Antoine Taly

Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system

Nicotinic receptors — a family of ligand-gated ion channels that mediate the effects of the neurotransmitter acetylcholine — are among the most well understood allosteric membrane proteins from a structural and functional perspective. There is also considerable interest in modulating nicotinic receptors to treat nervous-system disorders such as Alzheimers disease, schizophrenia, depression, attention deficit hyperactivity disorder and tobacco addiction. This article describes both recent advances in our understanding of the assembly, activity and conformational transitions of nicotinic receptors, as well as developments in the therapeutic application of nicotinic receptor ligands, with the aim of aiding novel drug discovery by bridging the gap between these two rapidly developing fields.

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.

A gating mechanism of pentameric ligand-gated ion channels.

Significance Pentameric ligand-gated ion channels (pLGICs) control membrane conductance in living systems from bacteria to humans. Molecular dynamics simulations based on the structures of the prokaryotic channels from Gloeobacter violaceus (GLIC) and Erwinia chrysanthemi (ELIC) and the eukaryotic channel from Caenorhabditis elegans (GluCl) show that the open-to-closed transition begins with a major quaternary (twisting) transition which is followed by tertiary relaxation of the pore-forming helices. The latter is initiated by the outward tilting of the extracellular β-sandwiches in response to agonist unbinding. The proposed atomic resolution mechanism for channel gating, which is in accord with the Monod–Wyman–Changeux model of allostery, is expected to be generally applicable to pLGICs. Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communication in the nervous system and are involved in fundamental processes such as attention, learning, and memory. They are oligomeric protein assemblies that convert a chemical signal into an ion flux through the postsynaptic membrane, but the molecular mechanism of gating ions has remained elusive. Here, we present atomistic molecular dynamics simulations of the prokaryotic channels from Gloeobacter violaceus (GLIC) and Erwinia chrysanthemi (ELIC), whose crystal structures are thought to represent the active and the resting states of pLGICs, respectively, and of the eukaryotic glutamate-gated chloride channel from Caenorhabditis elegans (GluCl), whose open-channel structure was determined complexed with the positive allosteric modulator ivermectin. Structural observables extracted from the trajectories of GLIC and ELIC are used as progress variables to analyze the time evolution of GluCl, which was simulated in the absence of ivermectin starting from the structure with bound ivermectin. The trajectory of GluCl with ivermectin removed shows a sequence of structural events that couple agonist unbinding from the extracellular domain to ion-pore closing in the transmembrane domain. Based on these results, we propose a structural mechanism for the allosteric communication leading to deactivation/activation of the GluCl channel. This model of gating emphasizes the coupling between the quaternary twisting and the opening/closing of the ion pore and is likely to apply to other members of the pLGIC family.

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

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.

A Putative Extracellular Salt Bridge at the Subunit Interface Contributes to the Ion Channel Function of the ATP-gated P2X2 Receptor

The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To reveal their significance for ion channel activation, we made systematic charge exchanges and measured the effects on ATP sensitivity. We found that charge reversals at the interfacial residues Glu63 and Arg274 produced gain-of-function phenotypes that were cancelled upon paired charge swapping. These results suggest that a putative intersubunit salt bridge formed between Glu63 and Arg274 contributes to the ion channel function. Engineered cysteines E63C and R274C formed redox-dependent cross-links in the absence of ATP. By contrast, the presence of ATP reduced the rate of disulfide bond formation, indicating that ATP binding might trigger relative movement of adjacent subunits at the level of Glu63 and Arg274, allowing the transmembrane helices to open the channel.

Optical control of an ion channel gate

Significance Engineered light-sensitive ion channels offer the opportunity to govern electrical activity of neurons. To date, developed strategies have relied on specific actions on either ligand-binding or permeation pathways. Here we developed a unique and versatile method, in which the gating machinery of an ATP-activated channel (purinergic P2X receptor) was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of the natural ligand (here ATP). We demonstrate photocontrol of neuronal activity by a light-gated P2X receptor, in which the natural sensitivity to ATP was genetically removed. These light-gated P2X receptors represent valuable tools for investigating their physiological functions. The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.