Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.

We present a three-dimensional model of the homopentameric alpha7 nicotinic acetylcholine receptor (nAChR), that includes the extracellular and membrane domains, developed by comparative modeling on the basis of: 1), the x-ray crystal structure of the snail acetylcholine binding protein, an homolog of the extracellular domain of nAChRs; and 2), cryo-electron microscopy data of the membrane domain collected on Torpedo marmorata nAChRs. We performed normal mode analysis on the complete three-dimensional model to explore protein flexibility. Among the first 10 lowest frequency modes, only the first mode produces a structural reorganization compatible with channel gating: a wide opening of the channel pore caused by a concerted symmetrical quaternary twist motion of the protein with opposing rotations of the upper (extracellular) and lower (transmembrane) domains. Still, significant reorganizations are observed within each subunit, that involve their bending at the domain interface, an increase of angle between the two beta-sheets composing the extracellular domain, the internal beta-sheet being significantly correlated to the movement of the M2 alpha-helical segment. This global symmetrical twist motion of the pentameric protein complex, which resembles the opening transition of other multimeric ion channels, reasonably accounts for the available experimental data and thus likely describes the nAChR gating process.

We constructed a three-dimensional model of the amino-terminal extracellular domain of three major types of nicotinic acetylcholine receptor, (␣7)5, (␣4)2(␤2)3, and (␣1)2␤1␥␦, on the basis of the recent x-ray structure determination of the molluscan acetylcholine-binding protein. Comparative analysis of the three models reveals that the agonist-binding pocket is much more conserved than the overall structure. Differences exist, however, in the side chains of several residues. In particular, a phenylalanine residue, present in ␤2 but not in ␣7, is proposed to contribute to the high affinity for agonists in receptors containing the ␤2 subunit. The semiautomatic docking of agonists in the ligand-binding pocket of (␣7)5 led to positions consistent with labeling and mutagenesis experiments. Accordingly, the quaternary ammonium head group of nicotine makes a -cation interaction with W148 (␣7 numbering), whereas the pyridine ring is close to both the cysteine pair 189 -190 and the complementary component of the binding site. The intrinsic affinities inferred from docking give a rank order epibatidine > nicotine > acetylcholine, in agreement with experimental values. Finally, our models offer a structural basis for potentiation by external Ca 2؉ .

The known seven mammalian receptor (R) subunits (P2X1-7) form cationic channels gated by ATP. Three subunits compose a receptor-channel. Each subunit is a polypeptide consisting of two transmembrane regions (TM1, TM2), intracellular Nand C-termini, and a bulky extracellular loop. Crystallization allowed the identification of the 3D-structure and gating cycle of P2XRs. The agonist binding pocket is located at the intersection of two neighboring subunits. In addition to the mammalian P2XRs their primitive ligand-gated counterparts with little structural similarity have also been cloned. Selective agonists for P2XR subtypes are not available, but medicinal chemistry supplied a range of subtype selective antagonists, as well as positive and negative allosteric modulators. Knockout mice and selective antagonists helped to identify pathological functions due to defective P2XRs, such as male infertility (P2X1), hearing loss (P2X2), pain/cough (P2X3), neuropathic pain (P2X4), inflammatory bone loss (P2X5), and faulty immune reactions (P2X7).

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Nicotinic acetylcholine receptors (nAChR) are pentameric ligandgated ion channels composed of subunits that consist of an extracellular domain that carries the ligand-binding site and a distinct ion-pore domain. Signal transduction results from the allosteric coupling between the two domains: the distance from the binding site to the gate of the pore domain is 50 Å. Normal mode analysis with a C␣ Gaussian network of a new structural model of the neuronal ␣7 nAChR showed that the lowest mode involves a global quaternary twist motion that opens the ion pore. A molecular probe analysis, in which the network is modified at each individual amino acid residue, demonstrated that the major effect is to change the frequency, but not the form, of the twist mode. The largest effects were observed for the ligand-binding site and the Cys-loop. Most (24͞27) of spontaneous mutations known to cause congenital myasthenia and autosomal dominant nocturnal frontal lobe epilepsy are located either at the interface between subunits or, within a given subunit, at the interface between rigid blocks. These interfaces are modified significantly by the twist mode. The present analysis, thus, supports the quaternary twist model of the nAChR allosteric transition and provides a qualitative interpretation of the effect of the mutations responsible for several receptor pathologies.allosteric transition ͉ nicotinic receptor ͉ pathological mutations ͉ normal mode perturbation scanning N icotinic acetylcholine receptors (nAChRs) play a central role in intercellular communications in the brain and at the neuromuscular junction. They are involved in nicotine addiction as well as in cognitive processes such as attention, access to consciousness, learning, and memory, and their pathologies include autism, schizophrenia, Parkinson's disease, and Alzheimer's disease (references in ref. 1). Understanding the functional organization of the nAChR at the atomic level thus is of considerable interest in itself and is a source of insights for the development of new drug therapies.nAChRs are members of the Cys-loop superfamily of ligandgated ion channels. They are hetero-or homopentameric integral membrane proteins with a fivefold axis of pseudosymmetry perpendicular to the membrane. Each subunit can be subdivided into two principal domains: extracellular and transmembrane. The extracellular domain (ECD) carries the acetylcholine (ACh) binding site at the boundary between subunits, and the transmembrane ion-pore domain (IPD) delineates an axial cation-specific channel (2, 3). These topologically distinct domains are coupled allosterically to each other. Therefore, nAChRs possess the structural elements necessary to convert a chemical signal, typically a local increase of extracellular ACh concentration, into an electrical signal generated by the opening of the ion channel.Electrophysiological analysis of nAChRs has shown that rapid delivery of ACh promotes fast opening of the channel, and that a prolonged application of ACh leads to a slow decrease of the re...

Neurotransmitters such as acetylcholine (ACh) and glycine mediate fast synaptic neurotransmission by activating pentameric ligandgated ion channels (LGICs). These receptors are allosteric transmembrane proteins that rapidly convert chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with an extracellular agonist-binding domain (ECD), triggering a tertiary͞quaternary conformational change in the protein that results in the fast opening of an ion pore domain (IPD). However, the molecular mechanism that determines the fast opening of LGICs remains elusive. Here, we show by combining whole-cell and single-channel recordings of recombinant chimeras between the ECD of ␣7 nicotinic receptor (nAChR) and the IPD of the glycine receptor (GlyR) that only two GlyR amino acid residues of loop 7 (Cys-loop) from the ECD and at most five ␣7 nAChR amino acid residues of the M2-M3 loop (2-3L) from the IPD control the fast activation rates of the ␣7͞Gly chimera and WT GlyR. Mutual interactions of these residues at a critical pivot point between the agonist-binding site and the ion channel fine-tune the intrinsic opening and closing rates of the receptor through stabilization of the transition state of activation. These data provide a structural basis for the fast opening of pentameric LGICs.allosteric proteins ͉ chimeric receptor ͉ Cys-loop receptor ͉ transition state P entameric ligand-gated ion channels (LGICs), such as the cationic nicotinic acetylcholine receptor (nAChR) and the anionic glycine receptor (GlyR), mediate fast excitatory or inhibitory chemical neurotransmission between neurons (1-6). A unique feature of these receptors is that they activate the ion channel, a process known as gating, in less than a ms. For nicotinic receptors, a detailed single-channel analysis has recently established a speed limit for the opening of the ion channel in the s time range (7). Perturbations of this rapid transmission pathway by natural mutants lead to severe diseases such as congenital myasthenic syndromes (8), hereditary hyperekplexia (9), or epileptic disorders (10).Pentameric LGICs, or Cys-loop receptors, are composed of five homologous subunits, sharing a common structural organization, arranged (pseudo)symmetrically around the central ion pore (1, 2). All subunits are made of two distinct topological domains: the extracellular (ECD) and the ion pore domains (IPD). First, the ECD is folded into a twisted ␤-sandwich core, as revealed by x-ray crystallographic studies of the mollusk acetylcholine-binding protein (AChBP), a soluble pentameric protein homologous to the extracellular domain of LGICs (11-14). Second, electron microscopy images of Torpedo nAChR at 4-Å resolution revealed that the four transmembrane segments (M1 to M4) of the IPD are folded into ␣-helices joined by linking loops of variable lengths (15). By combining these structural data, we built a 3D model of the full ␣7 nAChR (16). In this model, the coupling zone located at the interface between the two domains is framed by f...