# Vestibular Modulation by Stimulant Derivatives in a Pentameric Ligand-Gated Ion Channel

Emelia Karlsson, Olivia Andén, Chen Fan, Zaineb Fourati, Ahmed Haouz, Yuxuan Zhuang, Rebecca J Howard, Marc Delarue, Erik Lindahl

► To cite this version:

Emelia Karlsson, Olivia Andén, Chen Fan, Zaineb Fourati, Ahmed Haouz, et al. Vestibular Modulation by Stimulant Derivatives in a Pentameric Ligand-Gated Ion Channel. British Journal of Pharmacology, In press, 10.1101/2024.05.02.592243. pasteur-04769523v2

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Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH ARTICLE

BJP

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BRITISH
PHARMACOLOGICAL
SOCIETY

# Vestibular modulation by stimulant derivatives in a pentameric ligand-gated ion channel

Emelia Karlsson<fnref n="1" /> | Olivia Andén<fnref n="1" /> | Chen Fan<fnref n="2" /> | Zaineb Fourati<fnref n="3" /> |

Ahmed Haouz<fnref n="4" /> | Yuxuan Zhuang<fnref n="1" /> | Rebecca J. Howard<fnref n="1" />,<fnref n="2" /> | Marc Delarue<fnref n="5" /> |

Erik Lindahl<fnref n="1" />,<fnref n="2" />

**Correspondence**

Rebecca J. Howard and Erik Lindahl, Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, SE-17121, Sweden.

Email: rebecca.howard@dbb.su.se and
erik.lindahl@dbb.su.se

Marc Delarue, Architecture and Dynamics of Biological Macromolecules, Institut Pasteur, CNRS UMR 3528, Université Paris-Cité, Paris, FR-75015, France.

Email: delarue@pasteur.fr

**Funding information**

Swedish e-Science Research Centre; Sven och Lilly Lawskis Fond för Naturvetenskaplig Forskning; Vetenskapsrådet, Grant/Award Numbers: 2019-02433, 2021-05806; Stockholms Universitet, Grant/Award Number: FV-5.1.2-0523-19

**Background and Purpose:** Allosteric modulation of pentameric ligand-gated ion channels (pLGICs) are critical for the action of neurotransmitters and many psychoactive drugs. However, details of their modulatory mechanisms remain unclear, especially beyond the orthosteric neurotransmitter-binding sites. The recently reported prokaryotic symbiont of *Tevnia jerichonana* ligand-gated ion channel (sTeLIC), a pH-gated homologue of eukaryotic receptors in the pLGIC family, is thought to be modulated by aromatic compounds via a relatively uncharacterised modulatory site in the extracellular vestibule.

**Experimental Approach:** We have characterised the effects of psychostimulant derivatives on sTeLIC using two-electrode voltage-clamp electrophysiology in the presence and absence of engineered mutations, and determined X-ray and cryo-EM structures of the channel in both closed and open states.

**Key Results:** We have shown that sTeLIC is sensitive to potentiation by several amphiphilic compounds, which preferentially bind to a vestibular pocket in the contracted open-state extracellular domain.

**Conclusions and Implications:** This work provides a detailed structure-function mechanism for allosteric potentiation via a noncanonical ligand site, with potential conservation of the eukaryotic pentameric ligand-gated ion channels.

**KEYWORDS**

allosteric modulation, cryo-EM, druggable cavity, electrophysiology, ligand-gated ion channel, sTeLIC, X-ray crystallography

**Abbreviations:** BrAmp, 4-Bromoamphetamine; BrPEA, 4-Bromophenethylamine; CHAPS, 3-[(3-cholamidopropyl] dimethylammonio)-1-propanesulfonate; Cryo-EM, Cryogenic electron microscopy; DDM, n-Dodecyl-β-D-maltoside; FFC-8, Fluorinated fos-choline-8; pLGIC, Pentameric ligand-gated ion channel; sTeLIC, Symbiont of *Tevnia jerichonana* ligand-gated ion channel; WT, Wild type.

Olivia Andén, Chen Fan and Zaineb Fourati contributed equally to this work.

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2025 The Author(s). *British Journal of Pharmacology* published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.

<footnote n="1">¹Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden</footnote>

<footnote n="2">²Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden</footnote>

<footnote n="3">³Cibles Thérapeutiques et Conception de Médicaments (CiTCoM), CNRS UMR 8038, Université Paris-Cité, Paris, France</footnote>

<footnote n="4">⁴Plateforme de Cristallographie et de Cristallogènèse, Institut Pasteur, CNRS UMR 3528, Université Paris-Cité, Paris, France</footnote>

<footnote n="5">⁵Architecture and Dynamics of Biological Macromolecules, Institut Pasteur, CNRS UMR 3528, Université Paris-Cité, Paris, France</footnote>
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# 1 | INTRODUCTION

Pentameric ligand-gated ion channels (pLGICs) are a widely expressed family of membrane proteins that critically mediate electrochemical signal transduction across evolution (Corringer et al., 2012). In the animal nervous system, the chemical stimulus of neurotransmitters induces conformational changes, allowing selected ions to pass through the membrane-spanning pore (Nemecz et al., 2016). Members of the pLGIC family are targets in the treatment of neurological and gastrointestinal disorders including epilepsy (Macdonald et al., 2004), sleep disruption (Arnold et al., 2023), Alzheimer's and Parkinson's diseases (Bye et al., 2023), nausea and irritable bowel syndrome (Juza et al., 2020). Notably, eukaryotic pLGICs share structural architecture and complex pharmacology with prokaryotic channels (Sauguet et al., 2015), including conserved sensitivity to clinical drugs such as general anaesthetics (Weng et al., 2010), alcohols (Howard et al., 2011) and benzodiazepines (Spurny et al., 2012). Consequently, prokaryotic pLGICs constitute valuable model systems for structure-function studies of gating and modulation throughout the family (Jaiteh et al., 2016; Tasneem et al., 2005; Thompson et al., 2012).

The minimally conserved topology of each subunit includes an extracellular domain (ECD) comprising 10 β-strands (β1 to β10) and a transmembrane domain (TMD) composed of four α-helices (M1 to M4) (Jaiteh et al., 2016). Known sites of modulation are also conserved, including the so-called orthosteric sites at extracellular subunit interfaces, allosteric modulatory sites facing the outer or inner leaflets of the lipid bilayer and blocking sites in the ion pore (Figure 1a) (Corringer et al., 2012). Recently, small molecules have also been observed to bind in a vestibular site on the internal face of a single-subunit extracellular domain, such as the benzodiazepine flurazepam in Erwinia ligand-gated ion channel (ELIC; Spurny et al., 2012) and acetate in Gloeobacter violaceus ligand-gated ion channel (GLIC; Fourati et al., 2015).

A bacterial receptor known as symbiont of *Tevnia jerichonana* ligand-gated ion channel (sTeLIC) was recently shown to be functionally sensitive to alkaline pH and calcium (Hu et al., 2018), similar to several eukaryotic receptors (Garifulina et al., 2022; Krishek et al., 1996; Zimmermann et al., 2012). sTeLIC was crystallised in a wide-open state with a small synthetic potentiator in the vestibular site (Figure 1a and b), which shares structural features with some eukaryotic pLGICs (Brams et al., 2020; Hu et al., 2018). Based on these observations, we investigated whether other drugs might bind in the vestibular site and how they might modulate function.

Here, we answer these questions using a combination of oocyte electrophysiology, cryo-EM and X-ray crystallography experiments. We show that sTeLIC is sensitive to modulation by amphiphilic compounds including derivatives of amphetamine and phenethylamine (PEA) psychostimulants. We then used both the flexible solvation conditions of cryo-EM and anomalous diffraction available in X-ray crystallography to solve structures in both closed and open states, in lipid nanodiscs and detergent micelles, and in the absence and presence of modulators. From these data, combined with functional characterisation of engineered mutants, we find that ligands in the sTeLIC vestibular site preferentially stabilise the open state of the channel, likely by

## What is already known?

* Pentameric ligand-gated ion channels mediate electrochemical signal transduction across evolution.

* Small molecules potentiate the bacterial channel sTeLIC via a relatively unexplored vestibular site.

## What does this study add?

* sTeLIC is potentiated by amphiphilic compounds including phenethylamine psychostimulant derivatives.

* It extends our view of the complex allosteric gating landscape of membrane receptors.

## What is the clinical significance?

* We provide a mechanistic basis to target pharmacological tools against a relatively unexplored site.

strengthening inter-subunit interactions in the extracellular domain. Our results support a distinctive mechanism of allosteric potentiation via the extracellular domain vestibule, apparently specific to a subset of pLGICs including mammalian 5-HT3A receptors.

# 2 | METHODS

## 2.1 | Experimental protocols and design

### 2.1.1 | Mutagenesis design and generation

We used molecular visualisation and docking tools in UCSF Chimera 1.16 (Pettersen et al., 2004) to identify residues in the vestibular binding site in closed and open structures of sTeLIC. 4-Bromoamphetamine (BrAmp) was prepared for docking using the Dock Prep (Shapovalov & Dunbrack, 2011) tool with default settings, which adds polar hydrogens and charges. Local docking simulations were then conducted using the AutoDock Vina (Trott & Olson, 2010) module, with a 15 Å × 13 Å × 19 Å search volume enclosing and centred in the vestibular pocket of the closed cryo-EM structure or an open X-ray structure (PDB ID 6FL9; Hu et al., 2018). Mutations were generated in sTeLIC in the phospho-base *n*-methyltransferase 3 (pMT3) vector using the GeneArt Site-Directed Mutagenesis System (Thermo Fisher Scientific) and commercial mutagenic primers (IDT). Mutant cDNAs were then verified by Sanger sequencing (Eurofins Genomics) and purified on a larger scale using a HiSpeed Plasmid Midi Kit (Qiagen).
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<figure><img src="image_6.png" /><figcaption>**FIGURE 1** Functional enhancement of a model pentameric ligand-gated ion channel (pLGIC) by stimulant derivatives. (a) Views from the membrane plane (top) and extracellular side (bottom) of a previously published symbiont of *Tevnia jerichonana* ligand-gated ion channel (sTeLIC) X-ray structure (PDB ID 6FL9; Hu et al., 2018), highlighting orthosteric (orange) and vestibular (blue) sites known to bind ligands in at least some pLGIC types. (b) Ball-and-stick representation of 4-bromocinnamate (4-BrCA), a previously reported potentiator binding at the vestibular site. (c) Model as in Figure 1b of putative modulator BrAmp. (d) Model as in Figure 1b of putative modulator phenethylamine (PEA). (e) Representative electrophysiology recording from an oocyte expressing WT sTeLIC, showing potentiating effects of 30-s co-applications of BrAmp with pH 8.5 buffer. (f) Representative recording as in Figure 1e showing potentiating effects of PEA. (g) Concentration-response plot displaying the mean percent modulation ± SEM of sTeLIC by various concentrations of BrAmp (circles) and PEA (squares) (n = 5).</figcaption></figure>

## 2.1.2 | Oocyte preparation

Oocytes from female *Xenopus laevis* were injected via the animal pole with 6 ng/32.2 nl sTeLIC wild type (WT) or mutant cDNA using a Nanoject II microinjector (Drummond Scientific). After injection, oocytes were kept in a 48-well plate containing modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.91 mM CaCl₂, 0.5 mM theophylline, 0.1 mM G418, 17 mM streptomycin, 10,000 U I-1 penicillin, 2 mM sodium pyruvate (all from Merck Life Science AB, 16903 Solna, Sweden), 0.82 mM MgSO₄, 0.33 mM Ca[NO₃]₂ and 10 mM HEPES (all from Fisher Scientific, 41878 Gothenburg, Sweden), adjusted to pH 7.5) at 13°C.

## 2.1.3 | Oocyte electrophysiology

BrAmp-modulated currents were recorded 5-12 days after cDNA injection using a two-electrode voltage clamp (TEVC). The oocyte

membrane was voltage-clamped using two recording needles (5-50 MΩ). These needles, along with the ground stages, were filled with a 3 M KCl solution. The bath electrodes in the ground stages were connected to the bath chamber through 3 M KCl-agar bridges. Oocytes were perfused with the running buffer (123 mM NaCl, 2 mM KCl, 2 mM MgSO₄ and adjusted to pH 7.5) at a flow rate of roughly 1 ml min⁻¹. Oocytes were clamped at -40 mV using an OC-725C voltage clamp (Warner Instruments). A Digidata 1440A (Molecular Devices) was used to sample and digitise data using Clampex (Axon Instruments). Currents were filtered at a frequency of 1 kHz and analysed using Clampfit (Axon Instruments).

Before each recording occasion, we freshly prepared a 1-M stock solution of modulator (BrAmp, PEA, 4-bromophenethylamine [BrPEA], or fluorinated fos-choline-8 [FFC-8]) in dimethylsulfoxide (DMSO) - all from Merck Life Science AB. Solutions with varying modulator concentrations were then prepared in a buffer pH-adjusted to produce 20% maximal current (pH 8.5 for all variants).
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pH-activation buffer, in the absence or presence of modulator, was applied for 30 s, followed by 7 min washout. After each modulator application, the two following applications were solely ~EC₂₀ buffers to verify a return to baseline behaviour.

### 2.1.4 | sTeLIC expression and purification

sTeLIC was expressed and purified as described previously (Hu et al., 2018). Briefly, C43(DE3) *Escherichia coli* cells were transformed with vector pET20b containing a cDNA insert encoding maltose-binding protein fused to the N-terminus of sTeLIC. Cells were inoculated 1:100 into 2xYT media with 100 µg ml⁻¹ ampicillin, grown at 37°C to OD₆₀₀ = 0.8, induced with 0.1-0.4 mM isopropyl-β-D-1-thiogalactopyranoside and shaken overnight at 20°C. The overnight culture was harvested and resuspended in buffer A (20 mM Tris pH 8.0, 300 mM NaCl) supplemented with ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor (Thermo Fisher Scientific) and either benzonase nuclease or lysozyme plus DNase I. The cell membranes were solubilised in buffer A with 4% *n*-dodecyl-β-D-maltoside (DDM; Anatrace, Maumee, OH 43537, USA) at 4°C for 3-16 h. After membrane treatment, the extracted recombinant protein was purified on amylose resin in buffer A with 0.02% DDM, eluting with the addition of 20-50 mM maltose monohydrate and further purified by size exclusion on a Superose TM6 Increase 10/300 GL column (Cytiva). Maltose-binding protein was subsequently separated from sTeLIC by thrombin cleavage and a second round of gel filtration. Purified protein was pooled and concentrated to ~12 mg ml⁻¹.

The plasmid for SapA (saposin) expression was a gift from Salipro Biotech AB and its expression and purification followed previously published protocols (Lyons et al., 2017). Saposin nanodisc reconstitution started with mixing sTeLIC, saposin and polar brain-lipid extract at a molar ratio of 1:15:150, and then incubating the mixture on ice for 1 h. Bio-Beads SM-2 (BioRad) were added to the mixture and then gently shaken overnight at 4°C. The next day, the supernatant was collected and purified in buffer B (20 mM HEPES pH 7.5 (Fischer Scientific), 100 mM NaCl) by size-exclusion chromatography on a Superose TM6 Increase 10/300 GL column (Cytiva). Peak fractions were collected and concentrated to ~3 mg ml⁻¹.

### 2.1.5 | Crystallisation, data processing and structure determination

In order to produce large, well diffracting crystals, the purified protein was first mixed with 65 mM nonyl-β-D-glucose (NDG, Anatrace) at a 4:1 volume ratio and incubated on ice for 20 min. Co-crystallisation of sTeLIC with BrAmp or BrPEA was performed by mixing the nonyl-β-D-glucose-protein mixture with the desired ligand solubilised in 100% DMSO at a final concentration of 3 mM. Crystals were then grown by mixing the protein-ligand mixture with an equal volume of reservoir solution containing 100 mM Tris pH 8.0, 150 mM MgCl₂, 3% DMSO and 30-35% PEG 200 at a 1:1 volume ratio (all from

Merck). Mixtures were equilibrated against a 1-ml reservoir using the hanging drop method at 18°C. Co-crystals appeared within 2 weeks and grew to full size within a month. Prior to data collection, crystals were mounted on nylon loops and flash frozen in liquid nitrogen, using the crystallisation solution as a cryo-protectant.

Diffraction data were collected at SOLEIL (PROXIMA-1 and PROXIMA-2) and, for the 9EWA structure, processed using XDS (Kabsch, 2010). Because crystals grown with BrAmp were anisotropic, the 9EWL dataset was processed using STARANISO software (Global Phasing Limited) allowing extraction of the highest resolution batch from the dataset. While the conventionally estimated completeness was low, its value for the overall dataset was 99.7 with the ellipsoid cutoff surface used by STARANISO taken into account. The structure was solved by molecular replacement using a previously solved sTeLIC X-ray structure (PDB ID 6FL9; Hu et al., 2018) as a search model by Phaser-MR in Collaborative Computational Project 4 (CCP4; Winn et al., 2011). Refinement was performed using REFMAC5 (Refinement of Macromolecular Structures 5) software from the CCP4 suite with non-crystallographic symmetry (NCS) restraints applied. Anomalous maps were calculated using CAD software from the CCP4 suite and averaged in Coot (Emsley et al., 2010).

### 2.1.6 | Cryo-EM sample preparation and data acquisition

For saposin-nanodisc grids, 0.3 µL FFC-8 or 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonate (CHAPS) stock was added to 2.7 µL sample immediately before grid freezing to a final concentration of 2 mM. The mixtures were applied to glow-discharged R1.2/1.3 300 mesh Au grids (Quantifoil), blotted with force 0 for 2 s and plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) at 20°C. For the detergent sample at pH 7.5, 100 µM BrAmp was added to ~3 mg ml⁻¹ protein. Around 3 µl of the mixture was then applied to glow-discharged R1.2/1.3 300 mesh Cu grids (Quantifoil). For the detergent sample at pH 9, 3 µL of a ~7 mg ml⁻¹ sample was directly applied to R1.2/1.3 300 mesh Au grids (UltrAufoil) without any additives. For both detergent samples, the grids were then blotted with force 0 for 2 s and plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 100% humidity. Cryo-EM data were collected on a Titan Krios microscope (Thermo Fisher Scientific) operated at 300 kV with a K3 Summit detector (Gatan), using EPU automated collection software (Thermo Fisher Scientific). The total dose in each nanodisc dataset was ~45 electrons Å⁻² and the defocus range was set from -0.8 to -2.4 µm. The total dose in the detergent datasets were ~40 electrons Å⁻² (pH 7 with BrAmp) or ~36 electrons Å⁻² (pH 9), and the defocus range sets from -0.8 to -3 µm.

### 2.1.7 | Cryo-EM image processing

Dose-fractionated images in super-resolution mode were internally gain-normalised and binned by two in EPU (Thermo Fisher Scientific)
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

during data collection. Motion correction of the dose-fractionated images was performed in RELION 4.0 (Kimanius et al., 2021). Contrast transfer functions (CTFs) were estimated on the motion-corrected images using CTFFIND4.1 (Grant et al., 2018) and particles were automatically picked with 2D references generated from manually picked particles (detergent structures) or with Topaz 0.2.5 (Bepler et al., 2019) using the internal trained model (nanodisc structures). The rest of the processing was performed in RELION 4.0. First, the particles were binned by four and extracted. The 2D classification was run for the binned particles and then the classes with recognisable protein features were selected and re-extracted binned by two. For the detergent structures, the initial model was generated using ab initio reconstruction. For the nanodisc structures, the initial model was generated from the PDB of a previously published sTeLIC crystal structure (PDB ID 6FL9; Hu et al., 2018) using the molmap command in UCSF Chimera (Pettersen et al., 2004). The 3D classification was used to check the structural heterogeneity and further clean the dataset. High-quality classes were selected and re-extracted without binning, and these particles were then used for refinement with C5 symmetry imposed, followed by multiple rounds of CtfRefine and polishing to improve resolution.

## 2.1.8 | Cryo-EM model building

A previously published crystal structure (PDB ID 6FL9; Hu et al., 2018) was used as the starting model. The structure was first rigid body fitted into each cryo-EM map in ChimeraX (Pettersen et al., 2021) and then flexible fitted by ISOLDE (Croll, 2018). After that, each model was manually adjusted and ligands and lipids added in Coot 0.9.5 (Emsley et al., 2010). Models were optimised with PHE-NIX real-space refinement (Adams et al., 2010) and validated by Mol-Probity (Williams et al., 2018).

## 2.1.9 | Gating analysis

Extracellular domain twist, extracellular domain spread and M2-M1 (-) distance were calculated based on a previously published script (Bergh et al., 2021). Briefly, extracellular domain twist was defined by the dihedral angle among (a) the Cα-center of mass (COM) of a single-subunit extracellular domain, (b) the Cα-COM of the full extracellular domain, (c) the Cα-COM of the full transmembrane domain and (d) the Cα-COM of a single-subunit transmembrane domain. Extracellular domain spread was defined by the distance from the Cα-COM of the full extracellular domain to the Cα-COM of a single-subunit extracellular domain and M2-M1(-) distance by the distance between the COM of a full M2-helix, and that of the full complementary M1-helix. Specific loop movements were based on shifts in the Cα atoms of P51 (for the β2-β3 loop), F177 (for loop C), L157 (for loop F), or L250 (for the M2-M3 loop) between aligned closed and open states.

## 2.2 | Data and statistical analysis

Data and statistical analysis complied with the recommendations of the BJP on experimental design and analysis in pharmacology (Curtis et al., 2022). All statistical analyses of electrophysiology data were carried out in Prism 9.5.0 (GraphPad Software). The data analysed were derived from groups consisting of a minimum of five independent experiments. The results are presented as mean ± SEM and were evaluated using unpaired Student's t-tests, with significant effects set at $P < 0.05$. No data were excluded. In keeping with disciplinary practices, oocytes were not subjected to randomisation or blinding, as measured values were not considered substantially subject to genetic differences or bias. Normalised proton activation curves were calculated using the following equation:

$$Y = (R_{\text{basal}} + ((R_{\text{max}} - R_{\text{basal}})/(1 + 10^{\log(\text{EC}_{50} - X_i)})) * n_{\text{H}}$$

where Y is the activation response, $R_{\text{max}}$ is the maximal response, $R_{\text{basal}}$ is the baseline, X is the proton concentration, EC$_{50}$ is the proton concentration that induces 50% of maximal response and $n_{\text{H}}$ is the Hill coefficient. BrAmp modulation was calculated as

$$\% \text{modulation} = ((R_{\text{A}} - R_{\text{0}})/R_{\text{0}}) * 100$$

where $R_{\text{A}}$ represents the modulated peak response and $R_{\text{0}}$ is the response to activating pH measured immediately prior to the $R_{\text{A}}$ response. Normalisation was applied to control for standard variation in size and expression of individual oocytes; control values were not subjected to parametric analysis.

## 2.3 | Materials

All buffers and reagents of analytical grade, prepared with Milli-Q water, were stored at a temperature of 4°C. 1-M and 2-M stocks of BrAmp (National Measurement Institute), BrPEA (Sigma-Aldrich) and PEA (Thermo Fisher Scientific) were freshly prepared in DMSO. Oocytes from Xenopus laevis were purchased from EcoCyte Bioscience in Germany. The authors did not personally manage any frogs during the course of this project. Both FFC-8 and CHAPS were acquired from Anatrace. R1.2/1.3 300 mesh Au grids (Quantifoil), R1.2/1.3 300 mesh Cu grids (Quantifoil) and R1.2/1.3 300 mesh Au grids (UltrAufoil) were purchased from Electron Microscopy Science.

Details of other materials and suppliers are provided in the specific sections.

## 2.4 | Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY
<figure><img src="image_10.png" /></figure>

http://www.guidetopharmacology.org and are permanently archived
in the Concise Guide to PHARMACOLOGY 2023/24 (Alexander
et al., 2023).

3 | RESULTS

3.1 | Functional enhancement of a model pLGIC by stimulant derivatives

To investigate the activity of pharmaceutically relevant compounds via the vestibular site in a model pLGIC, we first tested the effect of BrAmp on sTeLIC in *Xenopus laevis* oocytes. This compound is similar to psychostimulants in common therapeutic and recreational use, with the inclusion of a bromine atom to facilitate structure determination by anomalous X-ray diffraction. We hypothesised that BrAmp would positively modulate sTeLIC, based in part on its structural similarity to 4-bromocinnamate (BrCA) (Figure 1b and c) (Hu et al., 2018). In two-electrode voltage clamp electrophysiology experiments, micromolar concentrations of BrAmp produced positive modulatory effects (Figure 1e and g), similar to 4-bromocinnamate (BrCA). We observed even greater potentiation, albeit at higher concentrations, with brominated and un-brominated forms of PEA (Figures 1d, f-g and S1A), a naturally occurring psychostimulant in bacteria, plants and animals, including in the human brain (Hill & Thomas, 2011; Irsfeld et al., 2013). There was an apparent decrease in potency at the highest accessible concentrations of BrAmp and BrPEA, possibly indicating additional modulatory interactions or experimental complications because of channel desensitisation or ligand insolubility. Consistent with previous reports, electrophysiology traces from this channel were generally stable over time in the presence or absence of drugs (Figure S1B) (Hu et al., 2018).

3.2 | Structures in lipid nanodiscs reveal a resting state

Given that all previous sTeLIC structures have been reported by X-ray crystallography in an open state (Hu et al., 2018), we sought a more complete view of the gating cycle by determining the structure in a closed state. Because crystallisation conditions were previously shown to favour activation, we pursued single-particle cryo-EM under resting conditions (pH 7.5). To further mimic a physiological environment, we reconstituted the receptor in asolectin with saposin nanodiscs. Interestingly, our initial micrographs nonetheless yielded a single reconstruction with an evidently open pore (Table S1 and Figures S2A and S3). Moreover, we found a distinct non-protein density in the vestibular pocket (Figure S1C). This density could reasonably accommodate fluorinated fos-choline-8 (FFC-8), which we had included in grid preparation to combat preferential particle orientation. Indeed, we subsequently found that micromolar concentrations of FFC-8 potentiated sTeLIC currents with even greater maximal effect than stimulant derivatives (Figure S1C), consistent with binding

in this site favouring the open state. This complex was otherwise com-
parable to other open structures (Cα RMSD < 1.4 Å versus PDB ID
6FLI; Hu et al., 2018), verifying that sTeLIC conformation is not sub-
stantially influenced by X-ray versus cryo-EM methods, nor by deter-
gent versus nanodisc reconstitution.

To minimise channel activation, we then substituted CHAPS for
FFC-8 during grid preparation. This approach resulted in a
C5-symmetric map at a nominal resolution of 2.4 Å (Table S1 and
Figures S2B and S3). The associated structure (Figure 2a and b) was
notably distinct from previous open structures of sTeLIC, with an
overall Cα root-mean-square deviation (RMSD) >4.3 Å (versus PDB ID
6FLI; Hu et al., 2018). Instead, the pore was comparable to apparent
resting structures of the related channel *Erwinia chrysanthemi* ligand-
gated ion channel (ELIC; Hilf & Dutzler, 2008; Pan et al., 2012), with a
minimal radius too narrow (<2 Å) to conduct ions (Figure 2c and d).
Local resolution varied between extracellular and intracellular ends,
with relatively poor definition in the outward-facing half of the extra-
cellular domain (Figure S2), reminiscent of the relative flexibility
observed in the extracellular domain of the resting-state bacterial
channel the *Gloeobacter violaceus* ligand-gated ion channel (GLIC;
Rovšnik et al., 2021).

3.3 | Vestibular ligand binding in open X-ray and Cryo-EM structures

To verify binding of psychostimulant derivatives to the open state of sTeLIC, we then took advantage of the anomalous X-ray diffraction signal available from the bromine moiety, solving a co-crystal structure of the complex with BrAmp. We co-crystallised the drug with the receptor according to previously established protocols in detergent (DDM and nonyl-β-D-glucose) at pH 8.0 (Hu et al., 2018). The resulting crystals diffracted up to 3.2 Å resolution, enabling structure determination by molecular replacement using a previously solved sTeLIC X-ray structure (PDB ID 6FL9; Hu et al., 2018) as a search model (Table S2). Anomalous signal from the bromine atom of BrAmp allowed definitive placement in the vestibular pocket of each subunit, with the bromine buried deep in the intrasubunit cavity (Figure 2a and b). We obtained a comparable structure with BrPEA to 3.0 Å resolution, verifying equivalent binding poses of the potentiators (Figure S1 and Table S2). Both structures were otherwise superimposable with previously reported open states of sTeLIC (Cα RMSD <0.5 Å versus PDB ID 6FLI; Hu et al., 2018), with a pore radius (≥ 6 Å) wider than that of open *Erwinia chrysanthemi* ligand-gated ion channel (ELIC; Petroff et al., 2022) and sufficient to conduct hydrated sodium ions (Figures 2c, d and S1A). To verify that these structures were not substantially influenced by crystallisation, we also collected cryo-EM data for sTeLIC in detergent with BrAmp. Although the ligand could not be definitively built without the benefit of anomalous diffraction, the protein density corresponded closely to the open X-ray structures (Table S1 and Figure S2C).

We also attempted to obtain an open-state structure without
modulatory ligands. Nanodisc samples behaved poorly in our
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**FIGURE 2** Structures of closed and open symbiont of *Tevnia jerichonana* ligand-gated ion channel (sTeLIC) in the absence and presence of modulators. (a) Extracellular and (b) membrane-plane views of sTeLIC in closed (red) and open (with BrAmp, X-ray, green) states. In each model, one subunit is shaded for clarity. In the open structure, BrAmp is represented as sticks in its molecular surface (beige). In Figure 2b, the dashed line at right estimates the axial span of the channel. (c) Pore surfaces (grey mesh) calculated using CHAP (Klesse et al., 2019) for the transmembrane domain (TMD) region. For clarity, only two opposing M2-helices are shown, with pore-facing residues labelled (absolute residue positions and prime notation) and shown as sticks. Pore diameters at the –1', 9' and 16' constriction points are indicated beside each model. (d) Pore radius profiles, plotted as a function of distance along the fivefold symmetry axis for closed sTeLIC (solid red), open sTeLIC (with BrAmp, X-ray, solid green), closed *Erwinia chrysanthemi* ligand-gated ion channel (ELIC; PDB ID 3RQU [Pan et al., 2012], dotted grey) and open ELIC (PDB ID 8D68 [Petroff et al., 2022], dashed grey). Dashed line at right corresponds to the axial span shown in Figure 2b. Relative to closed structures, the open structures contain wider TMD pores, notably at the –1', 9' and 16' constriction points.</figcaption></figure>

laboratory under the elevated-pH conditions expected to activate the channel; hence, supported by its evident insensitivity to membrane mimetic (described earlier), we prepared sTeLIC grids in detergent at pH 9.0. The resulting cryo-EM structure resolved to 2.6 Å (Table S1 and Figure S2D) and was superimposable with other open states (CaRMSD <0.6 Å versus PDB ID 6FLI; Hu et al., 2018). In contrast to the closed state, local resolution in all open cryo-EM structures was relatively high in the extracellular domain and lowest at the inward-facing end of the transmembrane domain (Figure S2). Even in the absence of known ligands, the vestibular cavities again contained nonprotein densities, which could reasonably accommodate the DDM detergent used for protein solubilisation (Figure S1D). A previously reported X-ray structure determined without known ligands also contained an unidentified density in this region (Hu et al., 2018). Thus, whereas the closed structure contained no apparent density in the extracellular

vestibule, it appears that the open-state cavity is prone to binding a variety of hydrophobic compounds, rendering the characterisation of a genuine apo open-state structure challenging.

## 3.4 | Structural transitions associated with alkaline gating

Comparison of our closed to open structures revealed not only expansion of the transmembrane domain pore but also rotation and contraction of the extracellular domain, including the extracellular vestibule (Figure 3). Overall, the extracellular domain contracted by 10% (25 to 23 Å extracellular domain spread, see Section 2 for measurement details) and rotated 9° anticlockwise relative to the transmembrane domain (extracellular domain twist). These relatively subtle
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

**FIGURE 3** Structural transitions associated with symbionts of *Tevnia jerichonana* ligand-gated ion channel (sTeLIC) gating. (a) Extracellular views of superimposed sTeLIC structures in closed (red) and open (green) states, highlighting conformational changes occurring in the extracellular domain (ECD; top) and transmembrane domain (TMD; bottom). A single-subunit ECD and the pore-lining M2-helices are darkened for clarity. Arrows indicate structural rearrangements upon activation, involving ECD contraction, and anticlockwise twist relative to the TMD (top) and TMD expansion (bottom). For reference, the ECD model includes Cα atoms at the midpoints of the highlighted β2-β3 loop and loop C as spheres (top). The TMD model includes the hydrophobic gate formed by L234 (9') as sticks and the midpoint of one M2-M3 loop as spheres (bottom). (b) Conformational changes in a single sTeLIC subunit, showing closed (red) and open (green) structures aligned on the ECD-TMD interface and viewed from the membrane plane. For reference, the closed-state complementary subunit is shown partially transparent. Arrows indicate ECD contraction and TMD expansion.

global motions were associated with notable local changes, including translation at the midpoint of each extracellular β2-β3 loop by 11 Å inward and for loop C by 5 Å towards the complementary subunit (Figure 3). At the extracellular domain-transmembrane domain interface, loop F also exhibited dramatic remodelling, moving 8 Å towards the conduction pathway. Interestingly, a similar motion was previously reported for the related bacterial channel, *Desulfofustis* deltaproteobacterium ligand-gated ion channel (DeCLIC), reorganising an intersubunit site for Ca²⁺ inhibition (Hu et al., 2020). In the transmembrane domain, the M2-M1(-) distance contracted by 9 Å (21 to 12 Å) and the M2-M3 loop moved 6 Å away from the pore (Figure 3). The closed pore profile (Figure 2d) was similar to those of *Erwinia chrysanthemi* ligand-gated ion channel (ELIC; Pan et al., 2012; Petroff et al., 2022) and other pLGICs (Figure S4A), while the open pore profile was notably wider (Figures 2D and S4B).

## 3.5 | Basis for positive allosteric modulation via the extracellular domain vestibule

In addition to the global extracellular domain motions described earlier, superposition of a single subunit from closed and open sTeLIC structures revealed local changes in and around the vestibular site

(Figure 4a). This site was defined in part by the so-called Ω-loop, a region spanning the β4-β5 strands that has been implicated in subtype-specific modulation (Hu et al., 2018), as well as the β6 strand on the inner face of the cavity. Whereas the closed state contained no apparent pocket capable of accommodating BrAmp (Figure 4b), all open structures contained such a site, sandwiched between residues including β4-W75 and β6-Y104 (Figure 4c). Specifically, the side chain of W75 occluded the pocket in the closed state but rotated outward to evacuate it in open structures. Whereas BrAmp could not be docked favourably (best pose 17 kcal mol⁻¹) in the closed state, it gave rise to favourable energy scores (best pose −4.8 kcal mol⁻¹) in the open state, mediated in part by apparent aromatic interactions with Y104 (Figure S5). Consistent with these predictions, substituting Ala or Val at either W75 or Y104 significantly reduced BrAmp potentiation of sTeLIC in oocyte recordings, without substantially altering pH sensitivity (Figure 4d and e).

In a plausible model for state-dependent binding, displacement of the W75 side chain from the vestibular pocket not only facilitates ligand binding but also enables it to make hydrophobic contacts with β5-L93 on the complementary subunit (Figure 4f). In the closed state, an equivalent rotation of W75 would not produce substantial contacts with the neighbouring subunit, because of the relative expansion of the extracellular domain. Indeed, multiple substitutions at residue L93
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>**FIGURE 4** Structural basis for positive allosteric modulation via the extracellular domain (ECD) vestibule. (a) Closed (red) and open (with BrAmp, X-ray, green) structures of symbionts of *Tevnia jerichonana* ligand-gated ion channel (sTeLIC), viewed from the extracellular side, showing relative contraction of the ECD upon activation. Inset, superimposing the structures on a single-subunit ECD and viewing from the vestibule (indicated by perspective eye) reveals reorientation of β4-W75 out of the pocket, which is instead occupied by BrAmp (beige) proximal to β6-Y104 in the open state. (b) Vestibular pocket in the closed state, viewed as in (Figure 4a, inset), including a semi-transparent molecular surface. BrAmp (beige) from the open X-ray structure is superimposed for perspective. (c) Vestibular pocket in the open structure (with BrAmp, X-ray), viewed as in Figure 4b including the bound BrAmp (beige) and a semi-transparent surface. (d) pH-response curves for WT and mutant receptors (n ≥ 5) recorded by two-electrode voltage clamp electrophysiology in *Xenopus laevis* oocytes. Normalised currents (I/Imax) ± SEM were fitted by nonlinear regression, yielding EC20 values for all constructs around pH 8.5 ± 0.2. (e) Percent modulation of sTeLIC current amplitudes upon coactivation of WT and mutant channels at pH 8.5 with BrAmp (n = 5), relative to currents activated without BrAmp immediately before treatment. Significance is relative to WT (*P* ≤ 0.05, **P** ≤ 0.01, ***P** ≤ 0.001, ****P ≤ 0.0001). (f) Rotated view of superposition (as in Figure 4a, inset), revealing interactions of β4-W75 with β5-L93 on the complementary subunit upon transitioning from closed (red) to open (green). (g) View as in Figure 4f of a mammalian 5-HT3A receptors, showing a cryo-EM structure without BrAmp (PDB ID 6DG8 [Basak et al., 2018], orange) and a computational model with BrAmp ([Haloi et al., 2023], teal). Residues V95 and P113, corresponding to sTeLIC W75 and L93, respectively, have been shown to mediate BrAmp modulation and are shown as sticks.</figcaption></figure>

(Ala, Val, Ser, Asp) prevented functional sTeLIC expression in oocytes, consistent with a critical role for this residue in channel gating and/or assembly. Although both W75 and L93 are poorly conserved among pLGICs, we recently reported that mutating equivalent positions in mammalian 5-HT₃A receptors (β4-V95, β6-N125, β5-P113) decreases sensitivity to both 5-HT activation and BrAmp potentiation (Figure 4g) (Haloi et al., 2023). This putative mechanism may be a specific feature of bacterial pLGICs and 5-HT₃A receptors, in which the vestibular pocket exhibits conserved architecture and intersubunit interactions (Figure S6A). In contrast, this region in eukaryotic

receptors for **GABA**, **glutamate**, **glycine** and **acetylcholine** is notably different, with even greater sequence diversity and a largely occluded pocket (Figure S6B).

4 | DISCUSSION

In this work, new cryo-EM and X-ray structures in combination with engineered site-directed mutations support the presence of a drug-gable pocket in the sTeLIC extracellular domain vestibule that binds
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**FIGURE 5** Proposed mechanism of allosteric enhancement via the vestibular site. (a) Cartoon of the extracellular domain (ECD) in a resting state, with an expanded pentamer, and W75 (yellow) occluding the vestibular site. Following deprotonation-induced activation, (b) modulator binding (reaction arrows) triggers ECD contraction (straight arrows), bringing the β4 and β5 strands on either end of the Ω-loop in closer proximity to one another. Binding of modulator at β6-Y104 (purple) is enabled by reorientation of β4-W75 (yellow) out from the vestibular site (curved arrows), enabling it to interact directly with β5-L93 (blue) on the complementary subunit and potentially stabilising the activated state. For clarity, inter-subunit contacts associated with the top-right subunit are rendered opaque.</figcaption></figure>

stimulants in a state-dependent manner (Figure 5). In a plausible mechanistic model, global contraction of the upper extracellular domain and local rearrangement of the W75 side chain facilitate contacts between Ω-loop residues (e.g. β4-W75 and β5-L93) and render the vestibular pocket accessible to ligands (mediated in part by β6-Y104), promoting a potentiated state. The precise mechanism of channel activation in the absence of vestibular potentiation remains unclear, as no open experimental structure has been captured thus far without evident ligand density in the vestibular pocket. Indeed, the vestibular site appears to accommodate a variety of amphiphilic ligands, including BrAmp, BrPEA, FFC-8 and DDM, all producing superimposable protein conformations despite variations in pH (7.5 or 9.0), membrane mimetic (detergent micelles or lipid nanodiscs) and structure determination method (X-ray crystallography or cryo-EM). In all cases, relative enhancement in local extracellular domain resolution in open versus closed cryo-EM structures (Figure S2) suggests that stabilisation in this region promotes channel activation, possibly via vestibular contacts. Interestingly, a similar correlation between activation and extracellular domain stabilisation has been reported for the homologous prokaryotic channel *Gloeobacter violaceus* ligand-gated ion channel (GLIC; Rovšnik et al., 2021). GLIC has also been shown to bind modulators such as dicarboxylates in its vestibular site, although the relative influence of vestibular versus orthosteric binding has been unclear (Fourati et al., 2020).

Psychostimulant derivatives including BrAmp, BrPEA and PEA act as positive vestibular modulators of sTeLIC, generally increasing in the low-to-high micromolar range. Although experimental constraints to oocyte two-electrode voltage clamp precluded definitive characterisation, a modest decrease in potency at the highest BrAmp and BrPEA concentrations tested (200 µM and 1 mM, respectively) may be related to confounding effects of desensitisation, ligand solubility and/or alternative binding sites. Nonetheless, BrPEA as well as FFC-8 was capable of enhancing sTeLIC currents more than 5000-fold (Figure S1A and

S1C), indicating the capacity of this modulatory site to dramatically favour channel activity. Both of the brominated compounds were more potent than PEA, indicating that bromine interactions deep in the vestibular pocket promote binding; interestingly, controlled substances including 4-bromo-2,5-dimethoxyphenethylamine (2C-B) contain a bromine atom at an equivalent aromatic position (Dean et al., 2013). Notably, several diverse ligands appear capable of binding and modulating this site in sTeLIC and its relatives, including FFC-8, dicarboxylates (in GLIC; Fourati et al., 2015) and flurazepam (in Erwinia chrysanthemi ligand-gated ion channel (ELIC; Spurny et al., 2012).

No structures of eukaryotic pLGICs have been captured thus far with ligands in the vestibular site. Recent work by Brams and colleagues indicates this pocket to be occluded in several eukaryotic pLGICs but selectively accessible to ligands in 5-HT₃A receptors (Brams et al., 2020). Their work further demonstrates that covalent labelling in the vestibular site modifies 5-HT₃A receptors currents, supporting a functional role in receptor modulation (Brams et al., 2020). In parallel with the present study, we have observed BrAmp, in particular, to potentiate 5-HT₃A receptors even more potently than sTeLIC (Haloi et al., 2023). Furthermore, BrAmp modulation of 5-HT₃A receptors is sensitive to mutations at positions equivalent to sTeLIC β4-W75, β5-L93 and β6-Y104, and appears to require a similar rearrangement of the β4 residue out of the vestibular site and in contact with the complementary β5 (Haloi et al., 2023). Taken together, these findings support a general mechanism of pocket opening and ligand modulation in a subset of pLGICs including mammalian 5-HT₃A receptors, which may constitute clinically important targets for drug development against the relatively unexplored vestibular site. Apparent inaccessibility and sequence diversity in this pocket among other pLGIC family members suggest that the vestibular mechanism is subtype-specific and of limited potential for off-target side effects. However, future studies may offer additional insights into the possibility of similarly cryptic pockets in other family members.
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

Our recordings and structures of sTELIC offer detailed mechanistic insights into stimulant modulation in a model system for clinically relevant pLGICs. In animals, amphetamines have long been known to increase **5-hydroxytryptamine (5-HT; serotonin)** levels by inhibiting presynaptic **monoamine transporters** and **monoamine oxidases**, enzymes responsible for 5-HT breakdown (Fischer & Cho, 1979; Iversen, 1971; Mantle et al., 1976; Seiden et al., 1993). However, psychostimulants can induce a range of side effects of unclear origin, including nausea, hypomania and restlessness (Nierenberg & Semprebon, 1993; Sporer, 1995; Sternbach, 1991). Indeed, 5-HT₃A receptors inhibitors include important treatments for chemotherapy-induced nausea; in contrast, stimulant potentiation could underlie undesirable gastrointestinal effects. Thus, our structure-function data offer a mechanistic basis to target or tailor pharmacological tools against a relatively unexplored site and to understand the complex allosteric gating landscape of membrane receptors.

## AUTHOR CONTRIBUTIONS

**Conceptualisation:** Emelia Karlsson, Rebecca J. Howard and Marc Delarue. **Electrophysiology:** Emelia Karlsson. **Cryo-EM structure determination and figure preparation:** Olivia Andén and Chen Fan. **X-ray structure determination and figure preparation:** Zaineb Fourati and Ahmed Haouz; **Model building support:** Yuxuan Zhuang. **Writing – original draft:** Emelia Karlsson, Marc Delarue, Chen Fan and Rebecca J. Howard. **Writing – review and editing:** Emelia Karlsson, Olivia Andén, Chen Fan, Rebecca J. Howard, Marc Delarue and Erik Lindahl. **Funding acquisition:** Erik Lindahl and Marc Delarue.

## ACKNOWLEDGEMENTS

E.K. and O.A. were supported by PhD fellowships from Sven och Lilly Lawskis fond. C.F. was supported by grant FV-5.1.2-0523-19 from Stockholm University, and R.J.H. and E.L. by grants from the Swedish Research Council (2019-02433, 2021-05806) and Swedish e-Science Research Centre. We also thank the staff of synchrotron SOLEIL, especially Pierre Legrand, for their help in data collection. Cryo-EM data were collected at the Cryo-EM Swedish National Facility funded by the Knut and Alice Wallenberg, Family Erling Persson and Kempe Foundations, SciLifeLab and Stockholm University.

## CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

## DATA AVAILABILITY STATEMENT

Cryo-EM densities, including sharpened and unsharpened maps, both half-maps, and the mask used for final FSC calculation, have been deposited in the EMDB at accession numbers EMD-50031 (closed), EMD-50030 (open FFC-8), EMD-50195 (open BrAmp), and EMD-50194 (open pH 9). Coordinates of cryo-EM and X-ray models have been deposited in the PDB at accession numbers 9EX6 (closed), 9EWL (open BrAmp, X-ray), 9EWA (open BrPEA, X-ray), 9F5O (open BrAmp, cryo-EM), 9EX4 (open FFC-8, cryo-EM), and 9F5N (open pH 9, cryo-EM).

# DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the **BJP** guidelines for **Design and Analysis** and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

## ORCID

Rebecca J. Howard https://orcid.org/0000-0003-2049-3378

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## SUPPORTING INFORMATION

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