Signal transduction through Cys-loop receptors is mediated by the nonspecific bumping of closely apposed domains.

One of the most fundamental questions in the field of Cys-loop receptors (pentameric ligand-gated ion channels, pLGICs) is how the affinity for neurotransmitters and the conductive/nonconductive state of the transmembrane pore are correlated despite the ∼60-Šdistance between the corresponding domains. Proposed mechanisms differ, but they all converge into the idea that interactions between wild-type side chains across the extracellular-transmembrane-domain (ECD-TMD) interface are crucial for this phenomenon. Indeed, the successful design of fully functional chimeras that combine intact ECD and TMD modules from different wild-type pLGICs has commonly been ascribed to the residual conservation of sequence that exists at the level of the interfacial loops even between evolutionarily distant parent channels. Here, using mutagenesis, patch-clamp electrophysiology, and radiolabeled-ligand binding experiments, we studied the effect of eliminating this residual conservation of sequence on ion-channel function and cell-surface expression. From our results, we conclude that proper state interconversion ("gating") does not require conservation of sequence-or even physicochemical properties-across the ECD-TMD interface. Wild-type ECD and TMD side chains undoubtedly interact with their surroundings, but the interactions between them-straddling the interface-do not seem to be more important for gating than those occurring elsewhere in the protein. We propose that gating of pLGICs requires, instead, that the overall structure of the interfacial loops be conserved, and that their relative orientation and distance be the appropriate ones for changes in one side to result in changes in the other, in a phenomenon akin to the nonspecific "bumping" of closely apposed domains.

Structural titration of receptor ion channel GLIC gating by HS-AFM.

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein-protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors.

Cys-loop receptors are neurotransmitter-gated ion channels that are essential mediators of fast chemical neurotransmission and are associated with a large number of neurological diseases and disorders, as well as parasitic infections. Members of this ion channel superfamily mediate excitatory or inhibitory neurotransmission depending on their ligand and ion selectivity. Structural information for Cys-loop receptors comes from several sources including electron microscopic studies of the nicotinic acetylcholine receptor, high-resolution X-ray structures of extracellular domains and X-ray structures of bacterial orthologues. In 2011 our group published structures of the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in complex with the allosteric partial agonist ivermectin, which provided insights into the structure of a possibly open state of a eukaryotic Cys-loop receptor, the basis for anion selectivity and channel block, and the mechanism by which ivermectin and related molecules stabilize the open state and potentiate neurotransmitter binding. However, there remain unanswered questions about the mechanism of channel opening and closing, the location and nature of the shut ion channel gate, the transitions between the closed/resting, open/activated and closed/desensitized states, and the mechanism by which conformational changes are coupled between the extracellular, orthosteric agonist binding domain and the transmembrane, ion channel domain. Here we present two conformationally distinct structures of C. elegans GluCl in the absence of ivermectin. Structural comparisons reveal a quaternary activation mechanism arising from rigid-body movements between the extracellular and transmembrane domains and a mechanism for modulation of the receptor by phospholipids.

A Crucial Role for Side-Chain Conformation in the Versatile Charge Selectivity of Cys-Loop Receptors.

Whether synaptic transmission is excitatory or inhibitory depends, to a large extent, on whether the ion channels that open upon binding the released neurotransmitter conduct cations or anions. The mechanistic basis of the opposite charge selectivities of Cys-loop receptors has only recently begun to emerge. It is now clear that ionized side chains-whether pore-facing or buried-in the first α-helical turn of the second transmembrane segments underlie this phenomenon and that the electrostatics of backbone atoms are not critically involved. Moreover, on the basis of electrophysiological observations, it has recently been suggested that not only the sign of charged side chains but also their conformation are crucial determinants of cation-anion selectivity. To challenge these ideas with the chemical and structural rigor that electrophysiological observations naturally lack, we performed molecular dynamics, Brownian dynamics, and electrostatics calculations of ion permeation. To this end, we used structural models of the open-channel conformation of the α1 glutamate-gated Cl- channel and the α1 glycine receptor. Our results provided full support to the notion that the conformation of charged sides chains matters for charge selectivity. Indeed, whereas some rotamers of the buried arginines at position 0' conferred high selectivity for anions, others supported the permeation of cations and anions at similar rates or even allowed the faster permeation of cations. Furthermore, we found that modeling glutamates at position -1' of the anion-selective α1 glycine receptor open-state structure-instead of the five native alanines-switches charge selectivity also in a conformation-dependent manner, with some glutamate rotamers being much more effective at conferring selectivity for cations than others. Regarding pore size, we found that the mere expansion of the pore has only a minimal impact on cation-anion selectivity. Overall, these results bring to light the previously unappreciated impact of side-chain conformation on charge selectivity in Cys-loop receptors.

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.

Progress in the discovery of small molecule modulators of the Cys-loop superfamily receptors.

The vertebrate Cys-loop family of ligand-gated ion channels (LGICs) are comprised of nicotinic acetylcholine (nAChR), serotonin type 3 (5-HT3R), γ-aminobutyric acid (GABAAR), and glycine (GlyR) receptors. Here, we review efforts to discover selective small molecules targeting one or more Cys-loop receptors, with a focus on state-of-the-art modulators that have been reported over the past five years. Several highlighted compounds offer robust oral bioavailability and central exposure and have thus been useful in delineating pharmacokinetic/pharmacodynamic relationships in pre-clinical disease models. Others offer high levels of subtype and/or inter-superfamily selectivity and have facilitated understanding of complex SAR and pharmacodynamics.

Atomistic insights into human Cys-loop receptors by solution NMR.

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) mediating fast neurotransmission in the central and peripheral nervous systems. They are important targets for many currently used clinical drugs, such as general anesthetics, and for allosteric modulators with potential therapeutic applications. Here, we provide an overview of advances in the use of solution NMR in structural and dynamic characterization of ion channels, particularly human Cys-loop receptors. We present challenges to overcome and realistic solutions for achieving high-resolution structural information for this family of receptors. We discuss how subtle structural differences among homologous channels define unique channel pharmacological properties and advocate the necessity to determine high-resolution structures for individual receptor subtypes. Finally, we describe drug binding to the TMDs of Cys-loop receptors identified by solution NMR and the associated dynamics changes relevant to channel functions.

Probing residues in the pore-forming (M2) domain of the Cys-loop receptor homologue GLIC reveals some unusual features.

Cys-loop receptors play important roles in signal transduction. The Gloeobacter ligand-gated ion channel (GLIC) pore binds similar compounds to Cys-loop receptor pores, but has the advantage of known structures in open and closed states. GLIC is activated by protons with a pEC50 of 5.4, and has a histidine residue (His 11') in its pore-forming α-helix (M2) which is involved in gating. Here we explore the role of this His and other M2 residues using two-electrode voltage clamp of mutant receptors expressed in oocytes. We show that 11'His is very sensitive to substitution; replacement with a range of amino acids ablates function. Similarly altering its location in M2 to the 8', 9', 10', 12', 13' or 14' positions ablated function. Most substitutions of Ser6' or Ile9' were also non-functional, although not Ile9'Leu and Ile9'Val. Unexpectedly, an Ile9'His substitution was constitutively active at pH 7, but closed as [H+] increased, with a pIC50 of 5.8. Substitution at 2', 5' and 7' had little effect on pEC50. Overall the data show Ser6' and His11' are critical for the function of the receptor, and thus distinguish the roles of these M2 residues from those of Cys-loop receptors, where substitutions are mostly well tolerated. These data suggest modellers should be aware of these atypical features when using the GLIC pore as a model for Cys-loop receptor pores.

Interaction of three-finger proteins from snake venoms and from mammalian brain with the cys-loop receptors and their models.

With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K D = 1.3 µM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.

Cysteine Modification: Probing Channel Structure, Function and Conformational Change.

Cysteine substitution has been a powerful tool to investigate the structure and function of proteins. It has been particularly useful for studies of membrane proteins in their native environment, embedded in phospholipid membranes. Among the 20 amino acids, cysteine is uniquely reactive. This reactivity has motivated the synthesis of a wide array of sulfhydryl reactive chemicals. The commercially available array of sulfhydryl reactive reagents has allowed investigators to probe the local steric and electrostatic environment around engineered cysteines and to position fluorescent, paramagnetic and mass probes at specific sites within proteins and for distance measurements between pairs of sites. Probing the reactivity and accessibility of engineered cysteines has been extensively used in Substituted Cysteine Accessibility Method (SCAM) investigations of ion channels, membrane transporters and receptors. These studies have successfully identified the residues lining ion channels, agonist/antagonist and allosteric modulator binding sites, and regions whose conformation changes as proteins transition between different functional states. The thousands of cysteine-substitution mutants reported in the literature demonstrate that, in general, mutation to cysteine is well tolerated. This has allowed systematic studies of residues in transmembrane segments and in other parts of membrane proteins. Finally, by inserting pairs of cysteines and assaying their ability to form disulfide bonds, changes in proximity and mobility relationships between specific positions within a protein can be inferred. Thus, cysteine mutagenesis has provided a wealth of data on the structure of membrane proteins in their functional environment. This data can complement the structural insights obtained from the burgeoning number of crystal structures of detergent solubilized membrane proteins whose functional state is often uncertain. This article will review the use of cysteine mutagenesis to probe structure-function relationships in ion channels focusing mainly on Cys-loop receptors.

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

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.

Introduction to thematic minireview series on celebrating the discovery of the cysteine loop ligand-gated ion channel superfamily.

The year 2012 marks the 25th anniversary of the discovery of the Cys loop ligand-gated ion channel superfamily of neurotransmitter receptors. This minireview series celebrates this with a series of articles reviewing current information for each of the family members, nicotinic acetylcholine receptors, glycine receptors, GABA(A) receptors, serotonin-3 (5-HT(3)) receptors, and glutamate-gated chloride ion channels of proteasome invertebrate phyla.

The nicotinic acetylcholine receptor: the founding father of the pentameric ligand-gated ion channel superfamily.

A critical event in the history of biological chemistry was the chemical identification of the first neurotransmitter receptor, the nicotinic acetylcholine receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and rational manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The nicotinic receptor has become the founding father of a broad family of pentameric membrane receptors, paving the way for their identification, including that of the GABA(A) receptors.

General anesthetics predicted to block the GLIC pore with micromolar affinity.

Although general anesthetics are known to modulate the activity of ligand-gated ion channels in the Cys-loop superfamily, there is at present neither consensus on the underlying mechanisms, nor predictive models of this modulation. Viable models need to offer quantitative assessment of the relative importance of several identified anesthetic binding sites. However, to date, precise affinity data for individual sites has been challenging to obtain by biophysical means. Here, the likely role of pore block inhibition by the general anesthetics isoflurane and propofol of the prokaryotic pentameric channel GLIC is investigated by molecular simulations. Microscopic affinities are calculated for both single and double occupancy binding of isoflurane and propofol to the GLIC pore. Computations are carried out for an open-pore conformation in which the pore is restrained to crystallographic radius, and a closed-pore conformation that results from unrestrained molecular dynamics equilibration of the structure. The GLIC pore is predicted to be blocked at the micromolar concentrations for which inhibition by isofluorane and propofol is observed experimentally. Calculated affinities suggest that pore block by propofol occurs at signifcantly lower concentrations than those for which inhibition is observed: we argue that this discrepancy may result from binding of propofol to an allosteric site recently identified by X-ray crystallography, which may cause a competing gain-of-function effect. Affinities of isoflurane and propofol to the allosteric site are also calculated, and shown to be 3 mM for isoflurane and 10 µM for propofol; both anesthetics have a lower affinity for the allosteric site than for the unoccupied pore.

