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PLOS Biology  2012 

Inhibition of the Prokaryotic Pentameric Ligand-Gated Ion Channel ELIC by Divalent Cations

DOI: 10.1371/journal.pbio.1001429

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The modulation of pentameric ligand-gated ion channels (pLGICs) by divalent cations is believed to play an important role in their regulation in a physiological context. Ions such as calcium or zinc influence the activity of pLGIC neurotransmitter receptors by binding to their extracellular domain and either potentiate or inhibit channel activation. Here we have investigated by electrophysiology and X-ray crystallography the effect of divalent ions on ELIC, a close prokaryotic pLGIC homologue of known structure. We found that divalent cations inhibit the activation of ELIC by the agonist cysteamine, reducing both its potency and, at higher concentrations, its maximum response. Crystal structures of the channel in complex with barium reveal the presence of several distinct binding sites. By mutagenesis we confirmed that the site responsible for divalent inhibition is located at the outer rim of the extracellular domain, at the interface between adjacent subunits but at some distance from the agonist binding region. Here, divalent cations interact with the protein via carboxylate side-chains, and the site is similar in structure to calcium binding sites described in other proteins. There is evidence that other pLGICs may be regulated by divalent ions binding to a similar region, even though the interacting residues are not conserved within the family. Our study provides structural and functional insight into the allosteric regulation of ELIC and is of potential relevance for the entire family.


[1]  Hille B (2001) Ion channels of excitable membranes, third edition. Sunderland, MA: Sinauer Associates Inc.
[2]  Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3: 102–114. doi: 10.1038/nrn731
[3]  Lester HA, Dibas MI, Dahan DS, Leite JF, Dougherty DA (2004) Cys-loop receptors: new twists and turns. Trends Neurosci 27: 329–336. doi: 10.1016/j.tins.2004.04.002
[4]  Sine SM, Engel AG (2006) Recent advances in Cys-loop receptor structure and function. Nature 440: 448–455. doi: 10.1038/nature04708
[5]  Sivilotti LG (2010) What single-channel analysis tells us of the activation mechanism of ligand-gated channels: the case of the glycine receptor. J Physiol 588: 45–58. doi: 10.1113/jphysiol.2009.178525
[6]  Miller PS, Smart TG (2010) Binding, activation and modulation of Cys-loop receptors. Trends Pharmacol Sci 31: 161–174. doi: 10.1016/
[7]  Thompson AJ, Lester HA, Lummis SC (2010) The structural basis of function in Cys-loop receptors. Q Rev Biophys 43: 449–499. doi: 10.1017/s0033583510000168
[8]  Yakel JL (2010) Gating of nicotinic ACh receptors: latest insights into ligand binding and function. J Physiol 588: 597–602. doi: 10.1113/jphysiol.2009.182691
[9]  Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 346: 967–989. doi: 10.1016/j.jmb.2004.12.031
[10]  Hilf RJ, Dutzler R (2009) A prokaryotic perspective on pentameric ligand-gated ion channel structure. Curr Opin Struct Biol 19: 418–424. doi: 10.1016/
[11]  Celie PH, van Rossum-Fikkert SE, van Dijk WJ, Brejc K, Smit AB, et al. (2004) Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41: 907–914. doi: 10.1016/s0896-6273(04)00115-1
[12]  Sabey K, Paradiso K, Zhang J, Steinbach JH (1999) Ligand binding and activation of rat nicotinic alpha4beta2 receptors stably expressed in HEK293 cells. Mol Pharmacol 55: 58–66.
[13]  Burzomato V, Beato M, Groot-Kormelink PJ, Colquhoun D, Sivilotti LG (2004) Single-channel behavior of heteromeric alpha1beta glycine receptors: an attempt to detect a conformational change before the channel opens. J Neurosci 24: 10924–10940. doi: 10.1523/jneurosci.3424-04.2004
[14]  Lester HA, Changeux JP, Sheridan RE (1975) Conductance increases produced by bath application of cholinergic agonists to Electrophorus electroplaques. J Gen Physiol 65: 797–816. doi: 10.1085/jgp.65.6.797
[15]  Rayes D, De Rosa MJ, Sine SM, Bouzat C (2009) Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors. J Neurosci 29: 6022–6032. doi: 10.1523/jneurosci.0627-09.2009
[16]  Beato M, Groot-Kormelink PJ, Colquhoun D, Sivilotti LG (2004) The activation mechanism of alpha1 homomeric glycine receptors. J Neurosci 24: 895–906. doi: 10.1523/jneurosci.4420-03.2004
[17]  Grosman C, Zhou M, Auerbach A (2000) Mapping the conformational wave of acetylcholine receptor channel gating. Nature 403: 773–776. doi: 10.1038/35001586
[18]  Taly A, Corringer PJ, Guedin D, Lestage P, Changeux JP (2009) Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nat Rev Drug Discov 8: 733–750. doi: 10.1038/nrd2927
[19]  Mohler H (2011) The rise of a new GABA pharmacology. Neuropharmacology 60: 1042–1049. doi: 10.1016/j.neuropharm.2010.10.020
[20]  Yamakura T, Bertaccini E, Trudell JR, Harris RA (2001) Anesthetics and ion channels: molecular models and sites of action. Annu Rev Pharmacol Toxicol 41: 23–51. doi: 10.1146/annurev.pharmtox.41.1.23
[21]  Lobo IA, Harris RA (2008) GABA(A) receptors and alcohol. Pharmacol Biochem Behav 90: 90–94. doi: 10.1016/j.pbb.2008.03.006
[22]  Hibbs RE, Gouaux E (2011) Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474: 54–60. doi: 10.1038/nature10139
[23]  Dani JA, Eisenman G (1987) Monovalent and divalent cation permeation in acetylcholine receptor channels. Ion transport related to structure. J Gen Physiol 89: 959–983. doi: 10.1085/jgp.89.6.959
[24]  Adams DJ, Dwyer TM, Hille B (1980) The permeability of endplate channels to monovalent and divalent metal cations. J Gen Physiol 75: 493–510. doi: 10.1085/jgp.75.5.493
[25]  Sine SM, Claudio T, Sigworth FJ (1990) Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities. J Gen Physiol 96: 395–437. doi: 10.1085/jgp.96.2.395
[26]  Mulle C, Lena C, Changeux JP (1992) Potentiation of nicotinic receptor response by external calcium in rat central neurons. Neuron 8: 937–945. doi: 10.1016/0896-6273(92)90208-u
[27]  Vernino S, Amador M, Luetje CW, Patrick J, Dani JA (1992) Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors. Neuron 8: 127–134. doi: 10.1016/0896-6273(92)90114-s
[28]  Peters JA, Hales TG, Lambert JJ (1988) Divalent cations modulate 5-HT3 receptor-induced currents in N1E-115 neuroblastoma cells. Eur J Pharmacol 151: 491–495. doi: 10.1016/0014-2999(88)90550-x
[29]  Niemeyer MI, Lummis SC (2001) The role of the agonist binding site in Ca(2+) inhibition of the recombinant 5-HT(3A) receptor. Eur J Pharmacol 428: 153–161. doi: 10.1016/s0014-2999(01)01251-1
[30]  Palma E, Maggi L, Miledi R, Eusebi F (1998) Effects of Zn2+ on wild and mutant neuronal alpha7 nicotinic receptors. Proc Natl Acad Sci U S A 95: 10246–10250. doi: 10.1073/pnas.95.17.10246
[31]  Smart TG, Xie X, Krishek BJ (1994) Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc. Prog Neurobiol 42: 393–441. doi: 10.1016/0301-0082(94)90082-5
[32]  Laube B, Kuhse J, Rundstrom N, Kirsch J, Schmieden V, et al. (1995) Modulation by zinc ions of native rat and recombinant human inhibitory glycine receptors. J Physiol 483 (Pt 3) 613–619.
[33]  Hubbard PC, Lummis SC (2000) Zn(2+) enhancement of the recombinant 5-HT(3) receptor is modulated by divalent cations. Eur J Pharmacol 394: 189–197. doi: 10.1016/s0014-2999(00)00143-6
[34]  Hsiao B, Mihalak KB, Magleby KL, Luetje CW (2008) Zinc potentiates neuronal nicotinic receptors by increasing burst duration. J Neurophysiol 99: 999–1007. doi: 10.1152/jn.01040.2007
[35]  Moroni M, Vijayan R, Carbone A, Zwart R, Biggin PC, et al. (2008) Non-agonist-binding subunit interfaces confer distinct functional signatures to the alternate stoichiometries of the alpha4beta2 nicotinic receptor: an alpha4-alpha4 interface is required for Zn2+ potentiation. J Neurosci 28: 6884–6894. doi: 10.1523/jneurosci.1228-08.2008
[36]  Hilf RJ, Dutzler R (2008) X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature 452: 375–379. doi: 10.1038/nature06717
[37]  Zimmermann I, Dutzler R (2011) Ligand activation of the prokaryotic pentameric ligand-gated ion channel ELIC. PLoS Biol 9: e1001101 doi:10.1371/journal.pbio.1001101.
[38]  Hilf RJ, Bertozzi C, Zimmermann I, Reiter A, Trauner D, et al. (2010) Structural basis of open channel block in a prokaryotic pentameric ligand-gated ion channel. Nat Struct Mol Biol 17: 1330–1336. doi: 10.1038/nsmb.1933
[39]  Hilf RJ, Dutzler R (2009) Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel. Nature 457: 115–118. doi: 10.1038/nature07461
[40]  Bocquet N, Nury H, Baaden M, Le Poupon C, Changeux JP, et al. (2009) X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature 457: 111–114. doi: 10.1038/nature07462
[41]  Pan J, Chen Q, Willenbring D, Yoshida K, Tillman T, et al. (2012) Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine. Nat Commun 3: 714. doi: 10.1038/ncomms1703
[42]  Arunlakshana O, Schild HO (1959) Some quantitative uses of drug antagonists. Br J Pharmacol 14: 48–58. doi: 10.1111/j.1476-5381.1959.tb00928.x
[43]  Colquhoun D (2007) Why the Schild method is better than Schild realised. Trends Pharmacol Sci 28: 608–614. doi: 10.1016/
[44]  Yuan P, Leonetti MD, Hsiung Y, MacKinnon R (2012) Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel. Nature 481: 94–97. doi: 10.1038/nature10670
[45]  Wu Y, Yang Y, Ye S, Jiang Y (2010) Structure of the gating ring from the human large-conductance Ca(2+)-gated K(+) channel. Nature 466: 393–397. doi: 10.1038/nature09252
[46]  Schumacher MA, Rivard AF, Bachinger HP, Adelman JP (2001) Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin. Nature 410: 1120–1124. doi: 10.1038/35074145
[47]  Auld DS (2009) The ins and outs of biological zinc sites. Biometals 22: 141–148. doi: 10.1007/s10534-008-9184-1
[48]  Vallee BL, Auld DS (1990) Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29: 5647–5659. doi: 10.1021/bi00476a001
[49]  Lu M, Chai J, Fu D (2009) Structural basis for autoregulation of the zinc transporter YiiP. Nat Struct Mol Biol 16: 1063–1067. doi: 10.1038/nsmb.1662
[50]  Galzi JL, Bertrand S, Corringer PJ, Changeux JP, Bertrand D (1996) Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor. The EMBO J 15: 5824–5832.
[51]  Eddins D, Sproul AD, Lyford LK, McLaughlin JT, Rosenberg RL (2002) Glutamate 172, essential for modulation of L247T alpha7 ACh receptors by Ca2+, lines the extracellular vestibule. Am J Physiol Cell Physiol 283: C1454–C1460. doi: 10.1152/ajpcell.00204.2002
[52]  Lyford LK, Sproul AD, Eddins D, McLaughlin JT, Rosenberg RL (2003) Agonist-induced conformational changes in the extracellular domain of alpha 7 nicotinic acetylcholine receptors. Mol Pharmacol 64: 650–658. doi: 10.1124/mol.64.3.650
[53]  Hosie AM, Dunne EL, Harvey RJ, Smart TG (2003) Zinc-mediated inhibition of GABA(A) receptors: discrete binding sites underlie subtype specificity. Nat Neurosci 6: 362–369. doi: 10.1038/nn1030
[54]  Thompson AJ, Lummis SC (2009) Calcium modulation of 5-HT3 receptor binding and function. Neuropharmacology 56: 285–291. doi: 10.1016/j.neuropharm.2008.07.009
[55]  Hu XQ, Lovinger DM (2005) Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2+. J Physiol 568: 381–396. doi: 10.1113/jphysiol.2005.092866
[56]  Colquhoun D (1998) Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125: 924–947. doi: 10.1038/sj.bjp.0702164
[57]  Auerbach A (2005) Gating of acetylcholine receptor channels: brownian motion across a broad transition state. Proc Natl Acad Sci U S A 102: 1408–1412. doi: 10.1073/pnas.0406787102
[58]  Mukhtasimova N, Lee WY, Wang HL, Sine SM (2009) Detection and trapping of intermediate states priming nicotinic receptor channel opening. Nature 459: 451–454. doi: 10.1038/nature07923
[59]  Auerbach A (1992) Kinetic behavior of cloned mouse acetylcholine receptors. A semi-autonomous, stepwise model of gating. Biophys J 62: 72–73. doi: 10.1016/s0006-3495(92)81783-6
[60]  Auerbach A (1993) A statistical analysis of acetylcholine receptor activation in Xenopus myocytes: stepwise versus concerted models of gating. J Physiol 461: 339–378.
[61]  Jadey S, Auerbach A (2012) An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors. J Gen Physiol 140: 17–28. doi: 10.1085/jgp.201210801
[62]  Amador M, Dani JA (1995) Mechanism for modulation of nicotinic acetylcholine receptors that can influence synaptic transmission. J Neurosci 15: 4525–4532.
[63]  Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Cryst 26: 795–800. doi: 10.1107/s0021889893005588
[64]  CCP4 (1994) Collaborative Computational Project Nr. 4. The CCP4 Suite: Programs for X-ray crystallography. Acta Crystallogr D 50: 760–763. doi: 10.1107/s0907444994003112
[65]  McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Cryst 40: 658–674. doi: 10.1107/s0021889807021206
[66]  Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66: 486–501. doi: 10.1107/s0907444910007493
[67]  Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, et al. (2002) PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58: 1948–1954. doi: 10.1107/s0907444902016657
[68]  Lorenz C, Pusch M, Jentsch TJ (1996) Heteromultimeric CLC chloride channels with novel properties. Proc Natl Acad Sci U S A 93: 13362–13366. doi: 10.1073/pnas.93.23.13362
[69]  Groot-Kormelink PJ, Beato M, Finotti C, Harvey RJ, Sivilotti LG (2002) Achieving optimal expression for single channel recording: a plasmid ratio approach to the expression of alpha 1 glycine receptors in HEK293 cells. J Neurosci Methods 113: 207–214. doi: 10.1016/s0165-0270(01)00500-3


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