Gating of Pentameric Ligand-Gated Ion Channels: Structural Insights and Ambiguities
2013; Elsevier BV; Volume: 21; Issue: 8 Linguagem: Inglês
10.1016/j.str.2013.06.019
ISSN1878-4186
AutoresCorrie J.B. daCosta, John E. Baenziger,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoPentameric ligand-gated ion channels (pLGICs) mediate fast synaptic communication by converting chemical signals into an electrical response. Recently solved agonist-bound and agonist-free structures greatly extend our understanding of these complex molecular machines. A key challenge to a full description of function, however, is the ability to unequivocally relate determined structures to the canonical resting, open, and desensitized states. Here, we review current understanding of pLGIC structure, with a focus on the conformational changes underlying channel gating. We compare available structural information and review the evidence supporting the assignment of each structure to a particular conformational state. We discuss multiple factors that may complicate the interpretation of crystal structures, highlighting the potential influence of lipids and detergents. We contend that further advances in the structural biology of pLGICs will require deeper insight into the nature of pLGIC-lipid interactions. Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic communication by converting chemical signals into an electrical response. Recently solved agonist-bound and agonist-free structures greatly extend our understanding of these complex molecular machines. A key challenge to a full description of function, however, is the ability to unequivocally relate determined structures to the canonical resting, open, and desensitized states. Here, we review current understanding of pLGIC structure, with a focus on the conformational changes underlying channel gating. We compare available structural information and review the evidence supporting the assignment of each structure to a particular conformational state. We discuss multiple factors that may complicate the interpretation of crystal structures, highlighting the potential influence of lipids and detergents. We contend that further advances in the structural biology of pLGICs will require deeper insight into the nature of pLGIC-lipid interactions. Cys-loop receptors, including nicotinic acetylcholine (nAChR), serotonin (5-HT3), glycine, and GABAA/GABAC receptors, mediate fast chemical to electrical transduction at synapses throughout the nervous system. Insight into Cys-loop receptor structure has expanded rapidly in recent years with structures of water-soluble homologs of the acetylcholine receptor agonist-binding domain (ABD) (Brejc et al., 2001Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.Nature. 2001; 411: 269-276Crossref PubMed Scopus (1278) Google Scholar, Celie et al., 2005Celie P.H. Klaassen R.V. van Rossum-Fikkert S.E. van Elk R. van Nierop P. Smit A.B. Sixma T.K. 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. 2005; 280: 26457-26466Crossref PubMed Scopus (135) Google Scholar, Dellisanti et al., 2007Dellisanti C.D. Yao Y. Stroud J.C. Wang Z.Z. Chen L. Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution.Nat. Neurosci. 2007; 10: 953-962Crossref PubMed Scopus (236) Google Scholar, Hansen et al., 2005Hansen S.B. Sulzenbacher G. Huxford T. Marchot P. Taylor P. Bourne Y. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations.EMBO J. 2005; 24: 3635-3646Crossref PubMed Scopus (395) Google Scholar, Li et al., 2011Li S.X. Huang S. Bren N. Noridomi K. Dellisanti C.D. Sine S.M. Chen L. Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.Nat. Neurosci. 2011; 14: 1253-1259Crossref PubMed Scopus (44) Google Scholar), the Torpedo nAChR (Unwin, 2005Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4A resolution.J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (993) Google Scholar), the prokaryotic pentameric ligand-gated ion channels (pLGICs), Erwinia ligand-gated ion channel (ELIC) (Hilf and Dutzler, 2008Hilf R.J. Dutzler R. X-ray structure of a prokaryotic pentameric ligand-gated ion channel.Nature. 2008; 452: 375-379Crossref PubMed Scopus (373) Google Scholar) and Gloebacter ligand-gated ion channel (GLIC) (Bocquet et al., 2009Bocquet N. Nury H. Baaden M. Le Poupon C. Changeux J.P. Delarue M. Corringer P.J. X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation.Nature. 2009; 457: 111-114Crossref PubMed Scopus (356) Google Scholar, Hilf and Dutzler, 2009Hilf R.J. Dutzler R. Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.Nature. 2009; 457: 115-118Crossref PubMed Scopus (294) Google Scholar), and the Caenorhabditis elegans glutamate-activated chloride channel (GluCl) (Hibbs and Gouaux, 2011Hibbs R.E. Gouaux E. Principles of activation and permeation in an anion-selective Cys-loop receptor.Nature. 2011; 474: 54-60Crossref PubMed Scopus (303) Google Scholar). These studies firmly establish the structural architecture of the pLGIC superfamily, in which all members adopt a similar quaternary structure formed from five subunits (Figure 1). The basic mechanism that emerges from accumulated structural, biochemical, and physiological studies is that agonist binding triggers a structural transition from a channel-closed to a channel-open conformation, followed by a relatively slow transition to an agonist-unresponsive desensitized state (see Figure 6A). Despite increasing structural data obtained in both the presence and absence of bound agonist, however, our understanding of channel activation remains clouded by our inability to unequivocally relate pLGIC structures to these canonical conformations. With this in mind, we introduce the pLGIC superfamily, review current pLGIC structures, and focus on the insight these structures provide into the conformational changes underlying agonist-induced channel gating. We then review evidence supporting the interpretation of each structure in terms of a specific conformation and discuss plausible causes for discrepancies. We also explore the possible consequences of detergent-solubilization on pLGIC structure. We conclude that a better understanding of the effects of lipids/detergents on pLGIC structure will be necessary to advance the structural biology of these important receptors. The identification of an abundant source of a "nicotinic receptive substance" in the electroplax tissues of both electric eels, Electrophorus, and electric fish, Torpedo (Changeux et al., 1970Changeux J.P. Kasai M. Lee C.Y. Use of a snake venom toxin to characterize the cholinergic receptor protein.Proc. Natl. Acad. Sci. USA. 1970; 67: 1241-1247Crossref PubMed Google Scholar, Miledi et al., 1971Miledi R. Molinoff P. Potter L.T. Isolation of the cholinergic receptor protein of Torpedo electric tissue.Nature. 1971; 229: 554-557Crossref PubMed Scopus (131) Google Scholar), originally led to the extensive biochemical characterization of the nAChR. The nAChR is an acetylcholine-gated, cation-selective ion channel. It is a heteropentamer, with a subunit stoichiometry of α2βγδ (Weill et al., 1974Weill C.L. McNamee M.G. Karlin A. Affinity-labeling of purified acetylcholine receptor from Torpedo californica.Biochem. Biophys. Res. Commun. 1974; 61: 997-1003Crossref PubMed Scopus (61) Google Scholar). Each subunit contains a large N-terminal ABD, a transmembrane domain (TMD) consisting of four membrane-spanning α helices (M1–M4), and a cytoplasmic domain, consisting of an amphipathic helix, MA, located between M3 and M4 (Finer-Moore and Stroud, 1984Finer-Moore J. Stroud R.M. Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor.Proc. Natl. Acad. Sci. USA. 1984; 81: 155-159Crossref PubMed Google Scholar). The M2 α helix from each subunit lines the ion channel pore (Akabas et al., 1994Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit.Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (314) Google Scholar, Akabas et al., 1992Akabas M.H. Stauffer D.A. Xu M. Karlin A. 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Key residues in the agonist binding site, including conserved aromatic residues and a vicinal disulfide, were located in loops "A" to "F" at the interfaces between the α/γ and α/δ subunits (Figure 2A). Further work led to the discovery that the Torpedo nAChR is part of a family of nicotinic receptors that includes both muscle and neuronal nAChRs (Changeux, 2012Changeux J.P. The nicotinic acetylcholine receptor: the founding father of the pentameric ligand-gated ion channel superfamily.J. Biol. Chem. 2012; 287: 40207-40215Crossref PubMed Scopus (33) Google Scholar, Taly and Changeux, 2008Taly A. Changeux J.P. Functional organization and conformational dynamics of the nicotinic receptor: a plausible structural interpretation of myasthenic mutations.Ann. N Y Acad. Sci. 2008; 1132: 42-52Crossref PubMed Scopus (9) Google Scholar). In humans, this nicotinic family is part of an even broader superfamily comprised of both excitatory (acetylcholine and 5-HT3) and inhibitory (glycine and GABAA/GABAC) channels, in which all members exhibit a similar pentameric architecture (Schofield et al., 1987Schofield P.R. Darlison M.G. Fujita N. Burt D.R. Stephenson F.A. Rodriguez H. Rhee L.M. Ramachandran J. Reale V. Glencorse T.A. et al.Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family.Nature. 1987; 328: 221-227Crossref PubMed Scopus (698) Google Scholar, Sine and Engel, 2006Sine S.M. Engel A.G. Recent advances in Cys-loop receptor structure and function.Nature. 2006; 440: 448-455Crossref PubMed Scopus (300) Google Scholar). Collectively, this superfamily is referred to as the "Cys-loop receptors," because each subunit contains a conserved 13 residue loop (the β6–β7, see below) in the ABD that is bracketed by two cysteine residues. The diversity of Cys-loop receptor subunits leads to a wealth of structural, functional, and pharmacological diversity. Several homopentameric prokaryotic homologs of the Cys-loop receptors have since been identified (Tasneem et al., 2005Tasneem A. Iyer L.M. Jakobsson E. Aravind L. Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels.Genome Biol. 2005; 6: R4Crossref PubMed Google Scholar). These bacterial counterparts lack the cysteine residues that frame the Cys-loop, hence the increasing use of the term pLGIC to describe the entire superfamily of channels. Despite considerable effort (Hertling-Jaweed et al., 1988Hertling-Jaweed S. Bandini G. Müller-Fahrnow A. Dommes V. Hucho F. Rapid preparation of the nicotinic acetylcholine receptor for crystallization in detergent solution.FEBS Lett. 1988; 241: 29-32Crossref PubMed Scopus (9) Google Scholar, Paas et al., 2003Paas Y. Cartaud J. 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Atomic resolution insight into the nAChR ABD began to emerge with the discovery of the so-called acetylcholine binding proteins (AChBP), water-soluble homologs of the nAChR ABD (Smit et al., 2001Smit A.B. Syed N.I. Schaap D. van Minnen J. Klumperman J. Kits K.S. Lodder H. van der Schors R.C. van Elk R. Sorgedrager B. et al.A glia-derived acetylcholine-binding protein that modulates synaptic transmission.Nature. 2001; 411: 261-268Crossref PubMed Scopus (358) Google Scholar). To date, structures have been solved for four AChBP orthologs in both the presence and absence of agonists and competitive antagonists (Billen et al., 2012Billen B. Spurny R. Brams M. van Elk R. Valera-Kummer S. Yakel J.L. Voets T. Bertrand D. Smit A.B. Ulens C. Molecular actions of smoking cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein.Proc. Natl. Acad. Sci. USA. 2012; 109: 9173-9178Crossref PubMed Scopus (25) Google Scholar, Brejc et al., 2001Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.Nature. 2001; 411: 269-276Crossref PubMed Scopus (1278) Google Scholar, Celie et al., 2005Celie P.H. Klaassen R.V. van Rossum-Fikkert S.E. van Elk R. van Nierop P. Smit A.B. Sixma T.K. 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. 2005; 280: 26457-26466Crossref PubMed Scopus (135) Google Scholar, Hansen et al., 2005Hansen S.B. Sulzenbacher G. Huxford T. Marchot P. Taylor P. Bourne Y. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations.EMBO J. 2005; 24: 3635-3646Crossref PubMed Scopus (395) Google Scholar). The AChBP structures are all homopentamers. Each subunit exhibits a conserved tertiary fold consisting of a short N-terminal α helix followed by a ten-strand β sandwich core. The β sandwich is formed from an inner β sheet of six strands (β1, β2, β3, β5, β6, and β8) that is located toward the central axis of the pentamer and an outer sheet of four strands (β4, β7, β9, and β10) that is located distally from the central axis (Figure 2). This same tertiary fold is conserved in structures of the water-soluble ABDs of the mouse muscle α1 subunit (Dellisanti et al., 2007Dellisanti C.D. Yao Y. Stroud J.C. Wang Z.Z. Chen L. Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution.Nat. Neurosci. 2007; 10: 953-962Crossref PubMed Scopus (236) Google Scholar), the prokaryotic pLGIC, GLIC (Nury et al., 2010Nury H. Bocquet N. Le Poupon C. Raynal B. Haouz A. Corringer P.J. Delarue M. Crystal structure of the extracellular domain of a bacterial ligand-gated ion channel.J. Mol. Biol. 2010; 395: 1114-1127Crossref PubMed Scopus (23) Google Scholar), and an α-7 nAChR ABD/AChBP chimera (Li et al., 2011Li S.X. Huang S. Bren N. Noridomi K. Dellisanti C.D. Sine S.M. Chen L. Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.Nat. Neurosci. 2011; 14: 1253-1259Crossref PubMed Scopus (44) Google Scholar). The same tertiary fold is also observed in the full-length structures of the prokaryotic pLGICs, GLIC and ELIC, and the C. elegans, GluCl. The agonist-bound structures of the AChBP confirm that the agonist-binding sites are formed by loops from both the principle and complementary subunits (Figure 2A) (Brejc et al., 2001Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.Nature. 2001; 411: 269-276Crossref PubMed Scopus (1278) Google Scholar, Sine, 2002Sine S.M. The nicotinic receptor ligand binding domain.J. Neurobiol. 2002; 53: 431-446Crossref PubMed Scopus (133) Google Scholar). Conserved aromatic residues, at positions analogous to those in loops A, B, and C on the principle face of the nAChR α subunit, form an "aromatic box" that surrounds the charged amine of bound agonists. AChBP-specific contributions to agonist binding are located on the complementary face of the agonist site and account for variations in agonist affinity between the different AChBP orthologs and between AChBP and different nAChRs (Hansen et al., 2004Hansen S.B. Talley T.T. Radic Z. Taylor P. Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica.J. Biol. Chem. 2004; 279: 24197-24202Crossref PubMed Scopus (106) Google Scholar, Rucktooa et al., 2009Rucktooa P. Smit A.B. Sixma T.K. Insight in nAChR subtype selectivity from AChBP crystal structures.Biochem. Pharmacol. 2009; 78: 777-787Crossref PubMed Scopus (47) Google Scholar, Smit et al., 2001Smit A.B. Syed N.I. Schaap D. van Minnen J. Klumperman J. Kits K.S. Lodder H. van der Schors R.C. van Elk R. Sorgedrager B. et al.A glia-derived acetylcholine-binding protein that modulates synaptic transmission.Nature. 2001; 411: 261-268Crossref PubMed Scopus (358) Google Scholar). AChBP has proven to be an excellent structural surrogate for probing agonist recognition (Cromer et al., 2002Cromer B.A. Morton C.J. Parker M.W. Anxiety over GABA(A) receptor structure relieved by AChBP.Trends Biochem. Sci. 2002; 27: 280-287Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, Reeves and Lummis, 2002Reeves D.C. Lummis S.C. The molecular basis of the structure and function of the 5-HT3 receptor: a model ligand-gated ion channel (review).Mol. Membr. Biol. 2002; 19: 11-26Crossref PubMed Scopus (149) Google Scholar, Rucktooa et al., 2009Rucktooa P. Smit A.B. Sixma T.K. Insight in nAChR subtype selectivity from AChBP crystal structures.Biochem. Pharmacol. 2009; 78: 777-787Crossref PubMed Scopus (47) Google Scholar), especially for the homopentameric α7 nAChR with which it shares a similar pharmacological profile (Brejc et al., 2001Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.Nature. 2001; 411: 269-276Crossref PubMed Scopus (1278) Google Scholar, Smit et al., 2001Smit A.B. Syed N.I. Schaap D. van Minnen J. Klumperman J. Kits K.S. Lodder H. van der Schors R.C. van Elk R. Sorgedrager B. et al.A glia-derived acetylcholine-binding protein that modulates synaptic transmission.Nature. 2001; 411: 261-268Crossref PubMed Scopus (358) Google Scholar). Agonist binding to AChBP elicits conformational change, suggesting that AChBP could serve as a surrogate for studying the structural rearrangements in the binding site that initiate pLGIC gating (Bourne et al., 2005Bourne Y. Talley T.T. Hansen S.B. Taylor P. Marchot P. Crystal structure of a Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors.EMBO J. 2005; 24: 1512-1522Crossref PubMed Scopus (185) Google Scholar, Hansen et al., 2002Hansen S.B. Radic' Z. Talley T.T. Molles B.E. Deerinck T. Tsigelny I. Taylor P. Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein.J. Biol. Chem. 2002; 277: 41299-41302Crossref PubMed Scopus (70) Google Scholar, Hansen et al., 2005Hansen S.B. Sulzenbacher G. Huxford T. Marchot P. Taylor P. Bourne Y. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations.EMBO J. 2005; 24: 3635-3646Crossref PubMed Scopus (395) Google Scholar). Support for this was obtained with the creation of a functional chimera between AChBP and the TMD of the 5-HT3 receptor (Bouzat et al., 2004Bouzat C. Gumilar F. Spitzmaul G. Wang H.L. Rayes D. Hansen S.B. Taylor P. Sine S.M. Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel.Nature. 2004; 430: 896-900Crossref PubMed Scopus (189) Google Scholar), although function was only obtained after replacement of the three TMD-facing loops, β1–β2, β6–β7, and β8–β9, of AChBP with the corresponding loops of the 5-HT3 receptor. Nevertheless, this chimera suggests that the conformational changes induced in AChBP upon agonist binding resemble those leading to gating in full-length pLGICs. The structural changes elicited by agonist binding to the AChBP orthologs, as well as to the α7/AChBP chimera, suggest common themes. First, residues in both the agonist-binding pocket and the two β sheets exhibit conformational flexibility but lock into a defined conformation in the agonist-bound state. This is most notable with the agonist-site C-loop, which in the absence of agonist extends tangentially from the protein exposing the binding site to solvent (Figure 3A). The precise position of the C-loop, however, varies both between structures and within subunits of individual structures (Li et al., 2011Li S.X. Huang S. Bren N. Noridomi K. Dellisanti C.D. Sine S.M. Chen L. Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.Nat. Neurosci. 2011; 14: 1253-1259Crossref PubMed Scopus (44) Google Scholar). In fact, the C-loop is most open when AChBP is complexed with neurotoxin antagonists, which are thought to stabilize the resting state (Baenziger et al., 1992Baenziger J.E. Miller K.W. Rothschild K.J. Incorporation of the nicotinic acetylcholine receptor into planar multilamellar films: characterization by fluorescence and Fourier transform infrared difference spectroscopy.Biophys. J. 1992; 61: 983-992Abstract Full Text PDF PubMed Google Scholar, Herz et al., 1987Herz J.M. Johnson D.A. Taylor P. Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding.J. Biol. Chem. 1987; 262: 7238-7247Abstract Full Text PDF PubMed Google Scholar, Moore and McCarthy, 1995Moore M.A. McCarthy M.P. Snake venom toxins, unlike smaller antagonists, appear to stabilize a resting state conformation of the nicotinic acetylcholine receptor.Biochim. Biophys. Acta. 1995; 1235: 336-342Crossref PubMed Scopus (38) Google Scholar). Upon agonist binding, the C-loop moves toward the channel pore, thus capping the bound agonist, as suggested by both molecular dynamics (MD) simulations and fluorescence quenching studies (Gao et al., 2005Gao F. Bren N. Burghardt T.P. Hansen S. Henchman R.H. Taylor P. McCammon J.A. Sine S.M. Agonist-mediated conformational changes in acetylcholine-binding protein revealed by simulation and intrinsic tryptophan fluorescence.J. Biol. Chem. 2005; 280: 8443-8451Crossref PubMed Scopus (100) Google Scholar). Agonist binding draws together aromatic residues to stabilize the quaternary ammonium of acetylcholine and recruits additional residues to mediate signal transduction (Li et al., 2011Li S.X. Huang S. Bren N. Noridomi K. Dellisanti C.D. Sine S.M. Chen L. Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.Nat. Neurosci. 2011; 14: 1253-1259Crossref PubMed Scopus (44) Google Scholar). In particular, aromatic residues in loops A (β7 Tyr91) and C (β7 Tyr184) move toward the anchoring loop B Trp residue (β7 Trp145) in the aromatic box. The F-loop in the complementary subunit moves toward the bound agonist. These local changes propagate to the rest of the protein primarily by subtle rotations of the outer β sheet, leading to a repacking of the β sandwich core. The repacking of the β sandwich is highlighted by a rotamer switch of a conserved aromatic (Phe or Tyr) residue in β10 (β7 Phe196). These rotations cause the outer β sheet to bend toward the central pore axis, while the position of the inner β sheet remains essentially unchanged. Although both the detected movements of the C-loop and the altered packing of the β sandwich core are conserved features in the gating of the Torpedo nAChR, a complete description of the conformational pathway leading from the agonist site to the TMD has, unfortunately, not been obtained. This is partly due to the fact that structures of AChBP cannot be unequivocally attributed to the resting and open conformations observed with full-length pLGICs. For example, some of the Apo AChBP structures exhibit solute electron density in the aromatic box, leading to the suggestion that they resemble more closely a desensitized state (Brejc et al., 2001Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.Nature. 2001; 411: 269-276Crossref PubMed Scopus (1278) Google Scholar, Celie et al., 2005Celie P.H. Klaassen R.V. van Rossum-Fikkert S.E. van Elk R. van Nierop P. Smit A.B. Sixma T.K. 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. 2005; 280: 26457-26466Crossref PubMed Scopus (135) Google Scholar, Grutter and Changeux, 2001Grutter T. Changeux J.P. Nicotinic receptors in wonderland.Trends Biochem. Sci. 2001; 26: 459-463Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Even Apo AChBP structures that lack solute electron density in the agonist site undergo a further opening of the C-loop upon binding toxins, which are thought to stabilize the resting nAChR (Figure 3B). In addition, the formation of a resting state-like AChBP/5-HT3 chimera was only achieved when AChBP was appropriately complexed with a complementary TMD (Bouzat et al., 2004Bouzat C. Gumilar F. Spitzmaul G. Wang H.L. Rayes D. Hansen S.B. Taylor P. Sine S.M. Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel.Nature. 2004; 430: 896-900Crossref PubMed Scopus (189) Google Scholar). As the presence of a TMD appears to influence the conformations of the ABD, soluble AChBPs cannot offer definitive insight into the structural changes leading to pLGIC gating. Nigel Unwin and colleagues have used cryo-electron microscopy (cryo-EM) since 1984 to image membrane-imbedded nAChR-enriched tubes formed from native Torpedo elelctroplaque membranes. Extensive studies culminated in a 4.0 Å resolution structure of the closed/resting nAChR pore in 2003 (Miyazawa et al., 2003Miyazawa A. Fujiyoshi Y. Unwin N. Structure and gating mechanism of the acetylcholine receptor pore.Nature. 2003; 423: 949-955Crossref PubMed Scopus (837) Google Scholar), which was subsequently refined by incorporating the AChBP crystal structure in 2005 (Unwin, 2005Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4A resolution.J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (993) Google Scholar). The 4.0 Å model provided the first direct insight into the structure of the TMD, confirming the existence of four membrane-spanning α helices in each subunit, as initially predicted by hydropathy plots (Schofield et al., 1987Schofield P.R. Darlison M.G. Fujita N. Burt D.R. Stephenson F.A. Rodriguez H. Rhee L.M. Ramachandran J. Reale V. Glencorse T.A. et al.Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family.Nature. 1987; 328: 221-227Crossref PubMed Scopus (698) Google Scholar). The α-helical nature of the TMD was subsequently reinforced by nuclear magnetic resonance (NMR) structures of the TMD of the neuronal α4β2 nAChR (Bondarenko et al., 2012Bondarenko V. Mowrey D. Tillman T. Cui T. Liu L.T. Xu Y. Tang P. NMR structures of the transmembrane domains of the α4β2 nAChR.Biochim. Biophys. Acta. 2012; 1818: 1261-1268Crossref PubMed Scopus (17) Google Scholar, Bondarenko et al., 2013Bondarenko V. Mowrey D. Liu L.T. Xu Y. Tang P. NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR.Biochim. Biophys. Acta. 2013; 1828: 398-404Crossref PubMed Scopus (7) Google Scholar) and by crystal structures of full-length prokaryotic pLGICs (see below). Unwin's refined model highlights the structurally distinct ABD, TMD, and cytoplasmic domains, although much of the latter structure is still unresolved. Unlike the AChBP, the nAChR is a heteropentamer formed from four different but homologous subunits. The conformations of the β-, γ-, and δ-subunit ABDs are similar to the agonist-bound AChBP protomer, whereas the ABDs of the two α subunits adopt a distinct "strained" conformation. In the α subunits, the inner β sheets are rotated counterclockwise relative to the other subunits (Figure 3B) (Unwin, 2005Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4A resolution.J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (993) Google Scholar, Unwin et al., 2002Unwin N. Miyazawa A. 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