Structural link between γ-aminobutyric acid type A (GABAA) receptor agonist binding site and inner β-sheet governs channel activation and allosteric drug modulation

Rapid opening and closing of pentameric ligand-gated ion channels (pLGICs) regulate information flow throughout the brain. For pLGICs, it is postulated that neurotransmitter-induced movements in the extracellular inner β-sheet trigger channel activation. Homology modeling reveals that the β4-β5 linker physically connects the neurotransmitter binding site to the inner β-sheet. Inserting 1, 2, 4, and 8 glycines in this region of the GABA(A) receptor β-subunit progressively decreases GABA activation and converts the competitive antagonist SR-95531 into a partial agonist, demonstrating that this linker is a key element whose length and flexibility are optimized for efficient signal propagation. Insertions in the α- and γ-subunits have little effect on GABA or SR-95531 actions, suggesting that asymmetric motions in the extracellular domain power pLGIC gating. The effects of insertions on allosteric modulator actions, pentobarbital, and benzodiazepines, have different subunit dependences, indicating that modulator-induced signaling is distinct from agonist gating.     

Desensitization mechanism in prokaryotic ligand-gated ion channel

Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.     

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals

The 5-HT₃A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT₃A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT₃A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.     

Hydrophobic and acidic moments of a nucleoplasmin NP-core chaperone

A recent crystal structure, at atomic resolution, of the NO38-core chaperone has revealed a decamer comprised of a dimer of pentamers, with each pentamer consisting of closely coupled eight-stranded beta-barrel monomers. This N-terminal core domain of the chaperone shares the Nucleoplasmin family fold and is presumed to assist the binding of the core histones in their assembly into nucleosomes during DNA replication and repair. The present work provides a measure of the hydrophobic residue burial about the different interfaces and centers of the NO38-core multimeric structure. While the hydrophobic "pentameric ring," comprised of the hydrophobic cores of the monomers and prevalence of non-polar residues at their interfaces is observed, a hydrophobic bias with respect to the center of the pentamer is also found, and consequently also expected to contribute to the thermostability of the multimer. Structural and chromatographic analysis had shown the NO38-core chaperone to bind (H3-H4)2 histone tetramers as well as H2A-H2B dimers. The acidic dipole, which reflects the spatial disposition of the acidic residues of the core monomer points to the lateral region of the monomers comprising the oligomers, and thereby, shows it to be the region of charge that would optimally complement the basic charge of the histones in their electrostatic binding to the chaperone. It is also pointed out that the prevalence of basic residues on the short helices of the histone cores also provides regions of charge that would complement histone binding to the chaperone.     

Contributions of conserved residues at the gating interface of glycine receptors

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.     

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.     

Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures

Nicotinic acetylcholine receptors are prototypes for the pharmaceutically important family of pentameric ligand-gated ion channels. Here we present atomic resolution structures of nicotine and carbamylcholine binding to AChBP, a water-soluble homolog of the ligand binding domain of nicotinic receptors and their family members, GABAA, GABAC, 5HT3 serotonin, and glycine receptors. Ligand binding is driven by enthalpy and is accompanied by conformational changes in the ligand binding site. Residues in the binding site contract around the ligand, with the largest movement in the C loop. As expected, the binding is characterized by substantial aromatic and hydrophobic contributions, but additionally there are close contacts between protein oxygens and positively charged groups in the ligands. The higher affinity of nicotine is due to a main chain hydrogen bond with the B loop and a closer packing of the aromatic groups. These structures will be useful tools for the development of new drugs involving nicotinic acetylcholine receptor-associated diseases.     

In glycine and GABA(A) channels, different subunits contribute asymmetrically to channel conductance via residues in the extracellular domain

Single-channel conductance in Cys-loop channels is controlled by the nature of the amino acids in the narrowest parts of the ion conduction pathway, namely the second transmembrane domain (M2) and the intracellular helix. In cationic channels, such as Torpedo ACh nicotinic receptors, conductance is increased by negatively charged residues exposed to the extracellular vestibule. We now show that positively charged residues at the same loop 5 position boost also the conductance of anionic Cys-loop channels, such as glycine (α1 and α1β) and GABA(A) (α1β2γ2) receptors. Charge reversal mutations here produce a greater decrease on outward conductance, but their effect strongly depends on which subunit carries the mutation. In the glycine α1β receptor, replacing Lys with Glu in α1 reduces single-channel conductance by 41%, but has no effect in the β subunit. By expressing concatameric receptors with constrained stoichiometry, we show that this asymmetry is not explained by the subunit copy number. A similar pattern is observed in the α1β2γ2 GABA(A) receptor, where only mutations in α1 or β2 decreased conductance (to different extents). In both glycine and GABA receptors, the effect of mutations in different subunits does not sum linearly: mutations that had no detectable effect in isolation did enhance the effect of mutations carried by other subunits. As in the nicotinic receptor, charged residues in the extracellular vestibule of anionic Cys-loop channels influence elementary conductance. The size of this effect strongly depends on the direction of the ion flow and, unexpectedly, on the nature of the subunit that carries the residue.     

GABA(C) receptors: a molecular view

In the central nervous system inhibitory neurotransmission is primarily achieved through activation of receptors for gamma-aminobutyric acid (GABA). Three types of GABA receptors have been identified on the basis of their pharmacological and electrophysiological properties. The predominant type, termed GABA(A), and a recently identified GABA(C) type, form ligand-gated chloride channels, whereas GABA(B) receptors activate separate cation channels via G proteins. Based on their homology to nicotinic acetylcholine receptors, GABA(C) receptors are believed to be oligomeric protein complexes composed of five subunits in a pentameric arrangement. To date up to five different GABA(C) receptors subunits have been identified in various species. Recent studies have shed new light on the biological characteristics of GABA(C) receptors, including the chromosomal localization of its subunit genes and resulting links to deseases, the cloning of new splice variants, the identification of GABA(C) receptor-associated proteins, the identification of domains involved in subunit assembly, and finally structure/function studies examining functional consequences of introduced mutations. This review summarizes recent data in view of the molecular structure of GABA(C) receptors and presents new insights into the biological function of this protein in the retina.     

Dirofilaria immitis: identification of a novel ligand-gated ion channel-related polypeptide

We have isolated a cDNA from Dirofilaria immitis that encodes a predicted ion channel subunit, Di-LGR-1. Secondary structure predictions and database searches reveal that Di-LGR-1 is distantly related to ligand-gated anion channels, such as the GABA(A) receptors, though there are marked differences in the sequences of the putative channel forming regions. Di-LGR-1 has 52% sequence identity to the Caenorhabditis elegans predicted polypeptide, T27A1.4: neighbour-joining trees show that these two polypeptides are the most divergent members of the nematode ligand-gated anion channel family. No close homologues are present in vertebrates, suggesting that their function may be specific to nematodes. RNAi experiments using a fragment of T27A1.4 with C. elegans failed to reveal any obvious phenotype, so the function of these channels remains unknown.     

Interaction of the M4 segment with other transmembrane segments is required for surface expression of mammalian α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors

Ionotropic glutamate receptors (GluRs) are ligand-gated ion channels with a modular structure. The ion channel itself shares structural similarity, albeit an inverted membrane topology, with P-loop channels. Like P-loop channels, prokaryotic GluR subunits (e.g. GluR0) have two transmembrane segments. In contrast, eukaryotic GluRs have an additional transmembrane segment (M4), located C-terminal to the ion channel core. However, the structural/functional significance of this additional transmembrane segment is poorly defined. Although topologically similar to GluR0, mammalian AMPA receptor (GluA1) subunits lacking the M4 segment do not display surface expression. This lack of expression is not due to the M4 segment serving as an anchor to the ligand-binding domain because insertion of an artificial polyleucine transmembrane segment does not rescue surface expression. Specific interactions between M4 and the ligand-binding domain are also unlikely because insertion of polyglycines into the linker connecting them has no deleterious effects on function or surface expression. However, tryptophan and cysteine scanning mutagenesis of the M4 segment, as well as recovery of function in the polyleucine background, defined a unique face of the M4 helix that is required for GluR surface expression. In the AMPA receptor structure, this face forms intersubunit contacts with the transmembrane helices of the ion channel core (M1 and M3) from another subunit within the homotetramer. Thus, our experiments show that a highly specific interaction of the M4 segment with an adjacent subunit is required for surface expression of AMPA receptors. This interaction may represent a mechanism for regulating AMPA receptor biogenesis.     

Ligand activation of the prokaryotic pentameric ligand-gated ion channel ELIC

While the pentameric ligand-gated ion channel ELIC has recently provided first insight into the architecture of the family at high resolution, its detailed investigation was so far prevented by the fact that activating ligands were unknown. Here we describe a study on the functional characterization of ELIC by electrophysiology and X-ray crystallography. ELIC is activated by a class of primary amines that include the neurotransmitter GABA at high micro- to millimolar concentrations. The ligands bind to a conserved site and evoke currents that slowly desensitize over time. The protein forms cation selective channels with properties that resemble the nicotinic acetylcholine receptor. The high single channel conductance and the comparably simple functional behavior make ELIC an attractive model system to study general mechanisms of ion conduction and gating in this important family of neurotransmitter receptors.     

Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition

Fast synaptic inhibition in the brain is largely mediated by GABA(A) receptors. These ligand-gated ion channels are crucial in the control of cell and network activity. Therefore, modulating their function or cell surface stability will have major consequences for neuronal excitation. It has become clear that the stability and activity of GABA(A) receptors at synapses can be dynamically modulated by receptor trafficking and phosphorylation. Here, we discuss these regulatory mechanisms, and their consequences for the efficacy of GABA(A) receptor mediated synaptic inhibition.     

Isoflurane alters the structure and dynamics of GLIC

Pentameric ligand-gated ion channels are targets of general anesthetics. Although the search for discrete anesthetic binding sites has achieved some degree of success, little is known regarding how anesthetics work after the events of binding. Using the crystal structures of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), which is sensitive to a variety of general anesthetics, we performed multiple molecular dynamics simulations in the presence and absence of the general anesthetic isoflurane. Isoflurane bound to several locations within GLIC, including the transmembrane pocket identified crystallographically, the extracellular (EC) domain, and the interface of the EC and transmembrane domains. Isoflurane also entered the channel after the pore was dehydrated in one of the simulations. Isoflurane disrupted the quaternary structure of GLIC, as evidenced in a striking association between the binding and breakage of intersubunit salt bridges in the EC domain. The pore-lining helix experienced lateral and inward radial tilting motion that contributed to the channel closure. Isoflurane binding introduced strong anticorrelated motions between different subunits of GLIC. The demonstrated structural and dynamical modulations by isoflurane aid in the understanding of the underlying mechanism of anesthetic inhibition of GLIC and possibly other homologous pentameric ligand-gated ion channels.     

Nematode cys-loop GABA receptors: biological function, pharmacology and sites of action for anthelmintics

Parasitic nematode infection of humans and livestock is a major problem globally. Attempts to control nematode populations have led to the development of several classes of anthelmintic, which target cys-loop ligand-gated ion channels. Unlike the vertebrate nervous system, the nematode nervous system possesses a large and diversified array of ligand-gated chloride channels that comprise key components of the inhibitory neurotransmission system. In particular, cys-loop GABA receptors have evolved to play many fundamental roles in nematode behaviour such as locomotion. Analysis of the genomes of several free-living and parasitic nematodes suggests that there are several groups of cys-loop GABA receptor subunits that, for the most part, are conserved among nematodes. Despite many similarities with vertebrate cys-loop GABA receptors, those in nematodes are quite distinct in sequence similarity, subunit composition and biological function. With rising anthelmintic resistance in many nematode populations worldwide, GABA receptors should become an area of increased scientific investigation in the development of the next generation of anthelmintics.     

The Validation of Nematode-Specific Acetylcholine-Gated Chloride Channels as Potential Anthelmintic Drug Targets

New compounds are needed to treat parasitic nematode infections in humans, livestock and plants. Small molecule anthelmintics are the primary means of nematode parasite control in animals; however, widespread resistance to the currently available drug classes means control will be impossible without the introduction of new compounds. Adverse environmental effects associated with nematocides used to control plant parasitic species are also motivating the search for safer, more effective compounds. Discovery of new anthelmintic drugs in particular has been a serious challenge due to the difficulty of obtaining and culturing target parasites for high-throughput screens and the lack of functional genomic techniques to validate potential drug targets in these pathogens. We present here a novel strategy for target validation that employs the free-living nematode Caenorhabditis elegans to demonstrate the value of new ligand-gated ion channels as targets for anthelmintic discovery. Many successful anthelmintics, including ivermectin, levamisole and monepantel, are agonists of pentameric ligand-gated ion channels, suggesting that the unexploited pentameric ion channels encoded in parasite genomes may be suitable drug targets. We validated five members of the nematode-specific family of acetylcholine-gated chloride channels as targets of agonists with anthelmintic properties by ectopically expressing an ivermectin-gated chloride channel, AVR-15, in tissues that endogenously express the acetylcholine-gated chloride channels and using the effects of ivermectin to predict the effects of an acetylcholine-gated chloride channel agonist. In principle, our strategy can be applied to validate any ion channel as a putative anti-parasitic drug target.     

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 β7–β10 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 recombinant glycine receptor fragment forms homo-oligomers distinct from its GABA(A) counterpart

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA(A) receptors are inhibitory. In this study, a fragment of the human glycine receptor alpha1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli. This fragment is highly homologous in sequence to the corresponding fragment of the GABA(A) receptor. The recombinant fragment was found to have stable beta-rich secondary structure, similar to that found for the homologous GABA(A) receptor fragment, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies suggest that the fragment forms trimers in solution. In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent. A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABA(A) receptor fragment reported by us earlier. The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo. Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily.     

Are chlorella viruses a rich source of ion channel genes?

Plaque-forming dsDNA (>330 kb) viruses that infect certain unicellular, eukaryotic chlorella-like green algae contain approximately 375 protein-encoding genes. These proteins include a 94 amino acid K+ channel protein, called Kcv, as well as two putative ligand-gated ion channels. The viruses also encode other proteins that could be involved in the assembly and/or function of ion channels, including protein kinases and a phosphatase, polyamine biosynthetic enzymes and histamine decarboxylase.     

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly,Trafficking, and Activation Mechanisms

Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus β subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors.