Abstract
Fast inhibitory neurotransmission is essential for nervous system function and is mediated by binding of inhibitory neurotransmitters to receptors of the Cys-loop family embedded in the membranes of neurons. Neurotransmitter binding triggers a conformational change in the receptor, opening an intrinsic chloride channel and thereby dampening neuronal excitability. Here we present the first three-dimensional structure, to our knowledge, of an inhibitory anion-selective Cys-loop receptor, the homopentameric Caenorhabditis elegans glutamate-gated chloride channel α (GluCl), at 3.3 Å resolution. The X-ray structure of the GluCl–Fab complex was determined with the allosteric agonist ivermectin and in additional structures with the endogenous neurotransmitter l-glutamate and the open-channel blocker picrotoxin. Ivermectin, used to treat river blindness, binds in the transmembrane domain of the receptor and stabilizes an open-pore conformation. Glutamate binds in the classical agonist site at subunit interfaces, and picrotoxin directly occludes the pore near its cytosolic base. GluCl provides a framework for understanding mechanisms of fast inhibitory neurotransmission and allosteric modulation of Cys-loop receptors.
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References
Coombs, J. S., Eccles, J. C. & Fatt, P. The specific ionic conductances and the ionic movements across the motoneuronal membrane that produce the inhibitory post-synaptic potential. J. Physiol. (Lond.) 130, 326–374 (1955)
Hille, B. Ion Channels of Excitable Membranes (Sinauer Associates, 2001)
Thompson, A. J., Lester, H. A. & Lummis, S. C. The structural basis of function in Cys-loop receptors. Q. Rev. Biophys. 43, 449–499 (2010)
Corringer, P. J. et al. Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues. J. Physiol. (Lond.) 588, 565–572 (2010)
Hilf, R. J. & Dutzler, R. A prokaryotic perspective on pentameric ligand-gated ion channel structure. Curr. Opin. Struct. Biol. 19, 418–424 (2009)
Miller, P. S. & Smart, T. G. Binding, activation and modulation of Cys-loop receptors. Trends Pharmacol. Sci. 31, 161–174 (2010)
Garcia, P. S., Kolesky, S. E. & Jenkins, A. General anesthetic actions on GABAA receptors. Curr. Neuropharmacol. 8, 2–9 (2010)
Cully, D. F. et al. Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans . Nature 371, 707–711 (1994)
Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)
Hilf, R. J. & Dutzler, R. Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel. Nature 457, 115–118 (2009)
Bocquet, N. et al. X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature 457, 111–114 (2009)
Hilf, R. J. & Dutzler, R. X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature 452, 375–379 (2008)
Brejc, K. et al. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269–276 (2001)
Celie, P. H. et al. Crystal structure of acetylcholine-binding protein from Bulinus truncatus reveals the conserved structural scaffold and sites of variation in nicotinic acetylcholine receptors. J. Biol. Chem. 280, 26457–26466 (2005)
Hansen, S. B. et al. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J. 24, 3635–3646 (2005)
Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J. Mol. Biol. 346, 967–989 (2005)
Campbell, W. C., Fisher, M. H., Stapley, E. O., Albers-Schonberg, G. & Jacob, T. A. Ivermectin: a potent new antiparasitic agent. Science 221, 823–828 (1983)
Aziz, M. A., Diallo, S., Diop, I. M., Lariviere, M. & Porta, M. Efficacy and tolerance of ivermectin in human onchocerciasis. Lancet 320, 171–173 (1982)
Arena, J. P., Liu, K. K., Paress, P. S. & Cully, D. F. Avermectin-sensitive chloride currents induced by Caenorhabditis elegans RNA in Xenopus oocytes. Mol. Pharmacol. 40, 368–374 (1991)
Adelsberger, H., Lepier, A. & Dudel, J. Activation of rat recombinant α1β2γ2s GABAA receptor by the insecticide ivermectin. Eur. J. Pharmacol. 394, 163–170 (2000)
Shan, Q., Haddrill, J. L. & Lynch, J. W. Ivermectin, an unconventional agonist of the glycine receptor chloride channel. J. Biol. Chem. 276, 12556–12564 (2001)
Krause, R. M. et al. Ivermectin: a positive allosteric effector of the α7 neuronal nicotinic acetylcholine receptor. Mol. Pharmacol. 53, 283–294 (1998)
Silberberg, S. D., Li, M. & Swartz, K. J. Ivermectin interaction with transmembrane helices reveals widespread rearrangements during opening of P2X receptor channels. Neuron 54, 263–274 (2007)
Etter, A., Cully, D. F., Schaeffer, J. M., Liu, K. K. & Arena, J. P. An amino acid substitution in the pore region of a glutamate-gated chloride channel enables the coupling of ligand binding to channel gating. J. Biol. Chem. 271, 16035–16039 (1996)
Ueno, S., Wick, M. J., Ye, Q., Harrison, N. L. & Harris, R. A. Subunit mutations affect ethanol actions on GABAA receptors expressed in Xenopus oocytes. Br. J. Pharmacol. 127, 377–382 (1999)
Young, G. T., Zwart, R., Walker, A. S., Sher, E. & Millar, N. S. Potentiation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site. Proc. Natl Acad. Sci. USA 105, 14686–14691 (2008)
Kao, P. N. et al. Identification of the α subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine receptor binding site. J. Biol. Chem. 259, 11662–11665 (1984)
Damle, V. N. & Karlin, A. Effects of agonists and antagonists on the reactivity of the binding site disulfide in acetylcholine receptor from Torpedo californica . Biochemistry 19, 3924–3932 (1980)
Celie, P. H. et al. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41, 907–914 (2004)
Mukhtasimova, N., Free, C. & Sine, S. M. Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor. J. Gen. Physiol. 126, 23–39 (2005)
Quiram, P. A., McIntosh, J. M. & Sine, S. M. Pairwise interactions between neuronal α7 acetylcholine receptors and α-conotoxin PnIB. J. Biol. Chem. 275, 4889–4896 (2000)
Lee, W. Y. & Sine, S. M. Principal pathway coupling agonist binding to channel gating in nicotinic receptors. Nature 438, 243–247 (2005)
Campos-Caro, A. et al. A single residue in the M2–M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors. Proc. Natl Acad. Sci. USA 93, 6118–6123 (1996)
Kusama, T., Wang, J. B., Spivak, C. E. & Uhl, G. R. Mutagenesis of the GABA rho 1 receptor alters agonist affinity and channel gating. Neuroreport 5, 1209–1212 (1994)
Lynch, J. W., Rajendra, S., Barry, P. H. & Schofield, P. R. Mutations affecting the glycine receptor agonist transduction mechanism convert the competitive antagonist, picrotoxin, into an allosteric potentiator. J. Biol. Chem. 270, 13799–13806 (1995)
Rajendra, S. et al. Mutation of an arginine residue in the human glycine receptor transforms β-alanine and taurine from agonists into competitive antagonists. Neuron 14, 169–175 (1995)
Takeuchi, A. & Takeuchi, N. A study of the action of picrotoxin on the inhibitory neuromuscular junction of the crayfish. J. Physiol. (Lond.) 205, 377–391 (1969)
Etter, A. et al. Picrotoxin blockade of invertebrate glutamate-gated chloride channels: subunit dependence and evidence for binding within the pore. J. Neurochem. 72, 318–326 (1999)
Fatima-Shad, K. & Barry, P. H. Anion permeation in GABA- and glycine-gated channels of mammalian cultured hippocampal neurons. Proc. R. Soc. Lond. B 253, 69–75 (1993)
Sunesen, M. et al. Mechanism of Cl- selection by a glutamate-gated chloride (GluCl) receptor revealed through mutations in the selectivity filter. J. Biol. Chem. 281, 14875–14881 (2006)
Wada, A. The α-helix as an electric macro-dipole. Adv. Biophys. 1–63 (1976)
Dutzler, R., Campbell, E. B., Cadene, M., Chait, B. T. & MacKinnon, R. X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion selectivity. Nature 415, 287–294 (2002)
Wilson, G. G., Pascual, J. M., Brooijmans, N., Murray, D. & Karlin, A. The intrinsic electrostatic potential and the intermediate ring of charge in the acetylcholine receptor channel. J. Gen. Physiol. 115, 93–106 (2000)
Keramidas, A., Moorhouse, A. J., Schofield, P. R. & Barry, P. H. Ligand-gated ion channels: mechanisms underlying ion selectivity. Prog. Biophys. Mol. Biol. 86, 161–204 (2004)
Mancinelli, R., Botti, A., Bruni, F., Ricci, M. A. & Soper, A. K. Hydration of sodium, potassium, and chloride ions in solution and the concept of structure maker/breaker. J. Phys. Chem. B 111, 13570–13577 (2007)
Akabas, M. H., Kaufmann, C., Archdeacon, P. & Karlin, A. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the α subunit. Neuron 13, 919–927 (1994)
Lee, D. J., Keramidas, A., Moorhouse, A. J., Schofield, P. R. & Barry, P. H. The contribution of proline 250 (P-2′) to pore diameter and ion selectivity in the human glycine receptor channel. Neurosci. Lett. 351, 196–200 (2003)
Reeves, D. C., Goren, E. N., Akabas, M. H. & Lummis, S. C. Structural and electrostatic properties of the 5-HT3 receptor pore revealed by substituted cysteine accessibility mutagenesis. J. Biol. Chem. 276, 42035–42042 (2001)
Barrantes, F. J. Structural basis for lipid modulation of nicotinic acetylcholine receptor function. Brain Res. Rev. 47, 71–95 (2004)
Sherrington, C. S. Integrative Action of the Nervous System (Yale Univ. Press, 1906)
Gensler, S. et al. Assembly and clustering of acetylcholine receptors containing GFP-tagged ε or γ subunits: selective targeting to the neuromuscular junction in vivo . Eur. J. Biochem. 268, 2209–2217 (2001)
Li, P., Slimko, E. M. & Lester, H. A. Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel. FEBS Lett. 528, 77–82 (2002)
Slimko, E. M. & Lester, H. A. Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels. J. Neurosci. Methods 124, 75–81 (2003)
Harlow, E. & Lane, D. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988)
Matthews, B. W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–497 (1968)
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)
The. CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)
Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)
Leslie, A. G. W. Joint CCP4 and ESF-EAMCB Newsletter on Protein Crystallography, No. 26. (1992)
Leslie, A. G. The integration of macromolecular diffraction data. Acta Crystallogr. D 62, 48–57 (2006)
Sauter, N. K., Grosse-Kunstleve, R. W. & Adams, P. D. Robust indexing for automatic data collection. J. Appl. Cryst. 37, 399–409 (2004)
Zhang, C. Y., Sauter, N. K., van den Bedem, H., Snell, G. & Deacon, A. M. Automated diffraction image analysis and spot searching for high-throughput crystal screening. J. Appl. Cryst. 39, 112–119 (2006)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)
Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201 (2006)
Cowtan, K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography. 31, 34–38 (1994)
Emsley, P. & Cowtan, P. Coot: Model-Building Tools for Molecular Graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)
Springer, J. P., Arison, B. H., Hirshfield, J. M. & Hoogsteen, K. The absolute stereochemistry and conformation of avermectin B2a aglycon and avermectin B1a. J. Am. Chem. Soc. 103, 4221–4224 (1981)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Collaborative Computational Project 4 The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–776 (1994)
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)
Pei, J., Kim, B. H. & Grishin, N. V. PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res. 36, 2295–2300 (2008)
Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994)
Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)
DeLano, W. L. The PyMOL Molecular Graphics System 〈http://www.pymol.org〉 (DeLano Scientific, 2002).
Smart, O. S., Goodfellow, J. M. & Wallace, B. A. The pore dimensions of gramicidin A. Biophys. J. 65, 2455–2460 (1993)
Liman, E. R., Tytgat, J. & Hess, P. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9, 861–871 (1992)
Alexander, C. et al. Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore. Biochemistry 48, 10078–10088 (2009)
Lamla, T. & Erdmann, V. A. The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins. Protein Expr. Purif. 33, 39–47 (2004)
Acknowledgements
We are grateful to H. Lester for providing the initial GluCl construct, to D. Cawley for monoclonal antibody production, to J. Michel for Fab fragment cloning and sequencing, to C. Alexander and D. C. Dawson for providing Xenopus oocytes, to M. Mayer for advice and equipment related to oocyte experiments, and to L. Vaskalis for help with illustrations. We thank the staff at the Advanced Photon Source beamline 24-ID-C for assistance with X-ray data collection. We are particularly appreciative of discussions with E.G. laboratory members and E. McCleskey. This work was supported by an individual NIH National Research Service Award (F32NS061404) to R.E.H. E.G. is an investigator with the Howard Hughes Medical Institute.
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Hibbs, R., Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011). https://doi.org/10.1038/nature10139
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DOI: https://doi.org/10.1038/nature10139
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