Pentameric ligand-gated ion channels (pLGICs), comprising nicotinic acetylcholine (nAChRs), serotonin (5-HT₃Rs), glycine (GlyRs), and gamma-amino butyric acid (GABA_ARs) receptors, are dynamic transmembrane proteins that mediate neuronal and non-neuronal communication (Nemecz et al., 2016). Among them, 5-HT₃Rs are cation-selective excitatory channels expressed in central and peripheral nervous systems, notably in the gut and gut-brain axis (Gibbs and Chakrapani, 2021). 5-HT₃Rs are encoded by five different genes (HTR3A, HTR3B, HTR3C, HTR3C, HTR3E), giving rise to five different subunits (A to E) that assemble as homo- or hetero-pentamers. A class of synthetic competitive antagonists called setrons target these receptors and are widely used in the clinic as antiemetics, notably to counteract vomiting and nausea induced by chemotherapy and radiotherapy. 5-HT₃Rs are also potential targets to treat irritable bowel syndrome and are implicated in neurological diseases such as schizophrenia, Parkinson’s disease, and depression. There is therefore much interest surrounding the molecular mechanisms governing 5-HT₃Rs function and pharmacological regulation (Mawe and Hoffman, 2013; Fakhfouri et al., 2019; Tsitsipa et al., 2022).

A hallmark of pLGICs, and among them 5-HT₃R, is that their action is mediated by varying population equilibrium between allosteric states that are differentially stabilized by the binding of ligands (Monod et al., 1965; Corradi et al., 2009). Agonists promote the allosteric transition between resting (closed channel) and active (open channel) states, while their prolonged application promotes a slower transition to desensitized (agonist-bound-closed channel) conformations.

The first high-resolution structure of mouse homo-pentameric 5-HT_3AR was solved by X-ray crystallography in complex with stabilizing nanobodies, followed by numerous structures solved by cryo-electron microscopy (cryo-EM) without ligands or in complex with the agonist 5-HT, a series of setron competitive antagonists and an allosteric modulator (Hassaine et al., 2014; Polovinkin et al., 2018; Basak et al., 2018b; Basak et al., 2018a; Basak et al., 2019; Basak et al., 2020; Zarkadas et al., 2020; Zhang et al., 2021). The 5-HT_3AR displays a typical pLGIC structure, where each subunit consists of a β-sandwich connected with loops composing the extracellular domain (ECD), four α-helices composing the transmembrane domain (TMD) and an intracellular domain. Agonists and competitive antagonists bind at the ECD interface between two subunits, at the orthosteric site composed of the so-called loops A, B, and C from the principal subunit and D, E, and F from the complementary subunit, with loop C bridging the subunit interface. In the presence of detergent C12E9 with or without added lipids, or in saposin nanodiscs, solved structures in the absence of orthosteric ligand (apo) were assigned as resting-like (Hassaine et al., 2014; Basak et al., 2018b; Zhang et al., 2021). Those in complex with setrons significantly diverged from the resting-like class and were called inhibited states (Polovinkin et al., 2018; Basak et al., 2019; Basak et al., 2020; Zarkadas et al., 2020). The structures captured with bound 5-HT all feature rather similar motions of the ECD, with 5-HT binding promoting a closure (in a capping motion) of loop C around the agonist, a global compaction, and a tilt of each subunit ECD. This generates a marked reorganization of the ECD-TMD interface, including in some cases an outward motion of the M2-M3 loop. However, the structures strongly diverge in the TMD, some featuring an apparently open pore and others a non-conducting pore (Polovinkin et al., 2018; Basak et al., 2018a). In addition, reconstitution into saposin-based nanodiscs captured distinct open conformations with symmetrical or asymmetrical conformation of the TMD (Zhang et al., 2021). Therefore, these atomic structures led to a complex landscape of more than the three putative conformations (resting, active, desensitized). Furthermore, 5-HT₃R’s wide conformational landscape accessibility seems to be governed by both orthosteric effectors and the lipids and/or detergents surrounding the TMD. Assigning these high-resolution structures to actual physiological states contributing to the electrophysiological response at the cell membrane remains to be established (Howard, 2021).

Complementary techniques that follow the structural reorganizations of the cell-expressed receptors are thus needed to identify structural motions contributing to gating (i.e. the pathway between resting and activated/open states) and to help annotate high-resolution structures to physiologically relevant conformational states (Howard, 2021). Voltage-clamp fluorometry (VCF), which monitors receptors expressed at the Xenopus oocyte plasma membrane, is well suited for this aim. VCF performs simultaneous measurement of channel opening by two-electrode voltage-clamp electrophysiology (TEVC) and of local protein motions by variation of fluorescence from covalently attached probes (Mannuzzu et al., 1996). This technique has been applied to various pLGICs, notably to human 5-HT_3AR with the generation of three sensors surrounding the orthosteric site as well as one sensor on the mouse 5-HT_3AR located at the extracellular entrance of the pore (Polovinkin et al., 2018; Munro et al., 2019).

In the present study, we have generated four fluorescent sensors grafted at new locations on the mouse 5-HT_3AR. We characterized their phenotypes following the binding of various agonists, partial agonists, and clinically relevant antagonists, with or without introduction of loss-of-function mutations. Simultaneous recordings of electrical currents and variations of fluorescence revealed different local and global reorganizations of the ECD depending on the pharmacological conditions. By following the conformational reorganizations leading to both electrophysiologically silent and active conformations, VCF data identify intermediate conformations within or outside the path of 5-HT₃R activation. These data provide unique structural information on membrane-embedded proteins to annotate known high-resolution structures to physiologically relevant allosteric states.

In this work, we undertook a screening of 19 mutants of the m5-HT_3AR with an aim to identify fluorescent sensors within the ECD. Around the orthosteric site, we find that cysteine incorporation often yields ΔF suitable for VCF investigation. In contrast, outside the orthosteric site all single-cysteine mutants investigated failed. This observation parallels a previous VCF screening on the h5-HT_3AR that identified four suitable sensors around the orthosteric site but none among the seven additional positions tested in the pre-M1 region (Munro et al., 2019). Of note, we have previously investigated a sensor on the top of M2 helices on m5-HT_3AR (Polovinkin et al., 2018). In the pLGIC family, the α1-GlyR has also been extensively studied by VCF and many positions were screened for cysteine incorporation (Shi et al., 2023; Pless et al., 2007; Pless and Lynch, 2009a; Pless and Lynch, 2009b; Han et al., 2013). In contrast to the 5-HT₃R, most positions show robust ΔF outside the orthosteric site. This suggests that the α1-GlyR undergoes gating reorganizations of larger amplitude than that of the 5-HT₃R. Still, we succeeded in designing two sensors at the vestibule and the ECD-TMD interface by implementing the tryptophan-induced quenching method, further illustrating that this technique is suitable to monitor subtle conformational changes when the fluorophore-quencher pair is properly engineered.

The mapping of the 5-HT₃R has enabled us to generate four fluorescence sensors of the protein conformations. They are located at three distant positions within the ECD 3D structure, thereby constituting reference points to infer its global conformation. Simultaneous steady-state concentration-response relationships for the fluorescence and current responses reveal important ligand-elicited protein motions and their relationship with activation.

We first show that agonists and antagonists elicit similar local reorganizations around orthosteric sensors. For both S204C and I160C/Y207W, all tested ligands at saturating concentrations elicit fluorescent dequenching signals. All agonists, partial agonists, granisetron, and ondansetron elicit ΔF of similar amplitude, while alosetron compared to metoclopramide display significantly higher and lower ΔF, respectively. These results are in remarkable concordance with cryo-EM structures. They show that both 5-HT and a series of setron antagonists promote locally a similar reorganization of the orthosteric site, with an inward displacement of the landmark loop C that caps the bound ligand (Figure 4—figure supplement 1; Polovinkin et al., 2018; Basak et al., 2018a). Furthermore, subjecting these structures to molecular dynamic simulations suggests that different antagonists promote different degrees of local conformational changes (Basak et al., 2019; Basak et al., 2020). Among setrons, alosetron is the one promoting the largest conformational effects, consistent with VCF data. Of note, the ligand-elicited dequenching signal of the I160C/Y207W is also consistent with the cryo-EM structures, since the capping of loop C is predicted to reduce the accessibility of the indole side chain to the rhodamine dye grafted on the other side of the loop. Interestingly, three other fluorescence sensors around the orthosteric site were already reported on the human 5-HT_3AR on loop C (M223C), D (Y89C), and E (Q146C). They also report that agonists and antagonists equally elicit fluorescence dequenching, with ΔF intensities strongly ligand-dependent for loops C and E, but not for loop D (Munro et al., 2019).

We also show that antagonist-elicited reorganizations do not spread to the vestibular and ECD-TMD sensors. Indeed, the sensors at the ECD-TMD interface (R219C/Y140W) and at the middle vestibule (V106C/L131W) show robust ΔF upon perfusion of agonists, but no effect for antagonists. Cryo-EM structures show no setron-elicited change at the ECD-TMD interface, especially no motion of the M2-M3 loop that lies in between the attachment points of the fluorophore and the indole of the tryptophan in R219C/Y140W (Figure 4—figure supplement 1; Polovinkin et al., 2018; Basak et al., 2020; Zarkadas et al., 2020). Likewise, setrons elicit small reorganizations of the architecture of the vestibule (measured as a slightly increased radius of the constriction at K108 and D105), while 5-HT elicits stronger reorganizations at this level. Overall, VCF data are in excellent agreement with cryo-EM data, providing evidence that high-resolution structures of setron-inhibited states represent pertinent snapshots of actual allosteric states at the plasma membrane.

Steady-state concentration-response relationships support that strong agonists elicit an apparently concerted reorganization of the global protein structure. Indeed, for most constructs, those agonists eliciting the maximal response currents among all tested agonists show no difference in ΔF and ΔI concentration-response curves. This is the case for 5-HT, that maximally activates the S204C, V106C/L131W, and R219C/Y140W sensors and for mCPBG that maximally activates the S204C and I160C/Y207W sensors. This suggests a concerted mechanism where local motions around the four sensors happen simultaneously with the pore opening. These data are thus accounted by a simple two-state model, where the receptor is in equilibrium between a resting and an active state. VCF measurements are also remarkably coherent with the 5-HT-bound structures showing an open pore: PDB 6HIN (called F), PDB 6DG8 (called state 2), and PDB Y5A (called serotonin-bound state). Indeed, all of them show symmetrical and global reorganizations of the ECD involving loop C capping, vestibular reorganization, and M2-M3 reorganization (Figure 4—figure supplement 1; Polovinkin et al., 2018; Basak et al., 2018a; Zhang et al., 2021).

However, we found that partial agonists stabilize additional intermediate conformations characterized by fluorescence variations but no current, especially when loss-of-function mutations are engineered. Intermediate conformations are observed for the two sensors outside the orthosteric site. Indeed, for R219C/Y140W sensor in combination with N101K, var elicits at saturation only 3% of 5-HT currents but promotes a robust ΔF signal corresponding to 38% of that of 5-HT. In addition, the ΔF concentration-response curve appears slightly left-shifted as compared to the ΔI curve, indicating that the ΔF/ΔI ratio is even higher at low var concentrations. These data clearly show that var stabilizes in majority intermediate conformations where the ECD-TMD interface has moved, generating a ΔF, but where the channel remains closed. A similar although milder ‘intermediate’ phenotype is seen for var on the V106C/L131W sensor.

Within the gating pathway, these intermediate conformations may a priori contribute either to the activation transition (pre-active state) or to the desensitization transition (fast or slow desensitized states). However, VCF data strongly argue for the former hypothesis. Indeed, for all experiments including var partial activation of V106C/L131W, the rise times of ΔF are in the same range to that of ΔI, desensitization being much slower and associated with no significant ΔF. This shows that the intermediate states are recruited during or concomitant with the activation phase. This is in line with the general observation that partial agonists currents are strongly increased by positive allosteric modulators, indicating that the electrophysiologically silent conformations they promote are activatable and therefore not desensitized (Felt et al., 2024).

Two 5-HT-bound structures of m5-HT_3A displaying a closed channel sustain the intermediate reorganizations seen by VCF. Indeed, conformations called I1 (PDB 6HIO) and state 1 (PDB 6DG7) feature rearrangements at the ECD and interface between ECD/TMD similar to that of conformation displaying an open channel, but with non-conducting pore (Figure 4—figure supplement 1; Polovinkin et al., 2018; Basak et al., 2018a). It is noteworthy that these states were initially proposed to represent either pre-active or desensitized states, and that these two possibilities could not be distinguished without ambiguity. However, indirect experiments of substituted cysteine accessibility method (SCAM) suggested that desensitization involves weak reorganizations of the upper part of the channel that holds the activation gate, arguing for the pre-active state hypothesis (Polovinkin et al., 2018). Combined SCAM and VCF data thus provide compelling evidence for annotation as pre-active state. Interestingly, during the revision of this article, several cryo-EM structures of m5-HT_3A in complex with partial agonists SMP-100 and ALB-148471 were published. In full agreement with our VCF data, partial agonists are found to stabilize the protein in both active and pre-active-like conformation (Felt et al., 2024).

Intermediate conformations are also observed on many occasions for the sensors near the orthosteric site. This is the case with the var partial agonist on S204C, which produces a lower maximum current variation (ΔImax) than that caused by 5-HT but a higher ΔFmax. The phenotype is exacerbated in the presence of N101K, 5-HT, and mCPBG showing intermediate conformations at sub-saturating concentrations, while var promote no current while still evoking robust ΔF. Of note, on this mutant, mCPBG is the most efficient agonist, but it remains possible that it has a partial agonistic character due to the strong loss-of-function N101K mutation. In this context, the sensor I160C/Y207W shows a loss-of-function phenotype resembling that of S204C/N101K with comparable relative position of the ΔI and ΔF dose-response curves. Correlating the motions at the orthosteric site with known cryo-EM structures is, however, difficult. Indeed, two hypotheses can be sustained by these results: the intermediate conformations revealed by these sensors can correspond either to those stabilized by setrons or to the pre-active conformations discussed above.

Altogether, VCF data highlight a progressive propagation of the signal following ligand binding. Setrons elicit local reorganizations shown by the sensors located around the orthosteric site, partial agonists elicit local reorganizations at the four sensors indicating a motion of the whole ECD with partial pore opening, and strong agonists elicit reorganizations detected by all four sensors together with channel opening. VCF data thus identify four families of conformations endowed with distinct ΔI/ΔF signatures contributing to signal transduction: resting-like apo, setron-inhibited, partial agonist-elicited pre-active and active states. The topological information given by the various sensors are remarkably consistent with the gallery of high-resolution structures solved thus far. Indeed, the data from fluorescence partners are consistent with respectively apo, setron-bound, 5-HT-bound-closed, and 5-HT-bound-open conformations. This provides important information allowing reasonable functional annotation of the various structures to physiological states in a membrane environment, at least regarding the ECD. Figure 4 shows a speculative four-state model as a framework integrating the whole set of data. In addition, VCF data give insights in the action of partial agonists, that do not exclusively stabilize the active state and document the phenotypes of various allosteric mutations.

Figure 4 with 1 supplement see all

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A key observation of the study is the identification of pre-active intermediates that are favored upon binding of partial agonists and/or in the presence of loss-of-function mutations. It is noteworthy that single-channel kinetic analyses of pLGICs early showed that the activation transition pathway involves multiple intermediated states. Analysis of numerous mutants of the muscle nAChR analyzed by REFERs (rate equilibrium linear free energy relationships) suggested a multistep reorganization that initiates in the orthosteric site and progressively spreads to the ion channel gate (Grosman et al., 2000). Analysis on GlyRs and nAChRs detected late intermediate states called ‘flip’ (Burzomato et al., 2004) or ‘primed’ (Mukhtasimova et al., 2009), that are favored by loss-of-function mutations (Plested et al., 2007; Lape et al., 2012), while a non-conducting intermediate state has been proposed from kinetic models of a high-conductance mutant of 5-HT₃R (Corradi et al., 2009).

More recently, fluorescence and VCF studies identified intermediate conformations for nAChRs, α1-GlyRs, and the bacterial homolog GLIC (Shi et al., 2023; Dahan et al., 2004; Mourot et al., 2008; Menny et al., 2017; Lefebvre et al., 2021). Of note, pre-active intermediates were unraveled by a fluorescence quenching pair at strictly homologous positions at the ECD-TMD interface of 5-HT₃R and α1-GlyRs. Indeed, residues R219C/Y140W are homologous to another sensor pair Q219C/K143W engineered on the α1-GlyR. MTS-TAMRA-labeled Q219C/K143W also reports a non-conducting intermediate in the pathway toward activation, with a fluorescence variation revealing an early reorganization of the ECD-TMD interface (Shi et al., 2023). Molecular dynamic simulations starting from a taurine-bound-closed cryo-EM structure was found to recapitulate the pharmacological properties observed by VCF, suggesting that the intermediate conformations correspond to a highly dynamic family of conformations featuring an active-like ECD but a resting-like pore (Yu et al., 2021). Altogether, VCF shows that virtually all pLGICs appear to share a common global mechanism of gating where the proteins visit a family of structurally dynamic intermediates showing an active-like ECD and a resting-like TMD conformation. The present work thus extends this idea to the 5-HT_3AR, together with providing structural blueprints for cryo-EM structural annotation.

In conclusion, by monitoring simultaneously electrophysiologically silent and active conformations of the 5-HT₃R, VCF allowed to characterize the mechanisms of action of allosteric effectors and allosteric mutations. We found that the strength of the agonist correlated to a degree with propagation of this fluorescence change beyond the local site of neurotransmitter binding, unraveling intermediate conformations allowing proposing a structure-based functional annotation of known high-resolution structures. Besides validating the mechanism underlying inhibition by setrons, these data unravel a unique mode of action of partial agonists to promote distinct intermediate conformations. The various fluorescence sensors developed will be valuable in future mutational analysis and characterization of drugs acting at the 5-HT₃R that hold promise for clinical use.

All the data generated and analyzed during this study have been included in the manuscript (Tables 1 and 2) and supporting files.

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Author details

1. Laurie Peverini

   Institut Pasteur, Université Paris Cité, CNRS UMR 3571, Channel-Receptors Unit, Paris, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   For correspondence

   laurie_peverini@hotmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7992-1245

2. Sophie Shi

   Institut Pasteur, Université Paris Cité, CNRS UMR 3571, Channel-Receptors Unit, Paris, France

   Contribution

   Validation, Investigation

   Competing interests

   No competing interests declared

3. Karima Medjebeur

   Institut Pasteur, Université Paris Cité, CNRS UMR 3571, Channel-Receptors Unit, Paris, France

   Contribution

   Validation, Investigation

   Competing interests

   No competing interests declared

4. Pierre-Jean Corringer

   Institut Pasteur, Université Paris Cité, CNRS UMR 3571, Channel-Receptors Unit, Paris, France

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   pjcorrin@pasteur.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4770-430X

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