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.