Molecular mechanism for the dual alcohol modulation of Cys-loop receptors.

Cys-loop receptors constitute a superfamily of pentameric ligand-gated ion channels (pLGICs), including receptors for acetylcholine, serotonin, glycine and γ-aminobutyric acid. Several bacterial homologues have been identified that are excellent models for understanding allosteric binding of alcohols and anesthetics in human Cys-loop receptors. Recently, we showed that a single point mutation on a prokaryotic homologue (GLIC) could transform it from a channel weakly potentiated by ethanol into a highly ethanol-sensitive channel. Here, we have employed molecular simulations to study ethanol binding to GLIC, and to elucidate the role of the ethanol-enhancing mutation in GLIC modulation. By performing 1-µs simulations with and without ethanol on wild-type and mutated GLIC, we observed spontaneous binding in both intra-subunit and inter-subunit transmembrane cavities. In contrast to the glycine receptor GlyR, in which we previously observed ethanol binding primarily in an inter-subunit cavity, ethanol primarily occupied an intra-subunit cavity in wild-type GLIC. However, the highly ethanol-sensitive GLIC mutation significantly enhanced ethanol binding in the inter-subunit cavity. These results demonstrate dramatic effects of the F(14')A mutation on the distribution of ligands, and are consistent with a two-site model of pLGIC inhibition and potentiation.

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.

Asymmetric gating of a human hetero-pentameric glycine receptor.

Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite intense investigations into their working mechanism and pharmaceutical potentials, how neurotransmitters activate these receptors remains unclear due to the lack of high-resolution structural information in the activated open state. Here we report near-atomic resolution structures resolved in digitonin consistent with all principle functional states of the human α1ß GlyR, which is a major Cys-loop receptor that mediates inhibitory neurotransmission in the central nervous system of adults. Glycine binding induces cooperative and symmetric structural rearrangements in the neurotransmitter-binding extracellular domain but asymmetrical pore dilation in the transmembrane domain. Symmetric response in the extracellular domain is consistent with electrophysiological data showing cooperative glycine activation and contribution from both α1 and ß subunits. A set of functionally essential but differentially charged amino acid residues in the transmembrane domain of the α1 and ß subunits explains asymmetric activation. These findings provide a foundation for understanding how the gating of the Cys-loop receptor family members diverges to accommodate specific physiological environments.

The structural basis of function in Cys-loop receptors.

Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT3, GABAA and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT3) and some are anion selective (e.g. GABAA and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience.In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them.

Cys-loop receptor channel blockers also block GLIC.

The Gloeobacter ligand-gated ion channel (GLIC) is a bacterial homolog of vertebrate Cys-loop ligand-gated ion channels. Its pore-lining region in particular has a high sequence homology to these related proteins. Here we use electrophysiology to examine a range of compounds that block the channels of Cys-loop receptors to probe their pharmacological similarity with GLIC. The data reveal that a number of these compounds also block GLIC, although the pharmacological profile is distinct from these other proteins. The most potent compound was lindane, a GABA(A) receptor antagonist, with an IC50 of 0.2 µM. Docking studies indicated two potential binding sites for this ligand in the pore, at the 9' or between the 0' and 2' residues. Similar experiments with picrotoxinin (IC50 = 2.6 µM) and rimantadine (IC50 = 2.6 µM) reveal interactions with 2'Thr residues in the GLIC pore. These locations are strongly supported by mutagenesis data for picrotoxinin and lindane, which are less potent in a T2'S version of GLIC. Overall, our data show that the inhibitory profile of the GLIC pore has considerable overlap with those of Cys-loop receptors, but the GLIC pore has a unique pharmacology.