-HT at the orthosteric binding site. The data throw further light on the importance of coupling interface in Cys-loop receptors and provide a platform for examining the mechanism of action of compounds that act in the extracellular domain of the 5-HT3A receptor and the transmembrane domain of ELIC. KEYWORDS: 5-HT3, ELIC, Erwinia, chimera, Cys-loop receptor, pLGIC, modulator, PAM

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INTRODUCTION 5-HT3 receptors, like nicotinic acetylcholine (nACh), GABAA, and glycine receptors, are members of the pentameric-ligandgated-ion-channel (pLGIC) superfamily. They are homomeric (5-HT3A) or heteromeric (5-HT3A with 5-HT3B-E) complexes that translate the binding of serotonin at the orthosteric site in the extracellular domain (ECD) to the opening of an integral cation-selective ion channel in the transmembrane domain (TMD).1 High-resolution structural details of full-length receptors first emerged from prokaryotic orthologues, the Erwinia (ELIC)2 and Gloeobacter (GLIC)3,4 ligand-gated ion channels. This provided further insight into the molecular mechanisms of ligand binding and gating. The more recent emergence of crystal-structure data from eukaryotic receptors has revealed a high degree of structural conservation, with conserved tertiary folding as well as conserved features, such as the rings of charges lining the pore. Nevertheless, there are many features of this important family of proteins that we do not yet fully understand, and one route to explore these is to use chimeric receptors. The modular nature of pLGICs, which have three distinct domains (extracellular, transmembrane, and intracellular), means that chimeric receptors can be excellent tools to determine the sites responsible for different aspects of receptor function and also have the potential to reveal information about the links between these domains. Prokaryotic−eukaryotic chimeras have proved to be especially useful, perhaps because many of these © XXXX American Chemical Society

are functional with minimal or no alterations, mostly replicating the WT characteristics of the relevant domain, whereas eukaryotic chimeras can be more challenging. For example, a chimera comprising the ECD of GLIC and the TMD of the glycine-receptor α1 subunit was shown to be activated by protons and potentiated by general anesthetics, alcohols, and ivermectin, characteristics of GLIC and the glycine receptor, respectively.5 An ELIC−nACh α7 and a GLIC−GABAρ chimera have revealed the importance of the ECD−TMD interface in enabling allosteric modulation, and other chimeras have probed the effects of adding the intracellular loops of eukaryotic receptors to GLIC.6−9 Chimeric receptors also have the possibility of being useful in identifying the binding sites of modulators. The 5-HT 3 receptor, like other pLGICs, is modulated by a range of compounds, including alcohols and volatile anesthetics,10,11 and a range of more selective modulators, such as PU0212 and 5chloroindole,13,14 have also been identified. Carvacrol, thymol, and trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA), positive allosteric modulators (PAMs) of the 5HT3 receptor, can also act as allosteric agonists of the receptor,15,16 showing similarities with the classes of so-called ago-PAMs that have been described for nACh receptors. In recent years, there has been considerable interest in these Received: January 19, 2018 Accepted: February 22, 2018

A

DOI: 10.1021/acschemneuro.8b00028 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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and GABA. Only one of these yielded responses to 5-HT, which we called 5-HT3ELIC (Figure 1). These data are consistent with previous studies that show that all of these regions contribute to transducing ligand-binding information to the channel gate, but the precise interactions are still unclear. Thus, obtaining a functional chimeric receptor usually requires testing a range of possibilities. Here, retaining the β1-β2 loop and the β10-M1 and C-terminal regions of the 5-HT3 receptor and combining them with the Cys and M2-M3 loops of ELIC resulted in a functional 5-HT-gated chimeric receptor (construct 5Ef, Table 1), whereas none of the other constructs responded to 5-HT or GABA. This chimeric receptor was further studied by electrophysiology. Its characteristics are discussed below. Concentration-dependent currents were obtained upon application of 5-HT (Figure 2), revealing an EC50 of 0.34 μM (pEC50 = 6.47 ± 0.06, n = 6), which was lower than that observed for wild-type 5-HT3A receptors (typically 1−3 μM17), and a Hill coefficient of 1.4 ± 0.2. The peak-current amplitudes were large, in the range 5−20 μA. 5-HT3ELIC also responded to mCPBG (pEC50 = 7.88 ± 0.05, EC50 = 13 nM, nH = 1.4 ± 0.3, n = 6); this EC50 was also significantly lower than previously reported values (0.3−0.5 μM).17,18 In contrast, the varenicline EC50 (pEC50 = 4.79 ± 0.06, EC50 = 16 μM, nH = 1.4 ± 0.2, n = 5) was similar to that found for 5-HT3 receptors (18 μM).19 Both of the latter two compounds were partial agonists, as they were for 5-HT3 receptors, although mCPBG had a lower Imax/Imax 5-HT (13 ± 3%, n = 5) compared with the mouse 5-HT3A receptor (∼90%);20,21 the values for varenicline were similar: 40 ± 8% (n = 4) for 5-HT3ELIC and 35% for the 5-HT3A receptor.19 It is intriguing that in the chimera, there are shifts in the EC50’s for 5-HT and mCPBG and a large effect on the Rmax of mCPBG compared with those of the 5-HT3A receptor, despite the binding site being unchanged, although there were no changes in the varenicline EC50 or Rmax. This may be due to differences in the abilities of these agonists to convert the receptor to an intermediate, preopen, “flip” state,22 which has been proposed as an explanation of the different efficacies of partial agonists. However, not all partial agonists may act similarly; for example, the low efficacy of tryptamine at the 5HT3 receptor is considered to be primarily due to its slow transition to the flip state, whereas the low efficacy of 2-methyl5-HT is thought to be due to channel block.23 Both of these mechanisms of action may be significantly altered in the chimera: the conversion to the flip state likely involves multiple regions of the protein, including not only the ECD but also the ECD−TMD interface, much of which differs from that of the WT receptor, whereas of course there is a completely different

modulators as tools to further understand allosteric receptor transitions and also as new therapeutics. However, the precise locations of their binding sites and their mechanisms of action are in many cases still unknown. The better chimeric pLGICs are understood, the greater our knowledge of the rules that govern the activation and modulation of this important protein family will become; this should ultimately allow us to design novel modulators with therapeutic properties. Here we describe the characteristics of a chimera comprising the ECD of the 5-HT3A receptor and the TMD of ELIC (Figure 1), which functions as a 5-HT-gated ion

Figure 1. 5-HT3−ELIC construct. (A) Diagram showing the changes made to the chimera composed of the mouse-5-HT3A-subunit ECD and the ELIC TMD. (B) Structural model of 5-HT3−ELIC created by combining the appropriate regions of the 5-HT3R (4PIR) and ELIC (3RQW) structures.

channel. We report on some of the interesting differences and similarities between this chimera and its WT counterparts and explore the effects of a range of modulators that act on ELIC or the 5-HT3A receptor.

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RESULTS AND DISCUSSION 5-HT3ELIC Activation. A range of constructs consisting of the extracellular domain of the mouse 5-HT3A receptor and the transmembrane domain of ELIC with differences in the β1-β2 loop, the Cys loop, the β10-M1 region, M2-M3 loop, and post M4 C-terminus (Table 1) were tested for activation with 5-HT Table 1. 5-HT3−ELIC Chimerasa

a

The chimeras (5Ea-g) were constructed by fusing the 5-HT3 receptor ECD to the ELIC TMD and then modifying the ECD-TMD interface loops as described. Sequences from the 5-HT3 receptor are in blue, and sequences from ELIC are in black. B

DOI: 10.1021/acschemneuro.8b00028 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Figure 2. Activation of 5-HT3ELIC by 5-HT, varenicline, and mCPBG. (A−C) Representative traces from a single oocyte showing the response of the chimera to (A) 0.1, 0.3, 1, and 3 μM 5-HT; (B) 1, 3, and 10 μM varenicline; and (C) 10, 30, and 100 nM mCPBG. (D) Concentration−response curves from a single oocyte showing that the maximum currents obtained with mCPBG and varenicline differ from those elicited by 5-HT. The data are typical of >4 oocytes.

bemesetron were 0.63 ± 0.08 μM (mean ± SEM, n = 3) and 38 ± 9 μM (mean ± SEM, n = 3), respectively (Figure 4).

channel in the chimera, and thus the characteristics of channel block will differ. Action of 5-HT3-Receptor-Competitive Antagonists at 5-HT3ELIC. The 5-HT3-selective antagonists ondansetron, granisetron, and bemesetron were potent antagonists of the 5-HT-induced response and conferred almost complete inhibition of the 5-HT-induced response at concentrations of 1 nM, 10 nM, and 1 μM, respectively (Figure 3). This is

Figure 4. Inhibition of ELIC by 5-HT3 receptor antagonists shown via concentration−inhibition curves of ondansetron and bemesetron coapplied with 10 mM GABA (mean ± SEM, n = 3). The inset shows the typical ELIC responses with (gray bar) and without (open bar) 1 μM ondansetron.

We observed no responses to GABA application (up to 100 mM), as expected from the absence of the ELIC extracellular domain, which contains the GABA binding site. Thymol and Ethanol Inhibition of 5-HT3ELIC. No allosteric-agonist activity or potentiation of 5-HT3ELIC was observed with thymol, but 10 μM thymol antagonized the 10 μM 5-HT elicited response to 49 ± 5% of its original value (mean ± SEM, n = 3, Figure 5A). Similar experiments in ELIC also revealed antagonism, with a decrease to 33 ± 8% of the response to 10 mM GABA (mean ± SEM, n = 3, Figure 5B) consistent with the effects of thymol being mediated via the TMD. The effects of thymol on 5-HT3 receptors are speciesspecific, with no effects being reported for mouse 5-HT3 receptors, consistent with our data, which showed 101 ± 5%

Figure 3. Inhibition of 0.3 μM 5-HT responses in 5-HT3ELIC by 5HT3-selective antagonists (bemesetron, granisetron, and ondansetron) at concentrations that would cause significant inhibition of responses in 5-HT3 receptors (mean ± SEM, n = 3−4).

consistent with the previously reported subnanomolar WT-5HT3A-receptor IC50’s of ondansetron (e.g., 0.44 nM).24 and granisetron (e.g., 0.2 nM)25 and slightly higher values for bemesetron (e.g., 30 nM or 0.77 μM)26,27 Exploring the effects of some of these 5-HT3 antagonists in ELIC revealed that they could also inhibit GABA-gated ELIC responses though at much higher concentrations; the IC50’s of ondansetron and C

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GLICECD−GlyRTMD chimera replicated the ethanol-potentiation phenotype of the glycine receptor rather than the inhibitory phenotype of GLIC,5 consistent with the current study. 5-Hydroxyindole and 5-Chloroindole Inhibition of 5HT3ELIC. We found that for the chimeric 5-HT3ELIC receptor, concentrations of up to 10 mM 5-hydroxyindole (5-HI) had no effect when applied in the absence of 5-HT and inhibited the response in a concentration-dependent manner when coapplied with 0.3 μM 5-HT (Figure 7). 5-HI has multiple effects on the

Figure 5. Representative traces showing (A) inhibition of a 10 μM 5HT (open bar) response by 10 μM thymol (gray bar) at 5-HT3ELIC and (B) inhibition of a 10 mM GABA (open bar) response by 10 μM thymol (gray bar) at ELIC.

of the 5-HT-elicited response in the presence of 10 μM thymol (n = 3), but with both agonist and potentiating effects being reported for human 5-HT3 receptors.15 This group provided evidence that thymol binds in a pocket formed by the M1 and M2 α-helices from one subunit and the M2 and M3 α-helices from a neighboring subunit,15 and it is possible that thymol occupies a similar position in ELIC. Alternatively, thymol could bind in a similar place to that proposed for its analogue propofol,28 which is between M3 and M4 and involves ELIC residues M265 and F308. Binding at either of these locations could restrict the flexibility and movement of M2, thereby inhibiting channel opening. Ethanol (10−100 mM) potentiates 5-HT3 receptors29,30 but inhibits ELIC stimulated by propylamine (EC20) and by GABA (5 mM), with IC50’s of 52 and 125 mM, respectively.31,32 Our chimera was found to reproduce the properties of ELIC; ethanol inhibited its responses to an EC50 concentration of 5HT with a pIC50 of 3.96 ± 0.08 μM, n = 3 (IC50: 110 μM, Figure 6). This suggests that the residues responsible for

Figure 7. Inhibition of 5-HT3ELIC by 5-HI. (A) Representative traces in which the open bars denote the application of 5-HT (10 μM), and the gray bar denotes the application of 5 mM 5-HI. (B) Concentration−inhibition curve showing inhibition of 0.3 μM 5-HT induced responses by 5-HI. The data are the means ± SEM (n = 3).

WT 5-HT3 receptor: at concentrations between 100 μM and 10 mM, 5-HI enhances the amplitude of 5-HT-evoked currents and slows the kinetics of desensitization, whereas at concentrations greater than 10 mM, it inhibits the response.33−35 Its complex mechanism of action has been shown to involve competitive and noncompetitive binding at two (or perhaps more) different sites.33 One of these is an orthosteric binding site, as high concentrations of 5-HI displace the competitive antagonist 3H-GR65630,33 suggesting that 5-HI exerts its inhibitory effects through direct competition with 5HT. 5-HI binding in the 5-HT3-receptor TMD may be responsible for the modulatory effects; a mutation of L293 in TM2 eliminates the positive modulation of 5-HI at the mouse 5-HT3 receptor.36 In ELIC, 10 mM 5-HI strongly potentiated GABA-induced responses of ELIC and caused a significant decrease in the EC50 from 4.0 mM to 1.3 mM (pEC50: 2.40 ± 0.04, n = 3, vs 2.90 ± 0.04, n = 4, Figure 8). 5-HI (10 mM) did not activate ELIC in the absence of GABA.

Figure 6. Inhibition of 5-HT3ELIC by ethanol. (A) Example traces showing the application of 0.3 μM 5-HT (open bar) and 300 μM ethanol (filled bar). (B) Concentration−inhibition curve of ethanol coapplied with 0.3 μM 5-HT revealing an IC50 of 110 μM. The data are the means ± SEM (n = 3).

ethanol inhibition in ELIC reside in the transmembrane domain. In support of this, ethanol potentiated rather than inhibited a chimera with the extracellular domain of ELIC and the transmembrane domain of the α1β3 GABAA receptor.31 Furthermore, crystal structures of ELIC suggest there are ethanol binding sites in both the extracellular domain and the channel pore (near the 6’ residue), but only mutagenesis near the channel site has an effect on inhibition.31 Additionally, a

Figure 8. 5-HI potentiates ELIC. (A) Representative traces in which the open bar indicates the application of 1.6 mM GABA (EC5), and the filled bar indicates the application of 10 mM 5-HI. (B) 5-HI (10 mM) causes a leftward shift of the concentration−response curve. The data are the means ± SEM (n = 3). D

DOI: 10.1021/acschemneuro.8b00028 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience These data suggest that in the chimera, 5-HI causes inhibition by binding at the orthosteric site in the 5-HT3receptor ECD, and the 5-HI binding site that causes the potentiation of ELIC is also in the ECD. However, we cannot rule out the possibility that 5-HI also binds in the TMD, but that suboptimal interactions with the ECD in the chimera preclude the allosteric transitions required to give an enhancement of the response. Interactions between the ECD and the TMD have been shown to be critical for correct receptor function,37−39 and consequently, functional receptor chimeras often require the substitution of some or all of the residues in the loops that form this interface.5,6,40 Tillman et al. found that a chimera composed of the ELIC ECD and the nACh α7 receptor TMD retained sensitivity to the PAMs ivermectin, PNU-120596, and TQS only when the ECD-TMD interface resembled that of the α7 nACh receptor rather than that of the ELIC receptor, despite both the α7-like and ELIC-like chimeras being functional and showing similar activation and desensitization kinetics.6 This suggests more stringent requirements for PAM activity compared with those of agonist activation. In support of this, human α7−5-HT3 chimeras with either 5-HT3 or α7 M2-M3 loops showed different responses to PAMs41 (e.g., potentiation by genistein42 required the α7 M2-M3 loop), and a recent study involving a GLIC−GABAA receptor chimera showed that proton activation and neurosteroid modulation were not simply dependent on residues in either the ECD or TMD but involved cross-domain interactions.43 5-Chloroindole (Cl-indole) is an analogue of 5-HI. It also inhibited responses to 5-HT in the chimeric receptor (Figure 9), and Cl-indole alone (100 μM) showed no activity. The

Figure 10. Cl-indole potentiates 5-HT3A receptors. (A) Traces showing enhancement of the response to 1 μM 5-HT (open bar) by coapplication with 30 μM Cl-indole (closed bar). (B) 5-HT concentration−response curves from 5-HT3A receptors showing the effects of different concentrations of Cl-indole.

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CONCLUSIONS In conclusion, we have created a 5-HT3−ELIC chimera that is activated by 5-HT3-receptor agonists, inhibited by 5-HT3receptor competitive antagonists, and modified by various pLGIC modulators (Table 2). The chimera requires three and Table 2. Data Summary 5-HT GABA thymol ethanol 5-HI 5-CI a

5-HT3

ELIC

5-HT3−ELIC

activation NR NR potentiation29,30 potentiation (0.1−10 mM) inhibition (>10 mM)33 potentiation

NRa activation inhibition inhibition potentiation

activation NR inhibition inhibition inhibition

inhibition

inhibition

NR indicates no response.

four 5-HT3A-receptor amino acid residues in the Cys-loop and C-terminal tail respectively (i.e., the regions that interact with the ECD) to be substituted into ELIC, supporting a range of other studies showing that a compatible ECD−TMD interface is crucial for agonist activation. The modulator studies reveal which domains encompass their site of action, which is a first step in understanding how they elicit their effects. Such knowledge is vital for the development of novel therapeutic agents that act at these sites.

Figure 9. Cl-indole inhibits 5-HT3ELIC. (A) Traces showing inhibition of the response to 10 μM 5-HT (open bar) by coapplication with 100 μM Cl-indole (gray bar). (B) Cl-indole concentration− inhibition curve at 0.3 μM 5-HT. The data are the means ± SEM (n = 3).

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METHODS

Oocyte Maintenance and Receptor Expression. Stage V and VI Xenopus oocytes were purchased from Ecocyte Bioscience (Castrop-Rauxel, Germany) and stored in ND96 containing 2.5 mM sodium pyruvate, 0.7 mM theophylline, and 50 μg mL−1 gentamicin. A 5-HT3ELIC chimera was constructed by fusing the extracellular domain (to R244) of the mouse 5-HT3A receptor to the transmembrane domain of ELIC (from R199 onward, Figure 1). A range of mutations were then made in loop 2, loop 7 (the Cys loop), the preM1 region, the M2-M3 region, and the post M4 C-terminus. In the successful construct, three residues in the Cys loop (IYN) were changed to their equivalents in ELIC (FRL), and the C-terminal residues IRGITL of ELIC were replaced with their 5-HT3A equivalents (WHYS). These changes were made using site-directed mutagenesis performed using QuikChange (Agilent, Santa Clara, CA). The ELIC and mouse 5-HT3A receptor constructs were used as described previously.17,44 cRNA was transcribed in vitro from a linearized pGEMHE cDNA template using the mMessage mMachine T7 kit (Ambion, Austin, TX). Stage V and VI oocytes were injected with 50 nL of ∼400 ng μL−1 cRNA, and currents were recorded 24−72 h postinjection.

effects of Cl-indole on the 5-HT3A receptor and ELIC, however, were quite different to those of 5-HI. For the 5-HT3A receptor, 10−30 μM caused potentiation of submaximal concentrations of 5-HT (Figure 10), whereas in ELIC, Clindole caused an inhibition of the response to GABA: coapplication of 100 μM resulted in 57 ± 10% (n = 6) inhibition of the EC50 response. Thus, our data show that the chimera resembles ELIC and suggests that the Cl-indolebinding site is in the TMD. This supports recent work reporting that this compound has a different mechanism of action from that of 5-HI,13,14 although there may be some differences between their human and our mouse 5-HT3A receptor as they suggest that Cl-indole acts as both an allosteric modulator and an orthosteric agonist, with both actions requiring the presence of 5-HT. E

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ACS Chemical Neuroscience Electrophysiology. Two-electrode voltage-clamp recordings of Xenopus oocytes were performed using Roboocyte (MultiChannel Systems, Reutlingen, Germany). The oocytes were clamped at −60 mV and perfused with ND96 containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.5, at a rate of 4 mL min−1. For ELIC and the 5-HT3 receptor, ND96 with no added CaCl2 was used. The analysis and curve fitting were performed using the fourparameter equation in Prism version 5 (GraphPad Software, La Jolla, CA). The concentration−response data for each oocyte were normalized to the maximum current for that oocyte.

