Thomas Grutter

A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family

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.

Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca2+-binding sites.

We constructed a three-dimensional model of the amino-terminal extracellular domain of three major types of nicotinic acetylcholine receptor, (α7)5, (α4)2(β2)3, and (α1)2β1γδ, 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 β2 but not in α7, is proposed to contribute to the high affinity for agonists in receptors containing the β2 subunit. The semiautomatic docking of agonists in the ligand-binding pocket of (α7)5 led to positions consistent with labeling and mutagenesis experiments. Accordingly, the quaternary ammonium head group of nicotine makes a π-cation interaction with W148 (α7 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 Ca2+.

Nicotinic receptors in wonderland

Abstract The structure of a soluble homopentameric homologue of the N-terminal extracellular domain of the nicotinic acetylcholine (ACh) receptor has recently been determined at the atomic level. These data reveal the three-dimensional structure of the binding site for ACh and nicotinic ligands. The ACh-binding sites are located at subunit boundaries in an equatorial position and are framed by residues previously identified in nicotinic receptors, by photoaffinity labelling and mutagenesis experiments, as being crucial for ligand binding. On this basis, a hypothetical mechanism for the allosteric transitions of the nicotinic receptors is suggested.

Implications of the quaternary twist allosteric model for the physiology and pathology of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand-gated 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 Cα Gaussian network of a new structural model of the neuronal α7 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 (24/27) 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.

Ligand-Gated Ion Channels: New Insights into Neurological Disorders and Ligand Recognition

Disorders and Ligand Recognition Damien Lemoine,‡ Ruotian Jiang,‡ Antoine Taly,† Thierry Chataigneau,‡ Alexandre Specht, and Thomas Grutter*,‡ ‡Laboratoire de Biophysicochimie des Rećepteurs Canaux, UMR 7199 CNRS, Conception et Application de Molećules Bioactives, Faculte ́ de Pharmacie, Universite ́ de Strasbourg, 67400 Illkirch, France Laboratoire de Chimie Bioorganique, UMR 7199 CNRS, Conception et Application de Molećules Bioactives, Faculte ́ de Pharmacie, Universite ́ de Strasbourg, 67400 Illkirch, France †Laboratoire de Biochimie Theórique, UPR 9080 CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France

An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors

The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H‐bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced α4 residues into the α7/5HT3 chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non‐additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole‐cell patch–clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5‐fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high‐affinity α‐bungarotoxin binding (100‐fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric–kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter‐backbone H‐bond between positions 152 and 193, revealed in α4 and in α7 double mutant but not in α7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.

Tightening of the ATP-binding sites induces the opening of P2X receptor channels

The opening of ligand‐gated ion channels in response to agonist binding is a fundamental process in biology. In ATP‐gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open ‘jaws’ induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

Moving through the gate in ATP-activated P2X receptors

P2X receptors are nonselective cation channels gated by extracellular ATP. They represent new therapeutic targets, and they form channels with a unique trimeric architecture. In 2009, the first crystal structure of a P2X receptor was reported, in which the receptor was in an ATP-free, closed channel state. However, our view recently changed when a second crystal structure was reported, in which a P2X receptor was bound to ATP and resolved in an open channel conformation. This remarkable structure not only confirms many key experimental data, including the recent mechanisms of ATP binding and ion permeation, but also reveals unanticipated mechanisms. Certainly, this new information will accelerate our understanding of P2X receptor function and pharmacology at the atomic level.

Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor

P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120–I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP–induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.

Agonist trapped in ATP-binding sites of the P2X2 receptor

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent “tethering” reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.