Cys-loop Receptor Superfamily Research Articles

Evolutionary conservation of Zinc-Activated Channel (ZAC) functionality in mammals: a range of mammalian ZACs assemble into cell surface-expressed functional receptors.

In contrast to the other pentameric ligand-gated ion channels in the Cys-loop receptor superfamily, the ZACN gene encoding for the Zinc-Activated Channel (ZAC) is exclusively found in the mammalian genome. Human ZAC assembles into homomeric cation-selective channels gated by Zn2+, Cu2+ and H+, but the function of the receptor in human physiology is presently poorly understood. In this study, the degree of evolutionary conservation of a functional ZAC in mammals was probed by investigating the abilities of a selection of ZACs from 10 other mammalian species than human to be expressed at the protein level and assemble into cell surface-expressed functional receptors in mammalian cells and in Xenopus oocytes. In an enzyme-linked immunosorbent assay, transient transfections of tsA201 cells with cDNAs of hemagglutinin (HA)-epitope-tagged versions of these 10ZACs resulted in robust total expression and cell surface expression levels of all proteins. Moreover, injection of cRNAs for 6 of these ZACs in oocytes resulted in the formation of functional receptors in two-electrode voltage-clamp recordings. The ZACs exhibited robust current amplitudes in response to Zn2+ (10mM) and H+ (pH 4.0), and the concentration-response relationships displayed by Zn2+ at these channels were largely comparable to that at human ZAC. In conclusion, the findings suggest that the functionality of ZAC at the molecular level may be conserved throughout mammalian species, and that the channel thus may govern physiological functions in mammals, including humans.

Diverse structural mechanisms of muscle-type nicotinic receptor antagonism

Nicotinic acetylcholine receptors are pentameric neurotransmitter-gated ion channels and are the archetypal members of the Cys-loop receptor superfamily. The nicotinic receptor subtype present in muscle fibers mediates the fast communication between motor neurons and skeletal muscles. This subtype is an important therapeutic target for myasthenic disorders and for neuromuscular blockers used during clinical anesthesia. However, our understanding of how this receptor is modulated is limited by a lack of high-resolution structural information.

Database of glutamate-gated chloride (GluCl) subunits across 125 nematode species: patterns of gene accretion and sequence diversification.

Glutamate-gated chloride channels belong to the Cys-loop receptor superfamily. Glutamate-gated chloride channels are activated by glutamate and form substrates for the antiparasitic drugs from the avermectin family. Glutamate-gated chloride channels are pentameric, and each subunit contains an N-terminal extracellular domain that binds glutamate and 4 helical transmembrane domains, which contain binding sites for avermectin drugs. In order to provide more insight into phylum-wide patterns of glutamate-gated chloride subunit gene expansion and sequence diversity across nematodes, we have developed a database of predicted glutamate-gated chloride subunit genes from 125 nematode species. Our analysis into this dataset described assorted patterns of species-specific glutamate-gated chloride gene counts across different nematodes as well as sequence diversity in key residues thought to be involved in avermectin binding.

Discovery and functional characterization of N-(thiazol-2-yl)-benzamide analogs as the first class of selective antagonists of the Zinc-Activated Channel (ZAC)

The Zinc-Activated Channel (ZAC) is an atypical member of the Cys-loop receptor (CLR) superfamily of pentameric ligand-gated ion channels, with its very different endogenous agonists and signalling properties. In this study, a compound library screening at ZAC resulted in the identification of 2-(5-bromo-2-chlorobenzamido)-4-methylthiazole-5-methyl ester (1) as a novel ZAC antagonist. The structural determinants for ZAC activity in 1 were investigated by functional characterization of 61 analogs at ZAC expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology, and couple of analogs exerting more potent ZAC inhibition than 1 were identified (IC50 values: 1–3 μM). 1 and N-(4-(tert-butyl)thiazol-2-yl)-3-fluorobenzamide (5a, TTFB) were next applied in studies of the functional properties and the mode of action of this novel class of ZAC antagonists. TTFB was a roughly equipotent antagonist of Zn+- and H+-evoked ZAC signaling and of spontaneous ZAC activity, and the slow on-set of its channel block suggested that its ZAC inhibition is state-dependent. TTFB was found to be a selective ZAC antagonist, exhibiting no significant agonist, antagonist or modulatory activity at 5-HT3A, α3β4 nicotinic acetylcholine, α1β2γ2S GABAA or α1 glycine receptors at 30 μM. 1 displayed largely non-competitive antagonism of Zn2+-induced ZAC signalling, and TTFB was demonstrated to target the transmembrane and/or intracellular domains of the receptor, which collectively suggests that the N-(thiazol-2-yl)-benzamide analog acts a negative allosteric modulator of ZAC. We propose that this first class of selective ZAC antagonists could constitute useful pharmacological tools in future explorations of the presently poorly elucidated physiological functions governed by this CLR.

Delineation of the functional properties exhibited by the Zinc-Activated Channel (ZAC) and its high-frequency Thr128Ala variant (rs2257020) in Xenopus oocytes

The signalling characteristics of the Zinc-Activated Channel (ZAC), a member of the Cys-loop receptor (CLR) superfamily, are presently poorly elucidated. The ZACN polymorphism c.454G>A encoding for the Thr128Ala variation in ZAC is found in extremely high allele frequencies across ethnicities. In this, the first study of ZAC in Xenopus oocytes by TEVC electrophysiology, ZACThr128 and ZACAla128 exhibited largely comparable pharmacological and signalling characteristics, but interestingly the Zn2+- and H+-evoked current amplitudes in ZACAla128-oocytes were dramatically smaller than those in ZACThr128-oocytes. While the variation thus appeared to impact cell surface expression and/or channel properties of ZAC, the similar expression properties exhibited by ZACThr128 and ZACAla128 in transfected mammalian cells indicated that their distinct functionalities could arise from the latter. In co-expression experiments, wild-type and variant ZAC subunits assembled efficiently into “heteromeric” complexes in HEK293 cells, while the concomitant presence of ZACAla128 in ZACThr128:ZACAla128-oocytes did not exert a dominant negative effect on agonist-evoked current amplitudes compared to those in ZACThr128-oocytes. Finally, the structural determinants of the functional importance of the 1-hydroxyethyl side-chain of Thr128 appeared to be subtle, as agonist-evoked current amplitudes in ZACSer128-, ZACVal128- and ZACIle128-oocytes also were substantially lower than those in ZACThr128-oocytes. In conclusion, the functional properties exhibited by ZAC in this work substantiate the notion of it being an atypical CLR. While the impact of the Thr128Ala variation on ZAC functionality in oocytes is striking, it remains to be investigated whether and to which extent this translates into an in vivo setting and thus could constitute a source of inter-individual variation in ZAC physiology.

Structure and gating mechanism of the α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.

Sleep-related hypermotor epilepsy associated mutations uncover important kinetic roles of α4β2- nicotinic acetylcholine receptor intracellular structures

Sleep-related hypermotor epilepsy (SHE) is a group of seizure disorders prominently associated with mutations in nicotinic acetylcholine receptors (nAChR). The most prevalent central nervous system nAChR subtype contains α4 and β2 subunits, in two ratios. (α4β2)2β2-nAChR have high agonist sensitivity (HS-isoform), whereas (α4β2)2α4-nAChR agonist responses exhibit a small high-sensitivity, and a predominant low-sensitivity, phase of function (LS-isoform). Multiple non-synonymous mutations in the second and third transmembrane domains of α4 and β2 subunits are associated with SHE. We recently demonstrated that two additional, SHE-associated, missense mutations in the major cytoplasmic loops of these subunits [α4(R336H) and β2(V337G)] cause increased macroscopic function-per receptor. Here, we use single-channel patch-clamp electrophysiology to show that these mutations influence single-channel amplitudes and open- and closed-state kinetics. Pure populations of HS- or LS-isoform α4β2-nAChR were expressed by injecting either 1:10 or 30:1 α4:β2 cRNA ratios, respectively, into Xenopus laevis oocytes. Functional properties of the resulting mutant α4β2-nAChR isoforms were compared to their wildtype counterparts. α4(R336H) subunit incorporation minimally affected single-channel amplitudes, whereas β2(V337G) subunit incorporation reduced them significantly in both isoforms. However, for both mutant subunits, increased function-per-receptor was predominantly caused by altered single channel kinetics. The α4(R336H) mutation primarily destabilizes desensitized states between openings. By contrast, the β2(V337G) mutation principally stabilizes receptor open states. The use of naturally-occurring and physiologically-impactful mutations has allowed us to define valuable new insights regarding the functional roles of nAChR intracellular domains. Further mechanistic context is provided by intracellular-domain structures recently published for other members of the Cys-loop receptor superfamily (α3β4-nAChR and 5-HT3AR).

A Computational Analysis of the Factors Governing the Dynamics of α7 nAChR and Its Homologs

The α7 nicotinic acetylcholine receptor is a homopentameric ion channel from the Cys-loop receptor superfamily targeted for psychiatric indications and inflammatory pain. Molecular dynamics studies of the receptor have focused on residue mobility and global conformational changes to address receptor function. However, a comparative analysis of α7 with its homologs that cannot trigger channel opening has not been made so far. To identify the residues involved in α7 activation, we ran triplicate 500-ns molecular dynamics simulations with an α7 extracellular domain homology model and two acetylcholine-binding protein homologs. We tested the effect of ligand binding and amino acid sequence on the structure and dynamics of the three proteins. We found that mobile regions identified based on root mean-square deviation and root mean-square fluctuation values are not always consistent among the individual α7 extracellular domain simulations. Comparison of the replica-average properties of the three proteins based on dynamic cross-correlation maps showed that ligand binding affects the coupling between the C-loop and the Cys-loop, vestibular loop, and β1–β2 loops. In addition, the main-immunogenic-region-like domain of α7 went through correlated motions with multiple domains of the receptor. These correlated motions were absent or diminished in α7 homologs, suggesting a unique role in α7 activation.

Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABAA receptor in complex with GABA.

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABAA receptors are heteromeric assemblies sensitive to many important drugs, from sedatives to anesthetics and anticonvulsant agents, with mutant forms of GABAA receptors implicated in multiple neurological diseases. Despite the profound importance of heteromeric GABAA receptors in neuroscience and medicine, they have proven recalcitrant to structure determination. Here we present the structure of a tri-heteromeric α1β1γ2SEM GABAA receptor in complex with GABA, determined by single particle cryo-EM at 3.1-3.8 Å resolution, elucidating molecular principles of receptor assembly and agonist binding. Remarkable N-linked glycosylation on the α1 subunit occludes the extracellular vestibule of the ion channel and is poised to modulate receptor assembly and perhaps ion channel gating. Our work provides a pathway to structural studies of heteromeric GABAA receptors and a framework for rational design of novel therapeutic agents.

Structure and Mechanism of Neuronal Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors are ligand gated ion channels that mediate fast chemical neurotransmission at the neuromuscular junction and play diverse signaling roles in the central nervous system. Here we describe a biochemical approach for characterization of subunit stoichiometry in heteromeric membrane proteins and present the first X-ray crystal structure of a nicotinic receptor. The α4β2 nicotinic receptor is the most abundant receptor subtype in the brain, is the principal target in nicotine addiction and its dysfunction is associated with familial epilepsy. The structure of the receptor in complex with the agonist nicotine reveals principles of ligand selectivity among different classes of subunit interfaces in the heteropentameric assembly. The receptor is stabilized by nicotine in a non-conducting, desensitized conformation. The constriction point in the permeation pathway is formed at the selectivity filter, located at the cytosolic end of the pore. The desensitized state of this channel provides a distinct structural reference point in the allosteric gating cycle of the larger Cys-loop receptor superfamily.

Activation and modulation of the nicotinic receptor

Nicotinic acetylcholine receptors (AChRs) are the best characterized ion channels representing the Cys-loop receptor superfamily. AChRs have all the machinery to recognize the neurotransmitter ACh and other agonists such as nicotine, and to transduce the agonist-induced conformational changes into the opening of the intrinsic cation channel. The gating machinery couples ligand binding, located at the extracellular portion, to the opening of the ion channel, located at the transmembrane region. The interface between the extracellular and the transmembrane domains is considered one of the most important structural and functional features for the process of gating. And finally, in the prolonged presence of agonists, the AChR becomes desensitized. Several drugs affect the functioning of these receptors. Among them, positive allosteric modulators (PAM) have acquired importance since are novel drugs for several neurological diseases. PAMs do not bind to the orthostetic binding sites but allosterically enhance the activity elicited by agonists by increasing the gating process and/or by decreasing desensitization. Instead, negative allosteric modulators (NAMs) produce the opposite effects. Interestingly, this negative effect is similar to that found for another class of allosteric drugs, i.e. non competitive antagonists (NCAs). However, the main difference between both categories of drugs is based on their distinct binding site locations. Although both NAMs and NCAs do not bind to the agonist sites, NACs bind to sites located in the ion channel, whereas NAMs bind to nonluminal sites. Interestingly, PAMs and NAMs might be developed as potential medications for the treatment of several diseases involving AChRs including, dementia-, skin-, and immunological-related diseases, drug addiction, and cancer. More exciting is the potential combination of specific agonists with PAMs. However, we are still in the beginning of understanding how these compounds act and how these drugs can be used therapeutically.

Structure and function development during evolution of pentameric ligand gated ion channels

Ligand Gated Ion Channels (LGICs) are receptors widely involved in vertebrate neuronal and non-neuronal signalling as well as muscle activation. Until very recently these receptors, were only known to exist in animal cells and were named the Cys-loop receptor superfamily because they all share this sub-substructure in their amino acid sequences. Nowadays, these receptors are known as pentameric LGICs (pLGICs), to differentiate from other LGICs such us tetrameric glutamate or trimeric ATP LGICs. The first phylogenetic analyses of pLGICs have been performed almost 20 years ago, where only eukaryotic members were known. Currently, we have many more sequences from invertebrate species, but most importantly, we know that these receptors are present in a variety of prokaryotic organisms, a fact predicted in those early studies. Equally important, we have information about the tertiary and quaternary structures from prokaryotic to eukaryotic members, which enlightened our evolutionary understanding of these fascinating and functionally important proteins. Surprisingly, the prokaryotic receptors lack the Cys-loop characteristic of eukaryote receptors which once gave the name to the superfamily. This review focuses on the history of the evolutionary processes of these receptors involved in very important physiological functions as well as in many diseases such as tobacco addiction and schizophrenia, to name just a few.

Identification and characterization of heptapeptide modulators of the glycine receptor

The glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (α1, α2, α3, and β) co-assemble to form pentameric channel proteins as either α homomers or αβ heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the α1β glycine receptor. This involved a panning procedure in which the phage library initially underwent subtractive panning against Human Embryonic Kidney (HEK) 293 cells expressing alternative glycine receptor subtypes before panning the remaining library over HEK 293 cells expressing the target, the α1β glycine receptor. Peptides were identified that act with selectivity on α1β and α3β, compared to α2β, glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol's enhancing actions at the receptor in the absence of zinc. Comparisons of the amino acid sequences of heptapeptides capable of potentiating glycine receptor function revealed several consensus sequences that may be predictive of a peptide's enhancing ability.

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.

Copper and protons directly activate the zinc-activated channel

The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn2+) but also copper (Cu2+) and protons (H+) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H+>Cu2+>Zn2+ and H+>Zn2+>Cu2+, respectively. The responses elicited by Zn2+, Cu2+ and H+ through ZAC are all characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H+ than for the two metal ions. The permeabilities of ZAC for Na+ and K+ relative to Cs+ are indistinguishable, whereas replacing all of extracellular Na+ and K+ with the divalent cations Ca2+ or Mg2+ results in complete elimination of Zn2+-activated currents at both negative and positive holding potentials. This indicates that ZAC is non-selectively permeable to monovalent cations, whereas Ca2+ and Mg2+ inhibit the channel. In conclusion, this is the first report of a Cys-loop receptor being gated by Zn2+, Cu2+ and H+. ZAC could be an important mediator of some of the wide range of physiological functions regulated by or involving Zn2+, Cu2+ and H+.

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates

We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that δD140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the α-, β-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.

Glycine Receptor: Molecular Organization and Pathology

Glycine receptor is the anion-selective channel, providing fast synaptic transmission in the central nervous system of vertebrates. Together with the nicotinic acetylcholine, GABA, and serotonin (5-HT3) receptors, it belongs to the superfamily of pentameric cys-loop receptors. In this review, we briefly describe main functions of these transmembrane proteins, their distribution and molecular architecture. Special attention is paid to recent studies on the molecular physiology of these receptors, as well as on presenting of molecular domains responsible for their dysfunction.

GLYCINE RECEPTOR: MOLECULAR ORGANIZATION AND PATHOLOGY

Glycine receptor is the anion-selective channel, providing fast synaptic transmission in the central nervous system of vertebrates. Together with the nicotinic acetylcholine, GABA and serotonin (5-HT3R) receptors, it belongs to the superfamily of pentameric cys-loop receptors. In this review we briefly describe main functions of these transmembrane proteins, their distribution and molecular architecture. Special attention is paid to recent studies on the molecular physiology of these receptors, as well as on presenting of molecular domains responsible for their dysfunction.

Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors.

Mutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol (EtOH) potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced EtOH and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for EtOH binding to GLIC. We directly compared EtOH modulation of the α1 GlyR and GLIC to a chimera containing the TM domain from human α1 GlyRs and the ligand-binding domain of GLIC using 2-electrode voltage-clamp electrophysiology of receptors expressed in Xenopus laevis oocytes. EtOH potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the EtOH potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50μM picrotoxin to the media, a technique that may have applications in expression of other ion channels. Our results are consistent with a TM mechanism of EtOH modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species.

Structural Requirements in the Transmembrane Domain of GLIC Revealed by Incorporation of Noncanonical Histidine Analogs

The cyanobacterial pentameric ligand-gated ion channel GLIC, a homolog of the Cys-loop receptor superfamily, has provided useful structural and functional information about its eukaryotic counterparts. X-ray diffraction data and site-directed mutagenesis have previously implicated a transmembrane histidine residue (His234) as essential for channel function. Here, we investigated the role of His234 via synthesis and incorporation of histidine analogs and α-hydroxy acids using invivo nonsense suppression. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used to monitor channel activity. We show that an interhelix hydrogen bond involving His234 is important for stabilization of the open state, and that the shape and basicity of its side chain are highly sensitive to perturbations. In contrast, our data show that two other His residues are not involved in the acid-sensing mechanism.