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(9) Goyal, R., Salahudeen, A. A., and Jansen, M. (2011) Engineering a prokaryotic Cys-loop receptor with a third functional domain. J. Biol. Chem. 286 (40), 34635−34642. (10) Davies, P. A. (2011) Allosteric modulation of the 5-HT3 receptor. Curr. Opin. Pharmacol. 11 (1), 75−80. (11) Walstab, J., Rappold, G., and Niesler, B. (2010) 5-HT3 receptors: role in disease and target of drugs. Pharmacol. Ther. 128 (1), 146−169. (12) Trattnig, S. M., Harpsøe, K., Thygesen, S. B., Rahr, L. M., Ahring, P. K., Balle, T., and Jensen, A. A. (2012) Discovery of a novel allosteric modulator of 5-HT3 receptors: inhibition and potentiation of Cys-loop receptor signaling through a conserved transmembrane intersubunit site. J. Biol. Chem. 287 (30), 25241−25254. (13) Newman, A. S., Batis, N., Grafton, G., Caputo, F., Brady, C. A., Lambert, J. J., Peters, J. A., Gordon, J., Brain, K. L., Powell, A. D., and Barnes, N. M. (2013) 5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor. Br. J. Pharmacol. 169 (6), 1228−1238. (14) Powell, A. D., Grafton, G., Roberts, A., Larkin, S., O’Neill, N., Palandri, J., Otvos, R., Cooper, A. J., Ulens, C., and Barnes, N. M. (2016) Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT3 A receptor. Br. J. Pharmacol. 173 (24), 3467−3479. (15) Lansdell, S. J., Sathyaprakash, C., Doward, A., and Millar, N. S. (2015) Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site. Mol. Pharmacol. 87 (1), 87−95. (16) Gasiorek, A., Trattnig, S. M., Ahring, P. K., Kristiansen, U., Frolund, B., Frederiksen, K., and Jensen, A. A. (2016) Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT3 receptors. Biochem. Pharmacol. 110−111, 92−108. (17) Price, K. L., Bower, K. S., Thompson, A. J., Lester, H. A., Dougherty, D. A., and Lummis, S. C. (2008) A hydrogen bond in loop A is critical for the binding and function of the 5-HT3 receptor. Biochemistry 47 (24), 6370−6377. (18) Duffy, N. H., Lester, H. A., and Dougherty, D. A. (2012) Ondansetron and granisetron binding orientation in the 5-HT(3) receptor determined by unnatural amino acid mutagenesis. ACS Chem. Biol. 7 (10), 1738−1745. (19) Lummis, S. C., Thompson, A. J., Bencherif, M., and Lester, H. A. (2011) Varenicline is a potent agonist of the human 5-hydroxytryptamine3 receptor. J. Pharmacol. Exp. Ther. 339 (1), 125−131. (20) Sepulveda, M. I., Lummis, S. C., and Martin, I. L. (1991) The agonist properties of m-chlorophenylbiguanide and 2-methyl-5hydroxytryptamine on 5-HT3 receptors in N1E-115 neuroblastoma cells. Br. J. Pharmacol. 104 (2), 536−540. (21) Absalom, N. L., Lewis, T. M., and Schofield, P. R. (2004) Mechanisms of channel gating of the ligand-gated ion channel superfamily inferred from protein structure. Experimental physiology 89 (2), 145−153. (22) Lape, R., Colquhoun, D., and Sivilotti, L. G. (2008) On the nature of partial agonism in the nicotinic receptor superfamily. Nature 454 (7205), 722−727. (23) Corradi, J., and Bouzat, C. (2014) Unraveling mechanisms underlying partial agonism in 5-HT3A receptors. J. Neurosci. 34 (50), 16865−16876. (24) Gill, C. H., Peters, J. A., and Lambert, J. J. (1995) An electrophysiological investigation of the properties of a murine recombinant 5-HT3 receptor stably expressed in HEK 293 cells. Br. J. Pharmacol. 114 (6), 1211−1221. (25) Paul, M., Callahan, R., Au, J., Kindler, C. H., and Yost, C. S. (2005) Antiemetics of the 5-hydroxytryptamine 3A antagonist class inhibit muscle nicotinic acetylcholine receptors. Anesth. Analg. 101 (3), 715−721. (26) Steward, L. J., Ge, J., Bentley, K. R., Barber, P. C., Hope, A. G., Lambert, J. J., Peters, J. A., and Blackburn, T. P. (1995) Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron. Br. J. Pharmacol. 116 (2), 1781−1788.

AUTHOR INFORMATION

Corresponding Author

*Tel.: (+44) 1223 765950. Fax (+44)1223 333345. E-mail: [email protected]. ORCID

Sarah C. R. Lummis: 0000-0001-9410-9805 Author Contributions

S.C.R.L. and K.L.P. participated in the research design, conducted experiments, performed data analysis, and wrote the manuscript. Funding

This work was supported by the Medical Research Council (grant MR L02/676). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS pLGIC, pentameric ligand-gated ion channel; ELIC, Erwinia ligand-gated ion channel; ECD, extracellular domain; TMD, transmembrane domain; 5-HI, 5-hydroxyindole; Cl-indole, 5chloroindole; nACh, nicotinic acetylcholine

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REFERENCES

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DOI: 10.1021/acschemneuro.8b00028 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX