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Research ArticleArticle

Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry

Lachlan Munro, Lucy Kate Ladefoged, Vithushan Padmanathan, Signe Andersen, Birgit Schiøtt and Anders S. Kristensen
Molecular Pharmacology December 2019, 96 (6) 720-734; DOI: https://doi.org/10.1124/mol.119.116657

Abstract

The 5-hydroxytryptamine (5-HT) type 3 receptor is a member of the cysteine (Cys)-loop receptor super family of ligand-gated ion channels in the nervous system and is a clinical target in a range of diseases. The 5-HT3 receptor mediates fast serotonergic neurotransmission by undergoing a series of conformational changes initiated by ligand binding that lead to the rapid opening of an intrinsic cation-selective channel. However, despite the availability of high-resolution structures of a mouse 5-HT3 receptor, many important aspects of the mechanistic basis of 5-HT3 receptor function and modulation by drugs remain poorly understood. In particular, there is little direct evidence for the specific conformational changes predicted to occur during ligand-gated channel activation and desensitization. In the present study, we used voltage-clamp fluorometry (VCF) to measure conformational changes in regions surrounding the orthosteric binding site of the human 5-HT3A (h5-HT3A) receptor during binding of 5-HT and different classes of 5-HT3 receptor ligands. VCF utilizes parallel measurements of receptor currents with photon emission from fluorescent reporter groups covalently attached to specific positions in the receptor structure. Reporter groups that are highly sensitive to the local molecular environment can, in real time, report conformational changes as changes in fluorescence that can be correlated with changes in receptor currents reporting the functional states of the channel. Within the loop C, D, and E regions that surround the orthosteric binding site in the h5-HT3A receptor, we identify positions that are amenable to tagging with an environmentally sensitive reporter group that reports robust fluorescence changes upon 5-HT binding and receptor activation. We use these reporter positions to characterize the effect of ligand binding on the local structure of the orthosteric binding site by agonists, competitive antagonists, and allosterically acting channel activators. We observed that loop C appears to show distinct fluorescence changes for ligands of the same class, while loop D reports similar fluorescence changes for all ligands binding at the orthosteric site. In contrast, the loop E reporter position shows distinct changes for agonists, antagonists, and allosteric compounds, suggesting the conformational changes in this region are specific to ligand function. Interpretation of these results within the framework of current models of 5-HT3 and Cys-loop mechanisms are used to expand the understanding of how ligand binding in Cys-loop receptors relates to channel gating.

SIGNIFICANCE STATEMENT The 5-HT3 receptor is an important ligand-gated ion channel and drug target in the central and peripheral nervous system. Determining how ligand binding induced conformational changes in the receptor is central for understanding the structural mechanisms underlying 5-HT3 receptor function. Here, we employ voltage-gated fluorometry to characterize conformational changes in the extracellular domain of the human 5-HT3 receptor to identify intrareceptor motions during binding of a range of 5-HT3 receptor agonists and antagonists.

Introduction

5-Hydroxytryptamine (5-HT) type 3 receptors in the central nervous system transduce fast synaptic transmission and are considered therapeutic targets for the treatment of chemotherapy-induced nausea and vomiting and psychiatric conditions such as schizophrenia and depression (Thompson and Lummis, 2007; Walstab et al., 2010). Furthermore, 5-HT3 receptors in the enteric nervous system regulate gut motility and are well-established targets for the treatment of irritable bowel syndrome (Lummis, 2012). 5-HT3 receptors belong to the pentameric ligand-gated ion channel superfamily, also known as cysteine (Cys)-loop receptors (Nemecz et al., 2016). Pentameric ligand-gated ion channels have as a structural hallmark five subunits forming a central ion-permeable pore in the transmembrane domain (TMD) (Fig. 1A) with orthosteric ligand binding sites located at subunit interfaces in the extracellular domain (ECD) (Fig. 1A). Pentameric ligand-gated ion channels are thought to share a common structural mechanism for how agonist binding in the ECD couples to channel activation and desensitization in the TMD (Grosman et al., 2000; Barnes et al., 2009; Corringer et al., 2010; Lummis, 2012; Keramidas and Lynch, 2013; Nys et al., 2013; Nemecz et al., 2016).

Fig. 1.
Fig. 1.

Identification of h5-HT3A residues as voltage-clamp fluorometry reporter positions. (A) Surface contoured cartoon representation of a homology model structure of the pentameric h5-HT3A receptor (left) from Ladefoged et al. (2018). The principal and complementary subunits are highlighted in cyan and green, respectively, with indications of the ECD and TMD. The cross-sectional view (right) illustrates the location of the five equivalent 5-HT binding pockets (dashed circles) formed at subunit interfaces in the ECD, with 5-HT indicated in yellow surface contour. The chemical structure of the endogenous agonist 5-HT is shown below. (B) Magnified view of the ECD interface structure in cartoon representation with the orthosteric binding site and the loop A to G regions highlighted in different colors and bound 5-HT in yellow surface contour. Positions where Cys residues were introduced by mutagenesis, and the endogenous Cys residues 157 and 171 are indicated as orange and yellow spheres, respectively. (C) Representative recording traces of parallel fluorescence and current measurements from uninjected oocytes and oocytes expressing WT h5-HT3A, Y89C, Q146C, F221C, and M223C mutants during application of 100 µM 5-HT (black line). Upper traces (red) show the membrane fluorescence signal (F), and lower traces (black) show the corresponding membrane currents (I). (D-E) Summary of fluorescence changes (ΔF) (D) and currents (E) evoked by application of 100 µM 5-HT. Shown are scatter plots of ΔF from individual uninjected oocytes and oocytes injected with WT h5-HT3A or Cys-mutant h5-HT3A. The ΔF value is calculated as the percentage difference between baseline fluorescence and steady-state fluorescence during 5-HT application (see Materials and Methods). Bars represent mean ΔF (±S.E.M): uninjected; −0.71 ± 0.28 (13 oocytes), WT; −0.81% ± 0.23% (22 oocytes), D74C; 1.1% ± 0.3% (nine oocytes), Y89C; 30% ± 2% (77 oocytes), Q146C; 11% ± 1% (80 oocytes), V196C; −1.2% ± 0.4% (nine oocytes), F221C; 4.6% ± 0.7% (48 oocytes), M223C; 14% ± 1% (83 oocytes), S225C; 1.6% ± 0.6% (seven oocytes), I238C; 0.63% ± 0.15% (eight oocytes), R239C; 1.8% ± 0.4% (eight oocytes), R241C; 0.39% ± 0.11% (six oocytes), L243C; −1.8% ± 0.3% (13 oocytes), V246C; 1.1% ± 0.2% (four oocytes), V247C; 0.66 ± 0.10 (five oocytes), L249C; and 0.56 ± 0.21 (five oocytes). * Denotes mean response significantly different from WT; P < 0.05 (ANOVA with Dunnet’s correction for multiple comparisons).

Detailed structural information is available for the 5-HT3 receptor in the form of X-ray crystal and cryo-electron microscopy structures of the mouse 5-HT3A (m5-HT3A) receptor (Hassaine et al., 2014; Basak et al., 2018a,b, 2019; Polovinkin et al., 2018). Also, the acetylcholine binding protein, which is a soluble pentameric eukaryotic protein homologous to the ECD of Cys-loop receptors (Brejc et al., 2001; Sixma and Smit, 2003), has been engineered to mimic the 5-HT3 receptor ligand binding profile (Kesters et al., 2013). Crystal structures of this construct (5-HT binding protein) in complex with 5-HT, granisetron (Kesters et al., 2013), palonosetron (Price et al., 2016), and varenicline (Price et al., 2015) provide atomic-level insight into potential ligand binding modes and local conformations of the ECD in the 5-HT3 receptor. However, the binding mode of 5-HT, as well as the local conformation of the binding sites observed in the 5-HT binding protein structure (Kesters et al., 2013), is different from the binding modes of 5-HT observed in recently reported cryo-electron microscopy structures of the mouse 5-HT3 receptor (Basak et al., 2018a; Polovinkin et al., 2018). Thus, there are still gaps in the understanding of how ligand binding in the orthosteric site can either induce or prevent the opening of the relatively distal ion channel; partly due to a lack of data describing specific intrareceptor motions during ligand binding.

Voltage-clamp fluorometry (VCF) is a technique that uses site-specific labeling of proteins with an environmentally sensitive fluorophore coupled with two-electrode voltage-clamp electrophysiology (Mannuzzu et al., 1996). VCF allows simultaneous real-time measurement of both the functional state of an ion channel and conformational changes around specific regions, which are reported as changes in fluorescence intensity (∆F) (Talwar and Lynch, 2015). Previously, VCF has been used to investigate conformational changes in the ECD of other Cys-loop receptors such as the glycine receptor (Pless and Lynch, 2009a; Lynagh and Lynch, 2012; Soh et al., 2017), GABAA receptor (Chang and Weiss, 2002; Khatri et al., 2009; Muroi et al., 2009; Akk et al., 2011; Eaton et al., 2014), and nicotinic acetylcholine receptor (Dahan et al., 2004), and very recently VCF has been used to track conformational changes in the ion pore region of the mouse 5-HT3 receptor associated with channel gating (Polovinkin et al., 2018).

In the present study, we use VCF to monitor conformational changes in the ECD around the orthosteric site of the human 5-HT3A (h5-HT3A) receptor induced by binding of agonists, antagonists, and allosteric modulators. At the 5-HT3 subunit interfaces, the orthosteric binding sites are formed by six segments (designated as loops A–F) (see Fig. 1B). Specifically, we identify reporter positions in loops C, E, and F where residues are amenable to replacement with cysteine and labeling with the fluorophore 2-([5(6)-tetramethyl-rhodamine] carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) and can report ligand-induced structural rearrangements as changes in tetramethyl-rhodamine (TAMRA) fluorescence. In agreement with VCF studies of other Cys-loop receptors, our findings indicate that the loop E reporter position displays distinct ∆F for agonists and antagonists. A loop D reporter position was found to report similar ∆F in response to all orthosteric binding ligands, regardless of function. We also observed that functionally and structurally similar antagonists can induce distinct ∆F at a reporter position in loop C.

Materials and Methods

Materials.

Chemicals were obtained from Sigma (St. Louis, MO) except when otherwise stated. DNA-modifying enzymes were obtained from New England Biolabs (Ipswich, MA). Granisetron, ondansetron, tropisetron, mirtazapine, thymol, and carvacrol were obtained from Tokyo Chemical Industry (Zwijndrecht, Belgium). m-Chlorophenyl biguanide (mCPBG) was obtained from Tocris Bioscience (Bristol, UK). MTS-TAMRA was obtained from Biotium (Hayward, CA).

Molecular Biology.

cDNA encoding h5-HT3A was kindly provided by Beate Niesler (Department of Human Molecular Genetics, University of Heidelberg, Heidelberg, Germany). For expression in Xenopus laevis oocytes, the h5-HT3A coding region was subcloned into the pXOON expression vector as described previously (Ladefoged et al., 2018). Cysteine mutations were introduced using the QuickChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). Successful mutagenesis was confirmed by sequencing of the entire coding region (GATC Biotech, Constance, Germany). mRNA was generated using the AmpliCap T7 High Yield Message Maker Kit (CellScript, Madison, WI) according to the manufacturer’s instructions.

Xenopus Laevis Oocyte Expression.

Defoliated stage V to VI oocytes from Xenopus laevis were prepared as described previously (Poulsen et al., 2014) and injected with 15 ng mRNA. The care and use of Xenopus laevis were in strict adherence to a protocol approved by the Danish Veterinary and Food Administration (license 2014-15-0201-00031). Injected oocytes were incubated at 18°C for 3–10 days in standard Barth’s medium containing 88 mM NaCl, 1 mM KCl, 0.41 mM CaCl2, 2.4 mM NaHCO3, 0.33 mM Ca(NO3)2, 0.82 mM MgSO4, and 5 mM Tris (pH 7.4) supplemented with 50 µg/ml gentamycin. Healthy oocytes that showed robust expression were stored at 4°C for up to five additional days.

Labeling of Oocytes.

Oocytes were transferred to frog Ringer’s solution [115 mM NaCl, 2 mM KCl, 5 mM HEPES, and 1.8 mM BaCl (pH 7.6)] containing 20 µM MTS-TAMRA and left for 30 seconds in the dark at room temperature. Oocytes were then rinsed three times in frog Ringer’s solution. Labeled oocytes were kept in Ringer’s solution in the dark for up to 2 hours before recording.

Electrophysiology and Voltage-Clamp Fluorometry.

VCF recordings were performed essentially as described previously (Söderhielm et al., 2015). A glass bottom perfusion chamber was mounted on an inverted epifluorescence microscope (Diaphot 300; Nikon, Japan) equipped with a 20× air objective and a G-1B filter set (Chroma 11002v2, excitation filter 541–551 nm, dichroic mirror >565 nm, emission >590 nm). Excitation was achieved using a light-emitting diode lamp with a nominal wavelength of 530 nm (M530L3; ThorLabs, Newton, NJ). Fluorescence intensity was measured with a D104 photomultiplier tube system (Photon Technologies International). The photomultiplier tube voltage was adjusted daily between 450 and 700 V such that baseline converted fluorescence output on labeled oocytes was between 0.5 and 5 V. Membrane current was measured under two-electrode voltage-clamp electrophysiology. Oocytes were voltage clamped at −20 mV and continuously perfused with frog Ringer’s. All ligands were dissolved in frog Ringer’s solution and applied using an automated programmable perfusion system (Bioscience Tools, San Diego, CA). Current, voltage, and fluorescence signals were digitized at 200 Hz (DigiData 1332; Molecular Devices, Sunnyvale, CA) and recorded using a PC running Clampex 10 software (Molecular Devices). For fluorescence antagonist dose-response curves, each concentration was applied for 2 minutes to allow stable fluorescence levels. The excitation light-emitting diode was pulsed for 200 milliseconds every second to limit the fluorophore photodestruction during the extended recording time. For fast-solution switching electrophysiology experiments to determine rates of activation, deactivation, and desensitization, we used a vertical flow chamber with a chamber volume of 200 µl (Joshi at al., 2004). With a constant flow rate of 20 ml/min, this chamber design results in complete solution exchange within ∼500 milliseconds as measured by recording open-tip pipette potential when the pipette is positioned just above the oocyte in response to exchange of high and low molarity.

Molecular Modeling.

A previously generated homology model of h5-HT3A (Ladefoged et al., 2018) was used as a starting point for the creation of models of h5-HT3A receptor Cys mutants with TAMRA conjugated. Specifically, for each reporter position (Tyr89, Met223, and Gln146), the wild-type residue was mutated to a Cys using the mutate tool within Maestro 10.1 (Schrödinger Suite, 2015 release). The TAMRA molecule including the linker was built in Maestro 10.1 and minimized using a conjugate-gradient algorithm in MacroModel 10.7. The protonation state of the TAMRA molecule was assessed using Epik 3.1 (Shelley et al., 2007; Greenwood et al., 2010) and was modeled in a zwitterionic state. The TAMRA structural conformation was optimized using MacroModel 10.7 and a 1000-step mixed torsional/low-mode sampling conformational search algorithm in which a 2500-step minimization followed each step with all other settings at default. Insertion of TAMRA and methanethiosulfonate linker at Cys residues was performed by structurally aligning S-Cβ-Cα atoms from the Cys side chain to S-CH2-CH3 heavy atoms from the linker. Duplicate atoms were deleted, and the atomic bond between the linker and the reporter position side chain was drawn manually in Maestro 10.1. The QuickTorsion tool available in Maestro was used to move the linker and TAMRA molecule out of direct steric clashes with the protein. The resulting TAMRA-linked h5-HT3A structure was further processed using the Protein Preparation Wizard (Sastry et al., 2013). Specifically, the conjugated TAMRA including linker and protein residues within 5 Å were subjected to energy minimization using a conjugate gradient algorithm and a conformational search using the substructure option in MacroModel 10.7, while residues in the 5–10 Å distance range were restrained by a force constant of 200 kJ/(mol·Å2). All remaining atoms were ignored during the calculation. Calculation of the accessible volume for TAMRA at each reporter position was performed using the Förster resonance energy transfer positioning and screening software (Kalinin et al., 2012). This software estimates the volume accessible to the methanethiosulfonate-linked TAMRA molecule at each reporter position using a geometric search algorithm. The fluorophore is approximated by a sphere, and the linker is simply treated as a distance. The search algorithm determines all possible orientations and positions the fluorophore can reach based on the applied linker that do not clash sterically with the protein. Before each calculation, the side chain of the reporter positions in subunit A (Met223 from the principal face) or E (Tyr89 and Gln146 from the complementary face) was removed to prevent the original residue from causing steric clashes with the fluorophore probe. Three parameters were used to describe MTS-TAMRA during the calculations: linker length (16 Å), linker width (3 Å), and dye radii (where x, y, and z are 7, 5, and 3 Å, respectively). The dimensions were estimated based on a computational model of MTS-TAMRA in a stretched conformation, and the linker length was the distance from the distal end of the linker to the geometric center of TAMRA.

Data and Statistical Analysis.

Data and statistical analyses were performed using pClamp 10 (Molecular Devices) or Prism 6.0 (GraphPad Inc., San Diego, CA) software. Calculation of ∆F was performed using the following equation:Embedded Imagewhere Fpeak is the peak fluorescence observed following application of ligand and Fbaseline is the fluorescence immediately before ligand application. For EC50 determination, current or fluorescence responses were normalized to the maximal ∆F or current response in each oocyte and then pooled. The composite concentration-response data was analyzed by iterative curve fitting using the following equation:Embedded Imagewhere Response is the ∆F or current response measured at a given concentration normalized to the maximum response; Y is the concentration of ligand that produces a half-maximal response (EC50); X is the logarithm of the concentration of the ligand; and nH is the Hill slope. Unless otherwise noted, data are reported as either mean ± S.E.M. or mean with 95% confidence intervals. Statistical analysis of pairwise or multiple comparisons was performed using ANOVA or Student’s t test as appropriate. A probability value of P < 0.05 was considered statistically significant.

Results

Identification of VCF Reporter Positions in h5-HT3A.

To investigate ligand-induced structural changes in 5-HT3 receptors, we first searched for residues in homomeric h5-HT3A receptors that were amenable to substitution with Cys and thiol-mediated fluorophore conjugation. However, a prerequisite of this type of site-specific fluorophore labeling is the absence of endogenous Cys residues that are extracellularly accessible and thiol reactive. Structures of the m5-HT3A receptor (Hassaine et al., 2014; Basak et al., 2018a,b, 2019; Polovinkin et al., 2018) show that the only Cys residues in the ECD are Cys157 and Cys171 in the eponymous Cys loop. Cys157 and Cys171 form a disulfide bond and thus should not be reactive toward MTS-TAMRA (Fig. 1B). Also, previous experiments utilizing thiol-reactive fluorophores on other Cys-loop receptors have found this pair of conserved Cys residues to be inaccessible to labeling (Dahan et al., 2004; Pless and Lynch, 2009a; Eaton et al., 2014). However, to verify that potential labeling of Cys157 and Cys171 did not produce background signals, we initially subjected oocytes expressing wild-type (WT) h5-HT3A receptors to MTS-TAMRA labeling and measured fluorescence during application of different ligands (see Materials and Methods). Following treatment with MTS-TAMRA, application of 100 µM 5-HT to WT h5-HT3A expressing oocytes showed no or less than 1% ∆F and was in general indistinguishable from fluorescence recordings at MTS-TAMRA–treated uninjected oocytes (Fig. 1, C and D). On this basis, we concluded that the WT h5-HT3A receptor formed a suitable background for the introduction of Cys residues for VCF.

We next sought to identify residues in the h5-HT3A receptor subunit useful as VCF reporter positions following substitution with Cys and MTS-TAMRA labeling. Criteria for a residue position to be a useful reporter included that: 1) the position must allow the side chain of the introduced Cys residue to be accessible to an extracellularly applied thiol-reactive fluorophore, 2) the Cys mutation does not interfere excessively with subunit assembly and normal receptor function, and 3) the Cys mutation must be capable of reporting ∆F in response to conformational changes at the labeled position. Extensive cysteine scans often yield only a small number of useful VCF reporter positions (Zhang et al., 2009; Talwar and Lynch, 2015). To improve the rate of identifying successful reporter residues, we used previously performed VCF work on other Cys-loop receptors as a guide to residue selection (Supplemental Fig. 1; Table 1). On this basis, seven h5-HT3A residues around the orthosteric binding site were selected for mutation to Cys (Fig. 1B; Table 1). Also, we sought to identify reporter positions for movements occurring outside the orthosteric binding site; potentially leading toward channel activation. The pre-M1 region is known to couple conformational changes between the orthosteric binding site and the channel and to rearrange before opening of the channel gate (Castaldo et al., 2004; Purohit et al., 2013), which via structures of the m5-HT3A receptor in various functional states have been identified to be formed by residues located roughly in the middle of transmembrane helix M2 that lines the pore (Basak et al., 2018a; Polovinkin et al., 2018). Therefore, we performed scanning Cys mutagenesis along the pre-M1 region; introducing Cys at seven more positions (Fig. 1B; Table 1).

View this table:
TABLE 1

Residues in h5-HT3A selected for cysteine substitution and their equivalents in other Cys-loop receptor subunits previously studied by fluorescence

We expressed Cys-mutant receptors individually in Xenopus oocytes, and following labeling with MTS-TAMRA we evaluated them for current and ∆F responses to the application of 100 µM 5-HT (see Materials and Methods) (Fig. 1B). As summarized in Fig. 1D, all tested mutants showed inward currents in response to 5-HT with amplitudes that were within the range observed for the WT receptor. Four mutants (Y89C, M223C, F221C, and Q146C) were found to report robust ∆F in response to 100 µM 5-HT (Fig. 1, C and D). These mutants all showed increased fluorescence in response to 5-HT with mean ∆F values of 30% ± 2% (Y89C), 11% ± 1% (Q146C), 4.6% ± 0.7% (F221C), and 14% ± 1% (M223C) (Fig. 1, B and C). The remaining mutants did not report detectable ∆F in response to 100 µM 5-HT or produce ∆F values that were less than 2-fold of background fluorescence, and thus were not further investigated. A caveat of VCF when using reporter positions near ligand binding sites is that fluorescence changes may theoretically indicate a change in microenvironment due to proximity to the ligand and not a conformational change in the protein. We determined the effect of 5-HT on TAMRA fluorescence in solution (see Materials and Methods). 5-HT caused a concentration-dependent decrease of TAMRA fluorescence intensity with no change in the peak emission wavelength (Supplemental Fig. 2). Thus, if bound 5-HT exerts a direct effect on the fluorescence of receptor-conjugated TAMRA, this would be reported as a decrease in fluorescence or negative ∆F. Since the ∆F observed during VCF at all positions were positive, we concluded that the observed changes in fluorescence are not caused by direct fluorophore-ligand interaction. Consequently, we assume that the ∆F observed in response to 5-HT indicates local or global conformational changes of the protein.

Characterization of Reporter Position Cys Mutants.

To evaluate if Cys mutation and TAMRA labeling change basal ligand-gated ion channel properties of the potential reporter Cys mutants, we performed electrophysiological characterization of 5-HT response kinetics and EC50 (Fig. 2; Table 2). First, we determined EC50 for 5-HT at the WT receptor and Y89C, M223C, Q146C, and F221C mutant receptors with and without MTS-TAMRA treatment (Fig. 2A). With and without labeling, the Y89C and Q146C mutants showed EC50 values that were within close range of the EC50 for the WT receptor (Table 2). In contrast, there was a profound decrease in 5-HT potency for the F221C mutant with a 47-fold increase in EC50, which upon TAMRA labeling further increased to more than 150-fold relative to WT (Table 2). The M223C mutant displayed a 3-fold increase in EC50 relative to WT and increased further to 12-fold upon TAMRA labeling (Table 2). The Phe221 and Met223 positions are located very near each other on loop C, and therefore likely report the same conformational changes. The VCF screening results suggested that the M223C mutant, in general, displayed stronger fluorescence signals compared with F221C (Fig. 1C). Therefore, due to the profound effect of Cys mutation and TAMRA labeling on the EC50 for 5-HT for the F221C mutant and its generally weaker fluorescence signals, we abandoned further work with the F221C mutant.

Fig. 2.
Fig. 2.

Characterization of effects of Cys-mutation and TAMRA labeling on h5-HT3A receptor function. (A) Concentration-response curves for 5-HT currents at WT and mutant h5-HT3A receptors expressed in oocytes with (red) and without (black) TAMRA labeling (see Materials and Methods). Data points represent the mean from three to five independent concentration-response experiments. Error bars are the S.E.M. and are shown when larger than symbol size. (B) Superimposed responses to 100-second applications of 1 mM 5-HT (black bar) recorded from oocytes injected with the indicated WT or Cys-mutant 5-HT3A subunit mRNA; before (black traces) and after TAMRA labeling (red traces). The onset of desensitization (left panels), activation (middle panels), and deactivation (right panels) are fitted with mono-exponential functions (shown as dotted lines) to determine time constants (τ). Note that for the middle and right panels that show the initial phase of the current response the superimposed responses are shown on a faster time scale compared with the left panel and that amplitudes for all responses are scaled to equal sizes. (C) Summary of time constants for desensitization (τdesensitization), activation (τactivation), and deactivation (τdeactivation). Data represent the mean and S.E.M. for 5–10 oocytes. * Denotes significantly different from unlabeled WT; P < 0.05 (ANOVA with Dunnet’s correction for multiple comparisons).

View this table:
TABLE 2

EC50 values for current and fluorescence responses to 5-HT and mCPBG at unlabeled and labeled WT and mutant h5-HT3A receptors

To further characterize the potential effects of Cys mutations and TAMRA labeling on receptor function, we performed fast ligand-application experiments to determine the 5-HT response kinetics. Specifically, using a vertical oocyte perfusion chamber that allows solution exchange rates in the 100–500 millisecond range (Joshi et al., 2004) (see Materials and Methods), we characterized the rate of activation, desensitization, and deactivation of currents evoked by application of a saturating concentration (1 mM) of 5-HT to oocytes expressing the WT receptor and Y89C, Q146C, and M223C mutant receptors (Fig. 2B). The obtained time constants for activation, deactivation, and desensitization were in good agreement with previously reported characteristics of 5-HT3A receptor currents recorded from oocytes ( Jackson and Yakel, 1995; Yakel, 1996; Davies et al., 1999; Dubin et al., 1999; Joshi et al., 2004). Furthermore, the results showed that the Cys mutations and TAMRA labeling had little or no effect on receptor kinetics; except for a modest 3-fold decrease in the activation rate for the Q146C mutant upon TAMRA labeling (Fig. 2C). In summary, the Y89C, Q146C, and M223C mutants overall retained receptor properties similar to the WT receptor; therefore, we selected all three mutants for further VCF experimentation.

Modeling TAMRA at the Three Reporter Positions.

To investigate how the Cys-conjugated TAMRA molecule at the 89, 146, and 223 positions relates to the orthosteric binding site, we generated models of the h5-HT3A receptor in the inactive conformation with the fluorophore attached at the three reporter sites (Fig. 3A) and calculated the volume accessible to the fluorophore at each position (see Materials and Methods). The models show that the fluorophore at the 89 and 146 positions in loops D and E, respectively, are well accommodated by the protein and can move freely around the complementary face of the binding site (Fig. 3, B and C). The accessible volume at these two reporter positions overlap directly in front of loops D and E; moreover, they are also close to loop C. However, only at position 89 can TAMRA reach loop B from the principal face and only at position 146 can TAMRA reach loops F and G (Fig. 3, B and C). Due to the flexible nature of the linker and loop C itself, TAMRA at position 223 can sample a large portion of both the principal and complementary subunit when loop C is in an extended conformation such as observed in structures thought to represent the receptor in an inactive state (Fig. 3D). In contrast, when loop C is closed in on the binding site, as is suggested to occur during activation, movement of the fluorophore is likely dynamically restricted by loop C since the Cys side chain at position 223 is facing the binding site. Importantly, based on the accessible volume calculations, the TAMRA molecule at position 223 is able to occupy the orthosteric binding site and possibly compete with the tested ligands, which may explain the increase in agonist EC50 observed for the M223C mutant following TAMRA labeling (Fig. 2A). In summary, the results from the TAMRA modeling indicate that the TAMRA conjugated at position 89, 146, and 223 collectively cover the space surrounding the orthosteric binding site.

Fig. 3.
Fig. 3.

Modeling of the orientation of conjugated fluorophores in the h5-HT3A receptor structure. (A) Side view of the structural models of TAMRA conjugation in the mutant Y89C, Q146C, and M223C h5-HT3A receptors. TAMRA is shown in stick representation (red) at position 89 in loop D, position 146 in loop E, and position 223 in loop C (see Materials and Methods). The agonist 5-HT is shown in yellow surface contour. (B–D) The predicted accessible volume for TAMRA conjugated at Cys residues inserted at positions 89 (B), 146 (C), and 223 (D) are shown in gray as observed frontally from within the membrane (top) as well as from the side (bottom) as calculated by the accessible volume method (see Materials and Methods). The receptor backbone of the principal and complement face of the ECD is shown as light gray ribbons and loops A–F are colored according to the color scheme introduced in Fig. 1.

Fluorometric Characterization of Ligand-Induced Conformational Changes.

At present, there is a complete lack of direct experimental evidence of the conformational changes in the ECD that are induced by different classes of 5-HT3 ligands and their correlation to ligand functional effects. Therefore, we measured ∆F in response to a range of structurally and functionally diverse 5-HT3 ligands (Fig. 4A) at the Y89C, M223C, and Q146C mutants. These ligands included the orthosteric ligands acting as agonists (5-HT and mCPBG) and competitive antagonists (granisetron, ondansetron, tropisetron, and encenicline) as well as the positive allosteric modulators thymol and carvacrol, which are suggested to bind at a transmembrane site (Lansdell et al., 2015) (Fig. 4A). Figure 4B shows the standard recording protocol used to compare the ∆F induced by different ligands. Briefly, the protocol employs an initial application of 100 µM 5-HT followed by a washout period to allow full recovery from desensitization of the receptors before the application of test ligand. Controls for nonspecific effects of ligands on TAMRA fluorescence included experiments on oocytes expressing WT h5-HT3A or uninjected oocytes subjected to similar MTS-TAMRA labeling protocols (Supplemental Fig. 3). These showed that saturating concentrations of all ligands evoked less than 1% ∆F at labeled uninjected or WT h5-HT3A expressing oocytes, except for thymol and carvacrol, which at 1 mM caused decreasing ∆F in the −1% to −3% range at both uninjected and WT expressing oocytes. However, these differences were considered small relative to the 5-HT–induced fluorescence changes at the reporter positions, and thus thymol and carvacrol were characterized for ∆F at the reporter positions.

Fig. 4.
Fig. 4.

Ligand-induced fluorescence changes in fluorophore-labeled h5-HT3A receptors. (A) Chemical structures of 5-HT3 receptor ligands characterized at the Y89C, Q146C, and M223C reporter mutants. (B) Representative traces illustrating the standard VCF recording protocol for measurement of fluorescence [(F), upper trace in red] and current [(I), lower trace in black] responses to ligand application from oocytes expressing h5-HT3A reporter mutants (see Materials and Methods). An oocyte expressing the Q146C mutant was exposed 5-HT (black bar; 100 µM) followed by a 6-minute wash period (gray bars) before application of a saturating concentration of encenicline (blue bar; 10 µM). Excitation light (green bars; 530 nM) was turned off during the wash period to minimize fluorophore photodestruction. Dotted boxes indicate regions of the fluorescence trace that are used for calculation of ΔF for 5-HT and ligand. (C and D) Representative parallel fluorescence and current responses to sequential application of 5-HT and ligand (encenicline, 10 µM; ondansetron, 3 µM; granisetron, 3 µM; and thymol, 1 mM) at the Y89C (C), Q146C (D), and M223C (E) reporter mutants.

Ligand-Specific Fluorescence Signals for the Loop D Mutant Y89C.

Tyr89 is located on loop D, which is part of a β-sheet motif forming the back wall of the orthosteric binding site (Fig. 3). For this reporter position, all tested ligands displayed ∆F responses to application of saturating concentrations that were very similar in amplitude and direction as the ∆F observed for 5-HT; except for thymol and carvacrol, whichdid not evoke detectable specific ∆F (Fig. 4C). Figure 5A summarizes the amplitude pattern of the ligand-specific ∆F responses normalized to the response amplitude of the ∆F evoked by the saturating concentration of 5-HT. The orthosteric ligands mCPBG, granisetron, ondansetron, tropisetron, and encenicline all induced ∆F with amplitudes that were not significantly different from those induced by 5-HT (Fig. 5A). These results indicate that the local environment surrounding loop D may experience similar changes in physicochemical properties in response to binding of orthosteric agonists and competitive antagonists.

Fig. 5.
Fig. 5.

Summary of fluorescence and current response patterns for the Y89C, Q146C, and M223C mutants. (A–C) Graphical summaries of ligand fluorescence response (ΔFligand) amplitudes normalized to the response amplitude to a previous application of 5-HT (ΔF5-HT) for the Y89C (A), Q146C (B), and M223C (C) reporter mutants. The 5-HT data represent recordings of a subsequent application of 5-HT following the same standard recording protocol illustrated in Fig. 4A. Data represent mean ± S.E.M. for 5–16 recordings of individual oocytes. *Denotes significantly different from 5-HT; P < 0.05 (ANOVA with Dunnet’ N.D., not determined.

Ligand-Specific Fluorescence Signals for the Loop E Mutant Q146C.

Gln146 is located on loop E, which like loop D forms part of the β-sheet at the back of the binding site (Fig. 3). All ligands tested at the TAMRA-labeled Q146C mutant caused robust increases in TAMRA fluorescence upon application, except thymol and carvacrol, which did not induce specific ∆F (Fig. 5B). 5-HT and the synthetic agonist mCPBG induced similar ∆F values, suggesting that both agonists induce similar local conformational changes around loop E upon binding and receptor activation. In contrast, all antagonists induced ∆F that was significantly larger than 5-HT (300%–600%) (Figs. 4B and 5B). Therefore, Q146C appears to report the regions around loop E to undergo conformational changes upon orthosteric ligand binding that are distinct for agonists and antagonists.

Ligand-Specific Fluorescence Signals for the Loop C Mutant M223C.

Met223 is located on loop C (Fig. 3), a region on the principal subunit that is thought to be flexible and adopt a contracted conformation upon agonist binding and an extended conformation when antagonists are bound (Kesters et al., 2013; Polovinkin et al., 2018). At TAMRA-labeled M223C mutant 5-HT3A receptors, all tested ligands reported ∆F in the same direction as 5-HT (Fig. 4E). However, in contrast to the relative ∆F patterns observed at the Y89C and Q146C mutant receptors, there was no clear trend in terms of the relative amplitude of the ∆F values for agonist or antagonist or positive allosteric modulators (Fig. 5C). mCPBG displayed ∆F values that were 34% of those for 5-HT (Fig. 5C). For the four antagonists, ondansetron and encenicline induced ∆F values that were 70%–130% larger than 5-HT, respectively, whereas granisetron and tropisetron displayed similar ∆F compared with the 5-HT application. Thymol and carvacrol evoked currents of similar amplitude as 5-HT, and both caused a small, but significant, ∆F with amplitudes of 22% and 15%, respectively, of the 5-HT evoked ∆F (Figs. 4E and 5C).

Concentration-Response Relationship of Ligand-Specific Fluorescence Signals.

The robust ligand-evoked ∆F responses for Y89C, Q146C, and M223C allowed the determination of steady-state concentration-response relationships for the fluorescence responses (Fig. 6; Table 2). Importantly, this enabled us to determine the EC50 value of the ∆F response for the electrically silent process of antagonist binding (Table 3). At Y89C receptors for the endogenous agonist 5-HT, we found that the fluorescence EC50 was increased by approximately 6-fold compared with the EC50 value obtained from the simultaneously determined current responses (Table 2). A similar result was obtained for the agonist mCPBG, which displayed a 4-fold increase in the EC50 value for ∆F responses compared with the EC50 value obtained from current responses (Table 2). M223C receptors reported similar EC50 values for current and ∆F in response to 5-HT, while mCPBG exhibited an EC50 value of ∆F responses that was approximately 2.5-fold larger than the current responses (Table 2). Q146C receptors showed the largest difference between fluorescence and current responses, with 5-HT and mCPBG showing EC50 values for fluorescence responses that were 12- and 45-fold larger than the EC50 values for current responses, respectively (Table 2). The concentration-response relationship for ∆F signals induced by the competitive antagonists granisetron, ondansetron, tropisetron, and encenicline showed ∆F response amplitudes to be concentration dependent (Fig. 6). In general, the obtained EC50 values for ∆F were within the 10-fold range of previously reported IC50 or Kd values for all of the antagonists (Hope et al., 1996; Lankiewicz et al., 1998; Ladefoged et al., 2018); except for the Q146C mutant that displayed decreased EC50 values for ∆F compared with Y89C and M223C mutants, which suggests that Cys mutation and/or TAMRA conjugation decreases ligand affinity. In general, we did not attempt to quantify the rate by which the fluorescence signal decayed back toward baseline during the ligand postapplication washout phase due to the design of our VCF recording chamber, which did not allow for solution exchange rates likely required to isolate the rate of ensemble receptor transitions upon removal of extracellular ligand. However, it is interesting to note that the fluorescence response at Q146C for ligands such as encenicline and ondansetron consistently decreased much faster during the washout phase compared with the responses at Y89C and M223C (Fig. 4, C and D); indicating that the ligand dissociation rate at TAMRA-labeled Q146C may be increased.

Fig. 6.
Fig. 6.

Concentration-response curves for agonist- and antagonist-induced fluorescence changes at the Y89C, Q146C, and M223C mutants. (A) Concentration-response curves for fluorescence and current responses evoked by the agonists 5-HT (upper curves) and mCPBG (lower curves). Data points represent the mean ± S.E.M. from five to eight individual experiments. Concentration-dependent increases in fluorescence. (B) Representative traces illustrating the standard recording protocol employed for fluorescence concentration-response experiments. Shown are 5-second segments from recording traces of membrane current (black traces) and fluorescence (red traces) from an oocyte expressing TAMRA-conjugated Y89C mutant receptor during application of increasing concentrations of ondansetron (indicated by black bars) (see Materials and Methods). The excitation light-emitting diode was pulsed for 200 milliseconds (indicated by green bars) every second to limit the fluorophore photodestruction during the extended recording time. Each ligand concentration was applied for 2 minutes to allow stable fluorescence levels. (C) Concentration-response curves for fluorescence responses evoked by setron-class antagonists at the Y89C, Q146C, and M223C mutants. Data points represent the mean ± S.D. from four to eight individual experiments.

View this table:
TABLE 3

EC50 values for fluorescence responses to antagonists at mutant h5-HT3A receptors

Discussion

Crystal and cryo-electron microscopy structures of the m5-HT3A receptor (Hassaine et al., 2014; Basak et al., 2018a,b, 2019; Polovinkin et al., 2018) and other Cys-loop receptors (Du et al., 2015; Morales-Perez et al., 2016; Phulera et al., 2018) have established structural conformations that likely reflect key functional receptor states, and thereby provide an essential framework for beginning to understand the structural mechanism of the 5-HT3 receptor. However, as static structures, these do not provide direct evidence for specific protein motions that occur during receptor state transitions. In our present study, we have used VCF to study conformational changes in the h5-HT3A receptor to provide direct experimental evidence of structural changes around the orthosteric ligand binding site of the h5-HT3A receptor during ligand binding and channel activation. We identify positions in loops C, D, and E where measurements of fluorescence from a conjugated environmentally sensitive fluorophore allow tracking of conformational changes in response to binding of a range of prototypical 5-HT3 ligands; including the endogenous agonist 5-HT. Interpretation of our fluorescence data in the context of presently available Cys-loop receptor structures presents an opportunity to evaluate and expand current structure-based models for the early steps in the 5-HT3 receptor activation mechanism. Specifically, the comparison of the patterns of fluorescence changes induced by different ligands provides insight into the differences in conformational changes that underlie agonist and antagonist ligand function. However, a potential concern for interpreting changes in fluorescence from the reporter fluorophore as the results of protein rearrangements is whether the fluorophore microenvironment is affected directly by the proximity of the bound ligands. In terms of direct ligand effects that produce decreases in fluorescence, examples include resonance energy transfer to the ligand or simple absorbance of emitted light by the ligand (Unruh et al., 2005; Pless and Lynch, 2009b; Talwar and Lynch, 2015). However, none of the ligands induced decreases in TAMRA fluorescence (Figs. 4C and 5); therefore, such direct quenching effects were not a concern in the present study. Direct ligand effects that increase fluorescence require that the presence of the ligand changes the physicochemical microenvironment around the fluorophore such that its quantum yield is increased. In the case of TAMRA, this most likely means increasing overall hydrophobicity; for example, by shielding the fluorophore from polar protein structure elements or water molecules or by direct steric effects that promote the fluorophore to orient toward a more hydrophobic environment. In this context, our modeling of TAMRA conjugated to the Cys-mutated Tyr89 and Gln146 positions in the h5-HT3A receptor showed that the fluorophore in these positions cannot access the binding site, and thus cannot be in direct contact with ligands (Fig. 3, B and C). In contrast, for the Met223 position, the side chain of the inserted Cys residue was facing into the binding pocket (Fig. 3D), and the accessible volume calculations showed that TAMRA from this position can at least partially occupy the binding pocket and thus potentially experience direct effects of orthosteric ligands; therefore, this aspect must be taken into account when interpreting the ΔF reported from this position. Another important issue for interpreting fluorescence data is that we used a steady-state recording methodology on a timescale that did not allow us to resolve receptor transitions (e.g., apo to preactive, active to desensitized); meaning that we cannot know whether a change in fluorescence is due to a local conformational change, such as a localized rearrangement of the orthosteric binding site upon ligand binding, or global conformational changes underlying transitions between receptor functional states.

At positions 89 and 146 in the D and E loops, respectively, the conjugated TAMRA consistently showed increases in fluorescence upon binding of all of the tested orthosteric ligands; thus, showing that conformational changes occur around these positions upon ligand binding. Principally, these changes can involve the local rearrangement of loops D and E, as well as the surrounding protein regions. Regarding the possibility of direct loop movements as a source for ΔF, then both loops E and D, together with loop G, form strands in a β-sheet structure that line the back of the orthosteric binding site. This β-sheet motif is considered to be rigid, and comparison of its conformation in recent structures of m5-HT3A and other Cys-loop receptors suggests that it undergoes little or no overall structural rearrangement during receptor state transitions (Nys et al., 2013; Hassaine et al., 2014; Du et al., 2015; Basak et al., 2018a,b, 2019; Polovinkin et al., 2018). Thus, it appears more likely that TAMRA at both positions reports movement of surrounding structural elements. Candidate elements that might influence fluorescence from TAMRA conjugated at loops E and D first and foremost include the C loop, which is positioned in close vicinity of both positions (Fig. 3). This is based on long-standing models for Cys-loop receptor function and involves the idea that ligand binding induces local movement of loop C around the orthosteric binding site (Chang and Weiss, 2002; Law et al., 2005; Pless and Lynch, 2009b; Kesters et al., 2013; Purohit and Auerbach, 2013; Du et al., 2015). However, for reporter position 89, potential structural rearrangement of loop F is also a possible candidate, and this loop region contains several residues that are suggested to be directly involved in agonist recognition (Thompson et al., 2006) and assume different conformations among the presently available 5-HT3 receptor structures (Hassaine et al., 2014; Basak et al., 2018a,b, 2019; Polovinkin et al., 2018). Likewise, reporter position 146 may be influenced by movements of loop B. Interestingly, although the N-terminal segment of loop B is locked in a β-strand motif and likely is overall rigid during state transitions, the C-terminal segment is not and may be able to adapt locally to ligand binding (Fig. 1B). Overall, this idea is supported by comparison of structures of apo and 5-HT–bound m5-HT3A, which show the ECD to adopt a more condensed conformation with 5-HT bound; most pronounced around the lower half of the ECD and loops B and C (Basak et al., 2018b; Polovinkin et al., 2018). In addition, a recent structure of m5-HT3A in complex with the antagonist granisetron also shows the ECD to condense when granisetron is bound; including a shift in the positions of loops B and C (Basak et al., 2019).

The Y89C mutant reported strikingly similar levels of ΔF for both agonists and competitive antagonists. This potentially means that if TAMRA conjugated to position 89 in loop D senses ligand-induced conformational changes, then these are distinct from those reported by TAMRA at position 146; despite the two reporter positions being closely located in the binding site (Fig. 3A). However, it should be noted that it is possible that different rearrangements upon agonist and antagonist binding can produce similar net effects on the environment around TAMRA and thus similar changes in fluorescence. Also, ligand binding may induce changes to the environment around TAMRA that are not directly related to conformational changes. Notably, albeit the accessible volume calculations show substantial overlap in the parts of the receptor surface that TAMRA can sample for both positions, only TAMRA at position 89 can sample the surface near loop D in the back of the pocket (Fig. 3, B and C). Instead of conformational changes, a possible source for the similar fluorescence response by the Y89C mutant to agonists and antagonists can be that ligand occupation of the binding pocket, in general, displaces one or more water molecules to increase general hydrophobicity and enhance TAMRA fluorescence.

Comparison of the ΔF observed at the Q146C mutant for agonists with those observed for antagonists shows a striking pattern where the agonists 5-HT and mCPBG induce similar ΔF, whereas the four competitive antagonists induce a distinct set of ΔF of likewise similar magnitude (Fig. 5B). VCF studies on other Cys-loop receptors have labeled positions in loop E and compared the effects of agonists and antagonists (Muroi et al., 2006; Pless and Lynch, 2009b; Akk et al., 2011). In GABAA receptors, Akk et al. (2011) observed that a fluorophore-labeled loop E position, corresponding to Gln146 in h5-HT3A, reported increases in fluorescence that were several fold larger in amplitude for the competitive antagonist gabazine than for the orthosteric agonists GABA and muscimol. Also, at the GABAA receptor, Chang and Weiss, (2002) found that the loop E position corresponding to position Gln146 in h5-HT3A reported distinct fluorescence changes for GABA and the competitive antagonist 3-aminopropyl methylphosphinic acid. Similarly, in glycine α1 receptors, Pless and Lynch, (2009b) reported that a loop E reporter position, which corresponds to Gln146 in h5-HT3A receptors (Supplemental Fig. 1; Table 1), showed a greater increase in fluorescence in response to the competitive antagonist strychnine than to the agonist glycine. Taken together with the results from the present study, which used a wider range of competitive antagonists, loop E of Cys-loop receptors seems to consistently report VCF results that reflect the functional properties of the ligand. Distinct conformational changes around loop E may thus be a hallmark feature of Cys-loop receptor agonism versus antagonism.

Met223 is located in loop C, and changes in fluorescence for TAMRA conjugated at this position might be ideal for tracking of movement of loop C during ligand binding as well as the ECD tightening occurring during receptor activation due to its close proximity to the interface of the principal and complementary subunits of each binding site. At this position, we did observe fluorescence changes for all ligands, but these displayed a range of relative ΔF magnitudes that did not follow any pattern correlated to ligand functionality (Fig. 5C). Movement of loop C has been proposed to have a central role in mechanistic models for Cys-loop receptor function. Specifically, binding of ligands that act as agonists have been proposed to contract loop C around the orthosteric binding site, while binding of ligands that act as antagonists do not induce constriction or induce a more extended orientation compared with the apo state (Celie et al., 2004; Hansen et al., 2005; (Brams et al., 2011), Kesters et al., 2013; Alix et al., 2016). This capping movement by loop C has been proposed as essential for promoting structural transitions toward the opening of the channel gate; either via a global transition or a sequence of local conformational changes of neighboring domains that eventually reach the channel gate. However, another model is that the varying loop C conformations in ligand-bound structures reflect the role of loop C residues in forming contacts with the ligand (Purohit and Auerbach, 2013). In this model, loop C capping correlates with activation not by inducing conformational change, but by local stabilization of the binding site in a conformation with high open probability. For the 5-HT3A receptor, these ideas can now be evaluated by a series of structures of the m5-HT3A receptor in the apo state and in complex with 5-HT (Basak et al., 2018b; Polovinkin et al., 2018) and the antagonists tropisetron and granisetron (Basak et al., 2019). Comparison of the orientation of the C loop between apo and 5-HT–bound structures indeed shows that the C loop contracts around the orthosteric binding site; moving toward loops E and D, whereas the orientation of loop C in the tropisetron-bound structure closely resembles the apo orientation; hereby supporting the model with loop C contraction as a key conformational change for receptor activation. However, the granisetron-bound structure also shows contraction of loop C around the binding site that is similar in direction to the 5-HT–bound structure; although 5-HT appears to induce the most contraction (Basak et al., 2019). We find binding of 5-HT, granisetron, and tropisetron to all produce increases in fluorescence from TAMRA conjugated to position 223 in loop C; with 5-HT and granisetron producing identical responses that are slightly higher in magnitude than tropisetron (Fig. 5B). Overall, these results suggest that loop C is closing around these ligands, which potentially indicates a more complex role of loop C movement for receptor activation or inhibition than suggested by the aforementioned existing models. However, as discussed previously, a significant concern is that TAMRA at position 223 may occupy the binding pocket and thus experience direct effects from ligand binding. Thus, further work is needed to explore other loop C positions that do not allow the fluorophore to potentially occupy the binding pocket while serving as reporters for loop C movement in the 5-HT3 receptor as well as the arrival of structures of 5-HT3A in complex with additional agonists and antagonists.

Thymol and carvacrol are structurally closely related positive allosteric modulators and channel activators of the h5-HT3A receptor. Their binding site is proposed to be located in the transmembrane domain at the subunit interface (Lansdell et al., 2015), which also has been suggested as the binding site for the positive allosteric modulator trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (Gasiorek et al., 2016; Polovinkin et al., 2018), which has not been explored in the present study. The M223C mutant showed slight, but significant increases in fluorescence in response to both compounds (Fig. 5). Since M223C is located on loop C, which connects directly into the TMD region, this result may demonstrate the allosteric coupling between the orthosteric site and the conformational changes that underlie channel activation as has previously been proposed (Du et al., 2015).

In summary, we have established a VCF approach for studies of conformational changes in the ECD region of the h5-HT3A receptor that contains the orthosteric binding site. We use this to compare the conformational effects of different classes of 5-HT3 receptor ligands . First, consistent with current predictions from structural and biochemical studies, our results support the idea that agonists and competitive antagonists induce distinct conformational changes in the orthosteric binding site. Second, the nature of the ligand-induced conformational changes around loop E appears to be a determinant for ligand functionality as either agonist or antagonist, most likely by assessing the intersubunit tightness in the ECD. Overall, our observations hereby provide insight into the initial steps of the conformational cycle that underly the ligand-gated ion channel function of the 5-HT3 receptor. We note that we were not successful in identifying reporter positions that could report structural transitions outside the immediate ECD regions containing the orthosteric binding site. Therefore, VCF work focused on identifying useful reporter positions in the ECD/TMD interfaces is warranted to enable tracking of structural changes further into the receptor functional cycle. Whereas our present work focused on the ECD segment, it is in this respect encouraging to note that recent work on the m5-HT3A receptor has reported a position (Ser296) in the upper region of the pore-lining M2 helix as a useful VCF reporter for structural rearrangements associated with gating transition (Polovinkin et al., 2018).

Acknowledgments

We thank Alexander B. J. Jensen for excellent technical assistance.

Authorship Contributions

Participated in research design: Munro, Ladefoged, Schiøtt, Kristensen.

Conducted experiments: Munro, Ladefoged, Padmanathan, Andersen, Kristensen.

Performed data analysis: Munro, Ladefoged, Kristensen.

Wrote or contributed to the writing of the manuscript: Munro, Ladefoged, Schiøtt, Kristensen.

Footnotes

    • Received March 24, 2019.
    • Accepted September 28, 2019.

  • This work was supported by the Danish Council of Independent Research for Medical Sciences [Grant DFF-404-00309], the Lundbeck Foundation [Grants 2017-1655 and 2012-12453], and the Carlsberg Foundation. Computations were made possible through allocations at the Centre for Scientific Computing Aarhus.

  • https://doi.org/10.1124/mol.119.116657.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

5-HT
5-hydroxytryptamine
Cys
cysteine
ECD
extracellular domain
∆F
fluorescence intensity
h5-HT3A
human 5-HT3A
mCPBG
m-chlorophenyl biguanide
m5-HT3A
mouse 5-HT3A
MTS-TAMRA
2-([5(6)-tetramethyl-rhodamine] carboxylamino) ethyl methanethiosulfonate
TAMRA
tetramethyl-rhodamine
TMD
transmembrane domain
VCF
voltage-clamp fluorometry
WT
wild type

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Ligand Specific Conformational Changes at the 5-HT3 Receptor

Lachlan Munro, Lucy Kate Ladefoged, Vithushan Padmanathan, Signe Andersen, Birgit Schiøtt and Anders S. Kristensen
Molecular Pharmacology December 1, 2019, 96 (6) 720-734; DOI: https://doi.org/10.1124/mol.119.116657
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