The high-resolution crystal structure of a pentameric ligand-gated ion channel reveals that hydroxylated residues and two water pentagon rings form an ion selectivity filter, explaining ion transport across hydrophobic constriction barriers.

The current understanding about ethanol effects on the ligand-gated ion channel (LGIC) superfamily has been restricted to identify potential binding sites within transmembrane (TM) domains in the Cys-loop family. Here, we demonstrate a key role of the TM3-4 intracellular loop and G␤␥ signaling for potentiation of glycine receptors (GlyRs) by ethanol. We discovered 2 motifs within the large intracellular loop of the GlyR ␣1 subunit that are critical for the actions of pharmacological concentrations of ethanol. Significantly, the sites were ethanol-specific because they did not alter the sensitivity to general anesthetics, neurosteroids, or longer n-alcohols. Furthermore, G␤␥ scavengers selectively attenuated the ethanol effects on recombinant and native neuronal GlyRs. These results show a selective mechanism for low-ethanol concentration effects on the GlyR and provide a mechanism on ethanol pharmacology, which may be applicable to other LGIC members. Moreover, these data provide an opportunity to develop new genetically modified animal models and novel drugs to treat alcohol-related medical concerns.pharmacology ͉ signal transduction ͉ glycine receptor ͉ alcoholism ͉ G proteins

Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.Gloeobacter violaceus | allosteric effector | evolution | fusion protein | membrane protein P entameric ligand-gated ion channels (pLGICs) mediate chemo-electric signal transduction in animals, thereby fulfilling key physiological functions, including neurotransmission. They are composed of five identical or homologous subunits and carry one to five agonist binding sites on their extracellular domain (ECD) that govern the opening/closing motion of the ion channel within the transmembrane domain (TMD, composed of four helices labeled M1-M4). In human, ≈40 genes code for pLGIC subunits that are organized into two phylogenetic subclasses: cationic excitatory channels, such as nicotinic acetylcholine (nAChR) and 5HT 3 receptors, and anionic inhibitory channels, such as glycine and GABA A receptors (1). More recently, several new members of the family have been discovered in prokaryotes (2), such as the homologous protein from Gloeobacter violaceus (GLIC), which forms a homopentamer functioning as a proton-gated ion channel (3).Eukaryotic and prokaryotic receptors clearly display homologous structures, characterized by a highly conserved β-sandwichfolded ECD coupled to an all-helix TMD, as described by electron microscopy observation of the Torpedo nAChR (TnAChR) (4), and the X-ray structures of the acetylcholine binding protein (...

We characterized the human Na ؉ -ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na ؉ -dependent transporter.

It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two ligand-gated ion receptor members, the GlyR adult ␣ 1 and embryonic ␣ 2 subunits, can be modified through selective mutations that rescued or impaired G␤␥ modulation. Even though both isoforms were able to physically interact with G␤␥, only the ␣ 1 GlyR was functionally modulated by G␤␥ and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in ␣ 2 GlyRs was enough to generate GlyRs modulated by G␤␥ and low ethanol concentrations. Interestingly, although we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a preopen-activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two ligand-gated ion receptor members based on selective G␤␥ modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs.Glycine receptors (GlyRs) 4 are members of the ligand-gated ion receptor (LGIC) superfamily, which includes the Cys-loop family composed of the inhibitory ␥-aminobutyric acid receptors and GlyRs and the excitatory nicotinic acetylcholine (nAChR) and 5-hydroxytryptamine receptors. These ionotropic receptors mediate fast synaptic transmission in the central nervous system (1, 2). Specifically, inhibitory GlyRs are critical for the control of excitability in the mammalian spinal cord and brain stem, regulating important physiological functions such as pain transmission, respiratory rhythms, motor coordination, and neuronal development (3-7).Like all Cys-loop receptors, GlyRs are heteropentameric complexes composed of ␣ and ␤ subunits, which can assemble to form homomeric (5␣) or heteromeric (2␣3␤) channels. To date, molecular cloning studies have demonstrated four isoforms of the ␣ GlyRs (␣ 1-4 ) and one ␤ isoform. Homomeric and heteromeric receptors share most of the GlyR general features, including a high percentage of identity between ␣ GlyRs (Ϸ75%). Nevertheless, biochemical, immunocytochemical, and in situ hybridization studies have shown that the expression of the subunits are developmentally and regionally regulated (3,4,8). For example, the ␣ 1 subunit expression increases after birth, whereas expression of the ␣ 2 subunit appears mainly restricted to early...

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein ␤␥ subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR ␣ 1 subunit that are critical for binding and functional modulation by G␤␥. Mutations within these sequences demonstrated that all of the residues detected are important for G␤␥ modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are ␣-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein ␤␥ subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.The ionotropic glycine receptors (GlyRs) 2 are members of the ligand-gated ion receptor superfamily, which includes inhibitory ␥-aminobutyric acid type A receptors and excitatory nAChR and 5-HT 3 receptors. These homologous receptors mediate fast synaptic transmission in the central nervous system (1, 2). Inhibitory GlyRs are critical for the control of excitability in the mammalian spinal cord and brain stem. Binding of glycine to the extracellular region induces a rapid increase in Cl Ϫ ion conductance, generating a hyperpolarization of the cell membrane (3-5). In neurons, the inhibitory action of GlyRs regulate several important physiological functions, like pain transmission, respiratory rhythms, motor coordination, and development (3-7). Like all members of the LGIC superfamily, GlyRs are pentamers composed of five subunits in which each subunit possesses four transmembrane domains arranged to form the ion pore. In this structure, the individual subunits provide extracellular and intracellular domains that play roles in ligand binding and intracellular modulation, respectively (1-3, 5).The function of GlyRs can be effectively modulated by extracellularly acting compounds like strychnine, picrotoxin, zinc ions, and ethanol (3-5, 8, 9). Furthermore, the receptor can also be modulated by intracellular signaling. One of the most studied and recognized pathways involved in regulation of ligandgated ion channel function are phosphorylation processes through protein kinases. Indeed, GlyR and other members of the LGIC superfamily are modulated by activation of cAMPdependent kinases and protein kinase C (10). This involves specific serine residues in the loop between transmembrane domains 3 and 4 (6, 10 -14). On the other hand, recent reports have shown that the activity of GlyRs and nAChRs can be modulated by G protein ␤␥ subunits in a phosphorylation-independent manner (15, 16). In both cases, activation of G proteins, using nonhydrolyzable GTP analogs or by application of purified G␤␥ dimers, generates a strong e...

The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α 1 GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α 1 GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α 1 GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a poreopening mechanism for the α 1 GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α 1 GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.glycine receptor | hyperekplexia | allostery

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