High-Throughput Venomics.

In this study, we present high-throughput (HT) venomics, a novel analytical strategy capable of performing a full proteomic analysis of a snake venom within 3 days. This methodology comprises a combination of RP-HPLC-nanofractionation analytics, mass spectrometry analysis, automated in-solution tryptic digestion, and high-throughput proteomics. In-house written scripts were developed to process all the obtained proteomics data by first compiling all Mascot search results for a single venom into a single Excel sheet. Then, a second script plots each of the identified toxins in so-called Protein Score Chromatograms (PSCs). For this, for each toxin, identified protein scores are plotted on the y-axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x-axis. These PSCs allow correlation with parallel acquired intact toxin MS data. This same script integrates the PSC peaks from these chromatograms for semiquantitation purposes. This new HT venomics strategy was performed on venoms from diverse medically important biting species; Calloselasma rhodostoma, Echis ocellatus, Naja pallida, Bothrops asper, Bungarus multicinctus, Crotalus atrox, Daboia russelii, Naja naja, Naja nigricollis, Naja mossambica, and Ophiophagus hannah. Our data suggest that high-throughput venomics represents a valuable new analytical tool for increasing the throughput by which we can define venom variation and should greatly aid in the future development of new snakebite treatments by defining toxin composition.

The structural basis for an on-off switch controlling Gßγ-mediated inhibition of TRPM3 channels.

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gßγ proteins exert on TRPM3 channels.

Acetylcholine-Binding Protein Affinity Profiling of Neurotoxins in Snake Venoms with Parallel Toxin Identification.

Snakebite is considered a concerning issue and a neglected tropical disease. Three-finger toxins (3FTxs) in snake venoms primarily cause neurotoxic effects since they have high affinity for nicotinic acetylcholine receptors (nAChRs). Their small molecular size makes 3FTxs weakly immunogenic and therefore not appropriately targeted by current antivenoms. This study aims at presenting and applying an analytical method for investigating the therapeutic potential of the acetylcholine-binding protein (AChBP), an efficient nAChR mimic that can capture 3FTxs, for alternative treatment of elapid snakebites. In this analytical methodology, snake venom toxins were separated and characterised using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and high-throughput venomics. By subsequent nanofractionation analytics, binding profiling of toxins to the AChBP was achieved with a post-column plate reader-based fluorescence-enhancement ligand displacement bioassay. The integrated method was established and applied to profiling venoms of six elapid snakes (Naja mossambica, Ophiophagus hannah, Dendroaspis polylepis, Naja kaouthia, Naja haje and Bungarus multicinctus). The methodology demonstrated that the AChBP is able to effectively bind long-chain 3FTxs with relatively high affinity, but has low or no binding affinity towards short-chain 3FTxs, and as such provides an efficient analytical platform to investigate binding affinity of 3FTxs to the AChBP and mutants thereof and to rapidly identify bound toxins.

Regulation of a pentameric ligand-gated ion channel by a semiconserved cationic lipid-binding site.

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure-function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABAA receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABAA receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs.

A lipid site shapes the agonist response of a pentameric ligand-gated ion channel.

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.

Structure of a transporter domain emerges.

Magnesium homeostasis relies on transporters like the CNNM family, but little information on these proteins' structure and regulation limits our understanding of their biology and functions in disease. New characterization of a conserved cytoplasmic domain now confirms the presence of a self-liganded architecture that is indispensable for Mg2+ efflux and suggests a possible role for a dimeric assembly.

An allosteric binding site of the α7 nicotinic acetylcholine receptor revealed in a humanized acetylcholine-binding protein.

Nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric ligand-gated ion channels and mediate fast excitatory transmission in the central and peripheral nervous systems. Among the different existing receptor subtypes, the homomeric α7 nAChR has attracted considerable attention because of its possible implication in several neurological and psychiatric disorders, including cognitive decline associated with Alzheimer's disease or schizophrenia. Allosteric modulators of ligand-gated ion channels are of particular interest as therapeutic agents, as they modulate receptor activity without affecting normal fluctuations of synaptic neurotransmitter release. Here, we used X-ray crystallography and surface plasmon resonance spectroscopy of α7-acetylcholine-binding protein (AChBP), a humanized chimera of a snail AChBP, which has 71% sequence similarity with the extracellular ligand-binding domain of the human α7 nAChR, to investigate the structural determinants of allosteric modulation. We extended previous observations that an allosteric site located in the vestibule of the receptor offers an attractive target for receptor modulation. We introduced seven additional humanizing mutations in the vestibule-located binding site of AChBP to improve its suitability as a model for studying allosteric binding. Using a fragment-based screening approach, we uncovered an allosteric binding site located near the ß8-ß9 loop, which critically contributes to coupling ligand binding to channel opening in human α7 nAChR. This work expands our understanding of the topology of allosteric binding sites in AChBP and, by extrapolation, in the human α7 nAChR as determined by electrophysiology measurements. Our insights pave the way for drug design strategies targeting nAChRs involved in ion channel-mediated disorders.

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine.

Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the ß8-ß9 loop in the extracellular ligand-binding domain. The ß8-ß9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the ß8-ß9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.

Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel.

KEY POINTS: Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na+ , K+ and Ca2+ channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K+ channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. ABSTRACT: Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K+ channels, in which mutations in S4 can create an analogous 'omega' pore, we performed site-directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non-canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway.

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.

Family of prokaryote cyclic nucleotide-modulated ion channels.

Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24-32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K(+) ions over Na(+) ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.

Different ligands of the TRPV3 cation channel cause distinct conformational changes as revealed by intrinsic tryptophan fluorescence quenching.

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.

Structure-function elucidation of a new α-conotoxin, Lo1a, from Conus longurionis.

α-Conotoxins are peptide toxins found in the venom of marine cone snails and potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs are cholinergic receptors forming ligand-gated ion channels in the plasma membranes of certain neurons and the neuromuscular junction. Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson disease, and Alzheimer disease. To expand the knowledge concerning cone snail toxins, we examined the venom of Conus longurionis. We isolated an 18-amino acid peptide named α-conotoxin Lo1a, which is active on nAChRs. To the best of our knowledge, this is the first characterization of a conotoxin from this species. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs expressed in Xenopus laevis oocytes. The three-dimensional solution structure of the α-conotoxin Lo1a was determined by NMR spectroscopy. Lo1a, a member of the α4/7 family, blocks the response to acetylcholine in oocytes expressing α7 nAChRs with an IC50 of 3.24 ± 0.7 µM. Furthermore, Lo1a shows a high selectivity for neuronal versus muscle subtype nAChRs. Because Lo1a has an unusual C terminus, we designed two mutants, Lo1a-ΔD and Lo1a-RRR, to investigate the influence of the C-terminal residue. Lo1a-ΔD has a C-terminal Asp deletion, whereas in Lo1a-RRR, a triple-Arg tail replaces the Asp. They blocked the neuronal nAChR α7 with a lower IC50 value, but remarkably, both adopted affinity for the muscle subtype α1ß1δε.

Structural basis of ligand recognition in 5-HT3 receptors.

The 5-HT(3) receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT(3) receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation-π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT(3) receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT(3) receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT(3) receptor.

Molecular actions of smoking cessation drugs at α4ß2 nicotinic receptors defined in crystal structures of a homologous binding protein.

Partial agonists of the α4ß2 nicotinic acetylcholine receptor (nAChR), such as varenicline, are therapeutically used in smoking cessation treatment. These drugs derive their therapeutic effect from fundamental molecular actions, which are to desensitize α4ß2 nAChRs and induce channel opening with higher affinity, but lower efficacy than a full agonist at equal receptor occupancy. Here, we report X-ray crystal structures of a unique acetylcholine binding protein (AChBP) from the annelid Capitella teleta, Ct-AChBP, in complex with varenicline or lobeline, which are both partial agonists. These structures highlight the architecture for molecular recognition of these ligands, indicating the contact residues that potentially mediate their molecular actions in α4ß2 nAChRs. We then used structure-guided mutagenesis and electrophysiological recordings to pinpoint crucial interactions of varenicline with residues on the complementary face of the binding site in α4ß2 nAChRs. We observe that residues in loops D and E are molecular determinants of desensitization and channel opening with limited efficacy by the partial agonist varenicline. Together, this study analyzes molecular recognition of smoking cessation drugs by nAChRs in a structural context.

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines.

GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA(A) receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

Multisite binding of a general anesthetic to the prokaryotic pentameric Erwinia chrysanthemi ligand-gated ion channel (ELIC).

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABA(A/C) receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the ß7-ß10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT(3)R.

Affinity comparison of p3 and p8 peptide displaying bacteriophages using surface plasmon resonance.

Ever increasing demands in sensitivity and specificity of biosensors have recently established a trend toward the use of multivalent bioreceptors. This trend has also been introduced in the field of bacteriophage affinity peptides, where the entire phage is used as a receptor rather than the individual peptides. Although this approach is gaining in popularity due to the numerous advantages, binding kinetics of complete phage particles have never been studied in detail, notwithstanding being essential for the efficient design of such applications. In this paper we used an in house developed fiber-optic surface plasmon resonance (FO-SPR) biosensor to study the affinity and binding kinetics of phages, displaying peptide libraries. By using either peptide expression on the p3 or on the p8 coat proteins, a corresponding density of 5 up to more than 2000 peptides on a single virus particle was obtained. Binding parameters of 26 different filamentous phages, displaying peptides selective for enhanced Green Fluorescent Protein (eGFP), were characterized. This study revealed a broad affinity range of phages for the target eGFP, indicating their potential to be used for applications with different requirements in binding kinetics. Moreover, detailed analysis of koff and kon values of several selected p3 and p8 phages, using the FO-SPR biosensor, clearly showed the correlation between the binding parameters and the density at which eGFP-peptides are being expressed. Consequently, although p3 and p8-based phages both revealed exceptionally high affinities for eGFP, two p8 phages were found to have the highest affinity with dissociation constants (Kd) in the femtomolar range.

Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor.

Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the cryo-EM structure of the lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators.