Gating of Pentameric Ligand-Gated Ion Channels: Structural Insights and Ambiguities

Structure Review

Corrie J.B. daCosta1 and John E. Baenziger2,* 1Receptor Biology Laboratory, Departments of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905, USA 2Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.06.019

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), pLGIC structure will be necessary to advance the structural serotonin (5-HT3), glycine, and GABAA/GABAC receptors, biology of these important receptors. mediate fast chemical to electrical transduction at synapses throughout the nervous system. Insight into Cys-loop receptor From the nAChR to Cys-Loop Receptors and the pLGIC structure has expanded rapidly in recent years with structures Superfamily of water-soluble homologs of the acetylcholine receptor The identification of an abundant source of a ‘‘nicotinic receptive agonist-binding domain (ABD) (Brejc et al., 2001; Celie et al., substance’’ in the electroplax tissues of both electric eels, Elec- 2005; Dellisanti et al., 2007; Hansen et al., 2005; Li et al., trophorus, and electric fish, Torpedo (Changeux et al., 1970; 2011), the Torpedo nAChR (Unwin, 2005), the prokaryotic pen- Miledi et al., 1971), originally led to the extensive biochemical tameric ligand-gated ion channels (pLGICs), Erwinia ligand- characterization of the nAChR. The nAChR is an acetylcholine- gated ion channel (ELIC) (Hilf and Dutzler, 2008) and Gloebacter gated, cation-selective ion channel. It is a heteropentamer, ligand-gated ion channel (GLIC) (Bocquet et al., 2009; Hilf and with a subunit stoichiometry of a2bgd (Weill et al., 1974). Each Dutzler, 2009), and the Caenorhabditis elegans glutamate-acti- subunit contains a large N-terminal ABD, a transmembrane vated chloride channel (GluCl) (Hibbs and Gouaux, 2011). These domain (TMD) consisting of four membrane-spanning a helices studies firmly establish the structural architecture of the pLGIC (M1–M4), and a cytoplasmic domain, consisting of an amphi- superfamily, in which all members adopt a similar quaternary pathic helix, MA, located between M3 and M4 (Finer-Moore structure formed from five subunits (Figure 1). and Stroud, 1984). The M2 a helix from each subunit lines the The basic mechanism that emerges from accumulated struc- ion channel pore (Akabas et al., 1994; Akabas et al., 1992; Imoto tural, biochemical, and physiological studies is that agonist et al., 1986), whereas M4 faces the lipid bilayer (Blanton and binding triggers a structural transition from a channel-closed to Cohen, 1992, 1994). Key residues in the agonist binding site, a channel-open conformation, followed by a relatively slow including conserved aromatic residues and a vicinal disulfide, transition to an agonist-unresponsive desensitized state (see were located in loops ‘‘A’’ to ‘‘F’’ at the interfaces between the Figure 6A). Despite increasing structural data obtained in both a/g and a/d subunits (Figure 2A). the presence and absence of bound agonist, however, our un- Further work led to the discovery that the Torpedo nAChR is derstanding of channel activation remains clouded by our part of a family of nicotinic receptors that includes both muscle inability to unequivocally relate pLGIC structures to these canon- and neuronal nAChRs (Changeux, 2012; Taly and Changeux, ical conformations. With this in mind, we introduce the pLGIC 2008). In humans, this nicotinic family is part of an even broader superfamily, review current pLGIC structures, and focus on the superfamily comprised of both excitatory (acetylcholine and insight these structures provide into the conformational changes 5-HT3) and inhibitory (glycine and GABAA/GABAC) channels, in underlying agonist-induced channel gating. We then review evi- which all members exhibit a similar pentameric architecture dence supporting the interpretation of each structure in terms of (Schofield et al., 1987; Sine and Engel, 2006). Collectively, this a specific conformation and discuss plausible causes for dis- superfamily is referred to as the ‘‘Cys-loop receptors,’’ because crepancies. We also explore the possible consequences of each subunit contains a conserved 13 residue loop (the b6–b7, detergent-solubilization on pLGIC structure. We conclude that see below) in the ABD that is bracketed by two cysteine residues. a better understanding of the effects of lipids/detergents on The diversity of Cys-loop receptor subunits leads to a wealth of

Figure 1. The Conserved Structural Architecture of pLGICs (A) Structure of the nicotinic acetylcholine receptor (nAChR) from Torpedo (Protein Data Bank [PDB] ID code 2BG9). A side view of the nAChR pentamer looking from the membrane plane (left) shows the extracellular agonist-binding (ABD, red), transmembrane pore (TMD, blue) and cytoplasmic (CD, green) domains. Side chains of residues forming part of the agonist-binding site (aW149) and the channel gate (aL251 and aV255, as well as analogous b-, g- and d-subunit residues) are shown as tan and yellow spheres, respectively. Views from the synaptic cleft are shown on the right for the ABD (top) and TMD (bottom) with color coding to highlight the different subunits (a, red; b, blue; g, purple; d, green). (B) Structures of the prokaryotic pLGICs, ELIC (left; PDB ID code 2VLO) and GLIC (center; PDB ID code 3EAM), and the C. elegans GluCl (PDB ID code 3RIF), colored as in (A) to highlight the ABD and TMD. Crystallization of GluCl required elimination of residues from both the N- and C termini, as well as replacement of the cytoplasmic domain (Hibbs and Gouaux, 2011). Residues forming the narrowest constriction of the pore in ELIC (L239 and F246) and at analogous positions in GLIC (I233 and I240/L241) and GluCl (L254 and A261/G262) are shown as yellow spheres. structural, functional, and pharmacological diversity. Several 2002). Conserved aromatic residues, at positions analogous to homopentameric prokaryotic homologs of the Cys-loop recep- those in loops A, B, and C on the principle face of the nAChR a tors have since been identified (Tasneem et al., 2005). These subunit, form an ‘‘aromatic box’’ that surrounds the charged bacterial counterparts lack the cysteine residues that frame the amine of bound agonists. AChBP-specific contributions to Cys-loop, hence the increasing use of the term pLGIC to agonist binding are located on the complementary face of the describe the entire superfamily of channels. agonist site and account for variations in agonist affinity between the different AChBP orthologs and between AChBP and different The Acetylcholine Binding Proteins nAChRs (Hansen et al., 2004; Rucktooa et al., 2009; Smit et al., Despite considerable effort (Hertling-Jaweed et al., 1988; Paas 2001). AChBP has proven to be an excellent structural surrogate et al., 2003; Padilla-Morales et al., 2011), the Torpedo nAChR for probing agonist recognition (Cromer et al., 2002; Reeves and has proven refractory to crystallization, limiting its use for X-ray Lummis, 2002; Rucktooa et al., 2009), especially for the homo- crystallographic studies of pLGIC structure. Atomic resolution pentameric a 7 nAChR with which it shares a similar pharmaco- insight into the nAChR ABD began to emerge with the discovery logical profile (Brejc et al., 2001; Smit et al., 2001). of the so-called acetylcholine binding proteins (AChBP), water- Agonist binding to AChBP elicits conformational change, sug- soluble homologs of the nAChR ABD (Smit et al., 2001). To gesting that AChBP could serve as a surrogate for studying the date, structures have been solved for four AChBP orthologs in structural rearrangements in the binding site that initiate pLGIC both the presence and absence of agonists and competitive gating (Bourne et al., 2005; Hansen et al., 2002; Hansen et al., antagonists (Billen et al., 2012; Brejc et al., 2001; Celie et al., 2005). Support for this was obtained with the creation of a func-

2005; Hansen et al., 2005). The AChBP structures are all homo- tional chimera between AChBP and the TMD of the 5-HT3 recep- pentamers. Each subunit exhibits a conserved tertiary fold con- tor (Bouzat et al., 2004), although function was only obtained sisting of a short N-terminal a helix followed by a ten-strand b after replacement of the three TMD-facing loops, b1–b2, b6– sandwich core. The b sandwich is formed from an inner b sheet b7, and b8–b9, of AChBP with the corresponding loops of the of six strands (b1, b2, b3, b5, b6, and b8) that is located toward 5-HT3 receptor. Nevertheless, this chimera suggests that the the central axis of the pentamer and an outer sheet of four conformational changes induced in AChBP upon agonist binding strands (b4, b7, b9, and b10) that is located distally from the resemble those leading to gating in full-length pLGICs. central axis (Figure 2). This same tertiary fold is conserved in The structural changes elicited by agonist binding to the structures of the water-soluble ABDs of the mouse muscle a1 AChBP orthologs, as well as to the a7/AChBP chimera, suggest subunit (Dellisanti et al., 2007), the prokaryotic pLGIC, GLIC common themes. First, residues in both the agonist-binding (Nury et al., 2010), and an a-7 nAChR ABD/AChBP chimera (Li pocket and the two b sheets exhibit conformational flexibility et al., 2011). The same tertiary fold is also observed in the full- but lock into a defined conformation in the agonist-bound state. length structures of the prokaryotic pLGICs, GLIC and ELIC, This is most notable with the agonist-site C-loop, which in the and the C. elegans, GluCl. absence of agonist extends tangentially from the protein The agonist-bound structures of the AChBP confirm that the exposing the binding site to solvent (Figure 3A). The precise agonist-binding sites are formed by loops from both the principle position of the C-loop, however, varies both between structures and complementary subunits (Figure 2A) (Brejc et al., 2001; Sine, and within subunits of individual structures (Li et al., 2011). In

Figure 2. The Agonist Binding Domain (A) Residues contributing to the agonist binding site of the Torpedo nAChR were identified by biochemical studies on noncontiguous loops A, B, and C from the principle (+) (Dennis et al., 1988; Fu and Sine, 1994; Galzi et al., 1990; Kao and Karlin, 1986; Middleton and Cohen, 1991; Silman and Karlin, 1969), and loops D, E, F from the complimentary (À)(Corringer et al., 1995; Czajkowski et al., 1993; Fu and Sine, 1994; Martin et al., 1996; O’Leary et al., 1994; Prince and Sine, 1996; Sine et al., 1995), subunits (top panel). Cartoon modified from Corringer et al. (2000). A close-up view of the agonist binding site of Lymnaea stagnalis AChBP with bound agonist; carbamylcholine (PDB ID code 1UV6) is shown in the lower panel, looking at the binding site from the side (same view as in B). The principle and complimentary faces are highlighted in yellow and blue, respectively. Interacting residues are highlighted as sticks. The polypeptide backbone is shown as a transparent cartoon. Carbamylcholine is highlighted as multicolored spheres. Aromatic residues interact with the quaternary amine of acetylcholine/ carbamylcholine via cation-p electron interactions (Dougherty, 1996; Fu and Sine, 1994). (B) Structure and tertiary fold of the ‘‘Holo’’ form of L. stagnalis AChBP (PDB ID code 1I9B) viewed from both the side and the top (top and bottom panels, respectively). Subunits are colored to highlight the pentameric quaternary structure. A HEPES molecule bound to one site is highlighted as multicolored spheres in the side view. (C) The tertiary fold of a single AChBP subunit shown in an orientation similar to that of the yellow subunit in the top panel of (B). The b strands comprising the inner and outer b sheets are highlighted in blue and red, respectively. The eponymous Cys-loop is green, whereas the C-loop is yellow. Both the vicinal disulfide bond in the C-loop and the disulfide bond closing off the Cys-loop are shown as orange sticks. A topology map of the AChBP protomer is shown in the bottom panel with the same color scheme, highlighting loops A–F. fact, the C-loop is most open when AChBP is complexed with of the outer b sheet, leading to a repacking of the b sandwich neurotoxin antagonists, which are thought to stabilize the resting core. The repacking of the b sandwich is highlighted by a rotamer state (Baenziger et al., 1992; Herz et al., 1987; Moore and switch of a conserved aromatic (Phe or Tyr) residue in b10 (b7 McCarthy, 1995). Upon agonist binding, the C-loop moves Phe196). These rotations cause the outer b sheet to bend toward toward the channel pore, thus capping the bound agonist, as the central pore axis, while the position of the inner b sheet re- suggested by both molecular dynamics (MD) simulations and mains essentially unchanged. fluorescence quenching studies (Gao et al., 2005). Although both the detected movements of the C-loop and the Agonist binding draws together aromatic residues to stabilize altered packing of the b sandwich core are conserved features in the quaternary ammonium of acetylcholine and recruits addi- the gating of the Torpedo nAChR, a complete description of the tional residues to mediate signal transduction (Li et al., 2011). conformational pathway leading from the agonist site to the TMD In particular, aromatic residues in loops A (b7 Tyr91) and C (b7 has, unfortunately, not been obtained. This is partly due to the Tyr184) move toward the anchoring loop B Trp residue (b7 fact that structures of AChBP cannot be unequivocally attributed Trp145) in the aromatic box. The F-loop in the complementary to the resting and open conformations observed with full-length subunit moves toward the bound agonist. These local changes pLGICs. For example, some of the Apo AChBP structures exhibit propagate to the rest of the protein primarily by subtle rotations solute electron density in the aromatic box, leading to the

Figure 3. Structural Transitions of the Agonist Binding Domain (A) Ligand-induced conformational change in the Aplysia californica AChBP, highlighting movement of the C-loop (colored). The structures on the top left (PDB ID code 2BYP) and top right (PDB ID code 2BYQ) are with the bound peptidic a-conotoxin antagonist, IMI, and the bound agonist, epibatidine, respectively. In both structures, loops and a helices at the top of each subunit have been removed to provide an unobstructed view of the C-loop. Below, the agonist sites of individual protomers in the presence of IMI, the absence of ligand (PDB ID code 2BYN), and in the presence of epibatidine are super- imposed with their C-loops colored blue, transparent gray, and red, respectively. Epibatidine is highlighted as multicolored spheres. The arrow shows the movement of the C-loop upon agonist binding. (B) Superposition of the Torpedo a- and d-subunit (PDB ID code 2BG9). Vibrant colors were chosen for the a subunit, whereas the corresponding pale colors were used for the d-subunit. Arrows show the movements of the inner and outer b sheets required to superimpose the non-a-subunit (in this case d) onto the ‘‘strained’’/unliganded a subunit. The top of the transmembrane a helices and the M2–M3 linker (red) are shown for the a subunit, with a conserved Pro residue (aP286) highlighted as red spheres. suggestion that they resemble more closely a desensitized state sheets are rotated counterclockwise relative to the other sub- (Brejc et al., 2001; Celie et al., 2005; Grutter and Changeux, units (Figure 3B) (Unwin, 2005; Unwin et al., 2002). In addition, 2001). Even Apo AChBP structures that lack solute electron den- the C-loops in the a subunits are extended, similar to that seen sity in the agonist site undergo a further opening of the C-loop in unliganded AChBP structures (Celie et al., 2004; Hansen upon binding toxins, which are thought to stabilize the resting et al., 2005; Unwin et al., 2002). These differences led to the sug- nAChR (Figure 3B). In addition, the formation of a resting state- gestion that agonist binding leads to closure of the C-loop and like AChBP/5-HT3 chimera was only achieved when AChBP relaxation of the inner b sheets to a more non-a/AChBP-like was appropriately complexed with a complementary TMD (Bou- conformation. Because the inner sheet of the ABD is directly zat et al., 2004). As the presence of a TMD appears to influence above the channel lining M2 a helix, it was originally suggested the conformations of the ABD, soluble AChBPs cannot offer that a change in structure of this b sheet couples directly to a definitive insight into the structural changes leading to pLGIC change in structure of the pore. Coupling was proposed to be gating. mediated primarily by interactions between aVal46 of the b1– b2 loop and aPro272 of the M2–M3 linker. More recent struc- The Torpedo nAChR tures, however, suggest that asymmetric motions of the different Nigel Unwin and colleagues have used cryo-electron micro- subunits ultimately lead to relatively large changes in conforma- scopy (cryo-EM) since 1984 to image membrane-imbedded tion of the b subunit, which plays the key role in transmitting nAChR-enriched tubes formed from native Torpedo elelctropla- changes from the agonist-binding site to the channel gate que membranes. Extensive studies culminated in a 4.0 A˚ resolu- (Unwin and Fujiyoshi, 2012). tion structure of the closed/resting nAChR pore in 2003 Several loops at the interface between the ABD and the TMD (Miyazawa et al., 2003), which was subsequently refined by are key to the allosteric transition that links the ABD to the chan- incorporating the AChBP crystal structure in 2005 (Unwin, nel gate (Andersen et al., 2011; Bouzat et al., 2004, 2008; Chak- 2005). The 4.0 A˚ model provided the first direct insight into the rapani et al., 2004; Grutter et al., 2005; Jha et al., 2007; Kash structure of the TMD, confirming the existence of four mem- et al., 2003; Lee et al., 2009; Lee and Sine, 2005; Reeves et al., brane-spanning a helices in each subunit, as initially predicted 2005; Shen et al., 2003; Xiu et al., 2005), as reviewed elsewhere by hydropathy plots (Schofield et al., 1987). The a-helical nature (Bouzat, 2012). The ABD is linked covalently to the TMD by a loop of the TMD was subsequently reinforced by nuclear magnetic between b10 and M1. The b1–b2, b6–b7 (Cys-loop), and the b8– resonance (NMR) structures of the TMD of the neuronal a4b2 b9 loops extend down toward the TMD, with the former two nAChR (Bondarenko et al., 2012, 2013) and by crystal structures directly contacting a short linker between the M2 and M3 trans- of full-length prokaryotic pLGICs (see below). Unwin’s refined membrane a helices, known as the M2–M3 linker. The M2–M3 model highlights the structurally distinct ABD, TMD, and cyto- linker is a well-established gating control element (Campos- plasmic domains, although much of the latter structure is still Caro et al., 1996; Grosman et al., 2000; Jha et al., 2007; Lee unresolved. and Sine, 2005; Lynch et al., 1997; Rovira et al., 1998, 1999). Unlike the AChBP, the nAChR is a heteropentamer formed Mutations within this linker affect nAChR apparent gating from four different but homologous subunits. The conformations kinetics without altering the nAChR’s affinity for agonist (Gros- of the b-, g-, and d-subunit ABDs are similar to the agonist-bound man et al., 2000). Unnatural amino acid substitutions suggest

AChBP protomer, whereas the ABDs of the two a subunits adopt that channel opening in the homologous 5-HT3 receptor is a distinct ‘‘strained’’ conformation. In the a subunits, the inner b directly related to the cis-trans conformational preference of an

rings of hydrophobic residues (aL251, aV255, aV259, and the corresponding residues from other subunits). At the level of aL251 and aV255, the pore constriction is both too narrow (6.0 A˚ diameter) and too hydrophobic to allow the passage of a hydrated sodium or potassium ion (8.0 A˚ ). By making it energetically unfavorable for a permeating ion to shed its solvating waters, these residues are thought to constitute a ‘‘hydrophobic gate’’ (Beckstein and Sansom, 2006; Ivanov et al., 2007; Miya- zawa et al., 2003). This constricted structure is stabilized by hydrophobic interactions between residues on adjacent M2 a helices, and structural rearrangements underlying gating are thought to break these interactions leading to a widening of the pore. Also, the narrowest constriction of the pore in the open state is closer to the cytoplasmic leaflet of the bilayer (Unwin and Fujiyoshi, 2012), consistent with a recent analysis of ion permeation through the prokaryotic homolog, GLIC (Sauguet et al., 2013b). Cryo-EM images recorded from nAChRs trapped in the open state at 9.0 A˚ resolution originally suggested that pore opening results from a clockwise rotation of the five pore lining M2 helices, causing them to collapse against the outer ring of TMD helices (Law et al., 2005; Unwin, 1995), although functional studies suggest rigid body tilting (Cymes and Grosman, 2008; Cymes et al., 2005; Paas et al., 2005). Both models are consistent with the observation that the relative stability of the open state is dependent upon the volume and stereochemistry of residues within M3 (Wang et al., 1999). More recent and higher resolution images suggest that although each of the pore lining M2 a Figure 4. The Transmembrane Pore helices move most in the extracellular leaflet, individual M2s (A) Right: side view surface diagram of the Torpedo nAChR pore (PDB ID code exhibit distinct and complex movements (Unwin and Fujiyoshi, 2BG9) with the g-subunit removed to provide an unobstructed view of the ion channel lumen. The M2 helices lining the pore are colored blue, whereas the 2012). Some helices tilt outward, whereas others straighten rings of negative/polar residues that play a role in ion selectivity are red. The from a ‘‘bowed’’ conformation. Although these rearrangements narrowest region of the pore corresponding to the hydrophobic gate is shown only increase the limiting diameter of the pore by about 1 A˚ , in yellow. Left: a ribbon representation of the aM2 transmembrane a helix they also diminish pore hydrophobicity by exposing previously showing the relative position of the highlighted residues lining the pore in the accompanying surface diagram. buried polar residues. These subtle, yet complex, movements (B) Top row: view looking toward the membrane surface of the transmembrane are consistent with those suggested by both MD simulations pore formed by the M2 a helices of each subunit in the Torpedo nAChR (left; and functional measurements examining gating (Beckstein and PDB ID code 2BG9), ELIC (center; PDB ID code 2VLO), and GLIC (right; PDB ID code 3EAM). The residues highlighted as yellow spheres in the nAChR and Sansom, 2004; Cymes and Grosman, 2008; Cymes et al., ELIC form the narrowest constriction of each pore. In GLIC, the highlighted 2005; Song and Corry, 2009; Wang et al., 2008, 2009). yellow residues are at positions analogous to those in ELIC. Bottom row: view Note that as the resolution of available structures has of both the pore and the interface between the agonist binding and trans- increased, so has the depth of understanding. In fact, structural membrane domains from within the plane of the membrane showing potential movements of the pore-lining a helices upon channel gating and/or uncoupling models of gating have evolved, with some original features main- (see text). For clarity, only two subunits are shown for each pLGIC (for the tained and others discarded. The evolution in our understanding nAChR two a subunits). For each subunit, the transmembrane a helices M2 of nAChR gating with increasing resolution of the solved struc- and M3 (blue), the M2–M3 linker (red), and both the b1–b2 (light blue) and b6– b7 (dark green) loops are shown. A conserved proline residue is highlighted as tures serves as a reminder that further improvements in resolu- red spheres in analogous positions of the M2–M3 linker in the nAChR (aP286), tion will likely lead to further insight. ELIC (P253), and GLIC (P247). pLGIC Crystal Structures and Comparison with the M2–M3 linker proline residue (aP272) (Lummis et al., 2005). Torpedo nAChR Unfortunately, the specific changes in conformation at the inter- A major breakthrough in the structural characterization of face between the ABD and TMD that lead to movement of the pLGICs arrived with the identification of several prokaryotic pore-lining TMD a helices remain poorly defined at the current pLGICs (Bocquet et al., 2007; Tasneem et al., 2005). Two of resolution of the open structure (6A˚ ). these, GLIC and ELIC, are cation-selective channels gated by The TMD pore is lined with the M2 a helices from each subunit. protons and primary amines (including GABA), respectively These pore lining a helices contain three rings of charged/polar (Bocquet et al., 2007; Zimmermann and Dutzler, 2011). Both residues that confer ion selectivity (aE241, aE262, aS266, and homopentamers are expressed in Escherichia coli at levels suf- the corresponding residues from other subunits) (Figure 4A). ficient for biochemical and structural studies. Both also exhibit In the nAChR’s closed conformation, the a helices bend toward drug sensitivities similar to other pLGICs (Thompson et al., the center of the pore leading to a narrow region formed by three 2012; Weng et al., 2010). GLIC and ELIC thus serve as excellent

models for probing both the mechanisms of channel activation The narrow constriction forms a hydrophobic, rather than a ste- and pLGIC-drug interactions. ric, barrier to ion flow (Beckstein and Sansom, 2006). In addition, The crystal structure of ELIC solved in 2008 (Hilf and Dutzler, the orientations of the pore lining a helices in the nAChR struc- 2008) was quickly followed by crystal structures of GLIC (Boc- ture are similar to the orientations of the pore-lining a helices in quet et al., 2009; Hilf and Dutzler, 2009) and the C. elegans, GluCl the structures of both GLIC and GluCl. In fact, the pore diameter (Hibbs and Gouaux, 2011). These structures revealed conserved of the ‘‘closed’’ nAChR resembles more closely that of the open tertiary and quaternary folds, as well as conserved motifs, such GLIC (and GluCl) than that of the closed ELIC (Figure 4B). as the rings of charged and hydrophobic residues that line the Furthermore, cryo-EM images of the nAChR in both the closed ion channel pore conferring ion selectivity and forming the chan- and open conformations suggest that the changes in orientation nel gate (Figure 4A). The prokaryotic pLGICs, however, lack both of the TMD a helices upon gating are subtle and lead to only the cysteine residues that bracket the Cys-loop and the cyto- minor changes in pore diameter—not the relatively large plasmic domain located between M3 and M4. The pore lining changes observed between the GLIC and ELIC structures (Boc- M2 a helices in the GLIC, ELIC, and GluCl structures also quet et al., 2009; Hilf and Dutzler, 2008, 2009). display a whole helix turn shift in register. Structures of ELIC, One possible interpretation is that the apparent conforma- GLIC, and GluCl have been solved in the presence of a variety tional ambiguities between the nAChR and ELIC/GLIC/GluCl of ligands (Corringer et al., 2012; Gonzalez-Gutierrez et al., structures reflect intrinsic structural differences between the 2012; Hilf et al., 2010; Nury et al., 2011; Pan et al., 2012a, pores and/or differences in the gating mechanisms of the 2012b; Sauguet et al., 2013a; Zimmermann and Dutzler, 2011; different orthologs. In an attempt to eliminate this ambiguity, Zimmermann et al., 2012). crystal structures of ELIC have been solved in both the presence The TMD Pore and absence of an activating ligand (Gonzalez-Gutierrez et al., The first crystal structure of the pLGIC, ELIC, was solved in the 2012; Zimmermann and Dutzler, 2011). Although additional elec- absence of agonist and attributed to the closed conformation tron density was observed in the agonist binding pockets of (Hilf and Dutzler, 2008). In agreement with this interpretation, ELIC, suggesting agonist binding, no structural rearrangements the TMD pore is occluded with hydrophobic side chains steri- were detected in the TMD pore. In fact, no movement of the cally blocking the pore (Figure 4B). Crystal structures of GLIC pore-lining M2 a helices was detected with mutants that prolong and GluCl were subsequently solved in the presence of acti- channel opening and exhibit no propensity to desensitize vating ligands and thus proposed to be stabilized in an open (Gonzalez-Gutierrez et al., 2012). It has been suggested that state (Bocquet et al., 2009; Hibbs and Gouaux, 2011; Hilf and the ELIC crystal structures reflect a state distinct from the ex- Dutzler, 2009). Consistent with this hypothesis, the C termini of pected closed, open, or desensitized states (Gonzalez-Gutierrez the pore-lining a helices in the GLIC and GluCl structures tilt and Grosman, 2010). away from the central channel axis (relative to the ELIC struc- The Coupling Interface between the ABD and TMD ture), leading to an increase in pore diameter suggestive of an Intimate interactions between structures at the interface open and conductive conformation (Figure 4B). In further sup- between the ABD and TMD mediate coupling of agonist binding port of an open conformation, disulfide-linked cystine mutants to channel gating. Given the ambiguity in the pore diameters of of GLIC designed to lock the channel in a closed ELIC-like ELIC/GLIC/GluCl versus the nAChR, it is intriguing to compare conformation reduce the pore diameter, demonstrating that the conformations of the loops at this interface. Subtle, but sig- closed conformations of GLIC with narrower pore diameters nificant, differences are observed between the closed ELIC exist. This latter closed structure may represent an intermediate and open GLIC/GluCl structures (Bocquet et al., 2009; Hibbs in the pathway, leading to channel gating (Prevost et al., 2012). and Gouaux, 2011; Hilf and Dutzler, 2009). Relative to ELIC, there GluCl was cocrystallized with the open channel blocker, picro- is a downward motion of the b1–b2 loop in GLIC/GluCl and a toxin. Electron density for the blocker was observed in the chan- displacement of both the b6–b7 loop and the M2–M3 linker nel pore, suggesting that the GluCl channel is open (Hibbs and away from the central pore axis so that the b1–b2 loop is now Gouaux, 2011). It should be mentioned that picrotoxin can be located on the proximal side (relative to the channel pore) of a trapped in the closed pore of the homologous GABAA receptor conserved proline (Torpedo aP272) in the M2–M3 linker (Bali and Akabas, 2007; Xu et al., 1995). Finally, a structure (Figure 4C). These movements are accompanied by an outward solved of GLIC at 2.4 A˚ reveals the detailed hydration of a sodium tilt of the C termini of the five M2 a helices, leading to an expan- ion within the pore of the open state. Computer simulations sion of the pore diameter. The largest changes in the diameter of describe the changes in hydration that occur as a sodium ion the pore occur in the extramembranous leaflet of the bilayer, transitions through the channel pore of this structure (Sauguet consistent with the most recent structural data for the open state et al., 2013b). Collectively, these data are consistent with the of the nAChR (Unwin and Fujiyoshi, 2012). assignment of the ELIC and the GLIC/GluCl structures to closed Surprisingly, the structural differences at this interface and open pore conformations, respectively. between the closed and open conformations of ELIC and GLIC The interpretations of the ELIC and the GLIC/GluCl structures are subtle in comparison to those between ELIC/GLIC and the in terms of closed and open conformations, however, are not nAChR (Figure 5A). In the nAChR, the extended side chains of easily reconcilable with the closed and open structures of the the b1–b2 and b6–b7 loops engage the M2–M3 linker in a fashion Torpedo nAChR (Figure 4B). The nAChR structure solved in the reminiscent of vice grips attached to a metal pipe. Specifically, absence of agonist is presumably in the closed conformation the side chains aE45 and aV46 in the b1–b2 loop form an inverted (Unwin, 2005). In contrast to ELIC, the closed nAChR structure ‘‘V’’ that interacts with both faces of aP272 in the M2–M3 linker. exhibits a channel pore that narrows to only 6 A˚ in diameter. The b6–b7 loop forms an inverted ‘‘Y’’ with side chains aV132

Figure 5. The Interface between the Agonist Binding and Transmembrane Domains (A–C) A single subunit of (A) the nAChR (a subunit) (PDB ID code 2BG9), (B) ELIC (PDB ID code 2VLO), and (C) GLIC (PDB ID code 3EAM) viewed from the membrane plane, highlighting the coupling interface (dashed box). In this orientation, the ion channel pore is located to the right of each subunit. The transmembrane a helices, the b1–b2 and b6– b7 loops, and the M2–M3 linker are colored as in Figure 4B. Post-M4 is colored orange. Residues potentially involved in post-M4/b6–b7 loop interactions are highlighted as sticks. Close-up views showing b1–b2/M2–M3 and b6–b7/M2–M3 interactions are shown on the left and right, respectively. The three views on the right are each from a slightly different angle that highlights the interactions between the b6–b7 loop and the M2– M3 linker in each protomer. (B) The terminal 5 residues of M4 are absent in the structure of ELIC. (C) The terminal 2 residues of M4 are absent in the structure of GLIC. A lipid that bridges post-M4 to the b6–b7 loop in GLIC is highlighted as tan spheres.

nAChR bear little resemblance to the conformations of the same loops in ELIC, GLIC, and GluCl, raising further questions regarding the conformational interpretation of the various structures. Signiﬁ- cantly, whereas the ELIC, GLIC, and GluCl structures suggest that gating results from increased interactions between the ABD and TMD upon agonist binding, the nAChR structure suggests that tight interactions between the two domains already exist in the agonist-free state.

Factors Contributing to the Conformational Ambiguity Based on both pore diameter and the geometry of the M2 a helices, the nAChR in the absence of agonist appears to be in a and aF135 extending to surround the polypeptide backbone of similar open structure to that of GLIC and GluCl, whereas the the M2–M3 linker. In contrast, substantially less contact ELIC structure appears to be closed. The assignment of the between the loops of the ABD and the M2–M3 linker of the nAChR to an open conformation, however, is not easily reconcil- TMD is observed in the ELIC, GLIC, and GluCl structures (Fig- able with the absence of an activating ligand. The relatively large ures 5B and 5C). In the latter, the b1–b2 and b6–b7 loops no differences in diameter of the channel pores between the closed longer surround the M2–M3 linker, which itself tilts down ELIC and open GLIC structures are also not consistent with the toward the membrane surface so that it approaches the b8–b9 subtle changes in nAChR pore diameter that are observed by loop on the complementary face of the adjacent subunit. The cryo-EM upon activation and that are further supported by func- side chains of the b1–b2 loop extend away from the M2–M3 tional studies and MD simulations (Beckstein and Sansom, 2004; linker ﬂattening the inverted ‘‘V’’ so that it interacts with only Cymes and Grosman, 2008; Cymes et al., 2005; Song and Corry, one surface of the conserved proline residue. In addition, the 2009; Unwin and Fujiyoshi, 2012; Wang et al., 2008, 2009). On b6–b7 loop forms an extended rod-like structure that interacts the other hand, based on the structures of the coupling interface with a single side of the M2–M3 linker. In the nAChR the b1– between the ABD and TMD, the nAChR adopts a conformation b2 loop forms numerous atom-atom contacts with the M2–M3 distinct from those of both the closed ELIC and the open linker proline that are %3.0 A˚ apart, whereas in ELIC, GLIC, GLIC/GluCl. Given that the interpretation of these structures and GluCl, the analogous contacts are typically R4.0 A˚ apart. impacts on our understanding of the mechanisms of channel These comparisons show that the conformations of the loops at activation, the question that arises is how to reconcile these the interface between the agonist-binding and TMDs in the structural differences.

One possible explanation might be the differences in resolu- GLIC did not desensitize in response to proton binding (Bocquet tion of the various structures. The closed and open nAChR struc- et al., 2007), this interpretation has been challenged (Gonzalez- tures were solved at 4.0 and 6A˚ resolution, respectively, Gutierrez and Grosman, 2010; Parikh et al., 2011; Velisetty whereas the structures of GLIC, ELIC, and GluCl were all et al., 2012). Membrane-reconstituted GLIC rapidly desensitizes, determined at higher resolution (2.4–3.3 A˚ ). Although the lower albeit at activating pH values that are below those used in crys- precision of the nAChR structures undoubtedly leads to some tallographic studies (Velisetty and Chakrapani, 2012). These ambiguity, it seems unlikely that the lower precision by itself recent findings raise the possibility that the agonist-bound struc- accounts for the relatively large differences in pore diameter tures of both GLIC and ELIC are influenced by desensitization between the closed nAChR and ELIC structures, as well as the (Parikh et al., 2011; Velisetty and Chakrapani, 2012; Velisetty substantial differences observed between the cryo-EM and et al., 2012). The possibility that GLIC and ELIC adopt a desen- crystal structures at the interface between the ABD and TMD. sitized conformation, however, does not explain the substantial In addition, the lower precision of the nAChR structures does structural differences between GLIC and ELIC, particularly in not explain the lack of structural change in the pore upon the diameters of their TMD pores. agonist-binding to ELIC (Gonzalez-Gutierrez et al., 2012; Zim- mermann and Dutzler, 2011). Lipid and Detergent Sensitivity of pLGICs Another possibility is that the structural discrepancies Another potential complication arises from the fact that the between the pLGICs reflect intrinsic structural differences and/ nAChR structure was solved by cryo-EM in its native Torpedo or distinct activation mechanisms. The open GluCl structures membrane, whereas the structures of ELIC, GLIC, and GluCl were solved in the presence of both agonist and the allosteric were each solved by X-ray diffraction in the detergent-solubilized ligand, ivermectin, which contributes to channel opening by state. Crystal packing forces may influence the conformations wedging between the M1 and M3 a helices of the TMD (Hibbs adopted by the crystallized pLGICs (Kollman et al., 2009). Deter- and Gouaux, 2011). The activating ligand of GLIC is a proton, gent molecules interact closely with the pore-lining M2 a helices which does not bind to the typical aromatic box found in the in structures of GLIC (Bocquet et al., 2009; Sauguet et al., 2013b). binding sites of other pLGICs. A protonation site in the TMD of Given that detergent-solubilization can influence the conforma- GLIC has also been implicated in gating (Wang et al., 2012), tion of any membrane protein extracted from its lipid environ- although a chimera formed between the ABD of GLIC and the ment, it is prudent to question whether the crystallized pLGIC TMD of the glycine receptor still responds to protons (Duret structures are influenced by detergent solubilization and whether et al., 2011). The activation mechanisms revealed by these struc- this might be the source of conformational ambiguity. In this tures may thus differ from the classical activation of the nAChR context, it should be noted that there are currently no published resulting from agonist binding to its canonical site. The similarity data directly characterizing the functional properties of ELIC, of the open structures of GLIC and GluCl, despite proton and GLIC, or GluCl in the detergent-solubilized state. In fact, it re- ivermectin binding to distinct sites, however, suggests a com- mains to be determined whether agonist-induced conformational mon activation mechanism for both GLIC and GluCl. Further- transitions even occur after detergent solubilization. more, the possibility of distinct activation mechanisms, and Although the conformational sensitivity of ELIC, GLIC, and thus potentially distinct closed states, does not explain the GluCl to lipids and detergent has not been extensively studied absence of conformational change in the pore structure of (Labriola et al., 2013; Velisetty and Chakrapani, 2012), lipids agonist-bound ELIC. are observed bound to the surface of crystallized GLIC In this light, it may be significant that the Torpedo nAChR is a (Figure 5C) and may thus play a role in GLIC function (Bocquet heteropentamer, whereas ELIC, GLIC, and GluCl are all homo- et al., 2009). Recent studies highlight the sensitivity of the pentamers. The proposed gating mechanism of the Torpedo GLIC TMD to allosteric modulators (Bro¨ mstrup et al., 2013; this nAChR suggests differential roles for the agonist-binding a issue of Structure). On the other hand, complex relations have versus the non-a subunits. In addition, the Torpedo nAChR re- been documented between lipids and the conformational land- quires only two bound agonist molecules for maximal stabiliza- scape of the Torpedo nAChR (Asmar-Rovira et al., 2008; Baen- tion of the open state, whereas an a7/5-HT3 homopentameric ziger and daCosta, 2013; Barrantes, 2002; Criado et al., 1984; chimera requires occupation of three agonist sites for maximal daCosta et al., 2009; Fong and McNamee, 1986; Hamouda opening (Rayes et al., 2009). There may be subtle differences et al., 2006; Rankin et al., 1997). Some reconstituted membranes in the activation mechanisms of hetero- versus homopentameric stabilize the nAChR in an activatable conformation, whereas channels. The fact that some subunits form both homo- and het- others favor either a desensitized or a distinct conformation eropentamers (Lummis, 2012), however, argues against distinct that binds agonist and other ligands with low resting-state like activation mechanisms in homomeric ELIC/GLIC/GluCl versus affinities but does not undergo agonist-induced conformational the heteromeric nAChR. change (daCosta and Baenziger, 2009). In the latter, binding The transient nature of the open state may also complicate of agonist is uncoupled from both channel gating and desensiti- interpretation of the pLGIC structures. The prolonged stabilities zation, as is apparently the case with the crystallized ELIC of the open conformations of crystallized GLIC, GluCl, and (Gonzalez-Gutierrez et al., 2012). A similar uncoupled conforma- ELIC in the presence of activating ligands have not been un- tion has been observed upon detergent solubilization of the equivocally demonstrated. ELIC undergoes desensitization after nAChR (Heidmann et al., 1980), raising the concern that deter- exposure to activating ligands, such as cystamine, although gent solubilization could have similar effects on other pLGICs. nondesensitizing mutants have been identified (Gonzalez- One defining property of the uncoupled nAChR is that re- Gutierrez et al., 2012). Although it was originally suggested that gions of the polypeptide backbone that are buried from solvent

Figure 6. The Uncoupled State (A) The nAChR from native Torpedo membranes undergoes agonist-induced conformational transitions from resting (R) to open (O) and then desensitized (D) conformations (scheme 2). The nAChR in reconstituted membranes also adopts an uncoupled conformation that binds agonist but typically does not transition to the open or desensitized states (scheme 1). (B) Schematic diagram showing the current model of lipid-dependent uncoupling, illustrated using a single nAChR subunit. The lipid exposed M4 likely plays a key role in sensing the lipid bilayer. In unfavorable membrane environments, the conformation of M4 may change, thus weakening interactions between the b1–b2 (light blue)/b6–b7 (green) loops of the ABD and the M2–M3 linker (red) of the TMD (for details, see daCosta and Baenziger, 2009).

in the resting and desensitized conformations become A lipid-dependent uncoupled conformation of ELIC would exposed to solvent in the uncoupled state (daCosta and explain both the lack of agonist-induced structural change in Baenziger, 2009; Me´ thot et al., 1995). Given the importance the crystal structures of ELIC (Gonzalez-Gutierrez et al., 2012) of the coupling interface in nAChR function, it has been sug- and would largely reconcile the conformational ambiguities gested that lipid-dependent uncoupling results from weakened between available pLGIC structures. Rather than representing interactions, and increased physical separation, between the the canonical closed state, the conformation of the pore in the ABD and TMD (daCosta, 2006; daCosta and Baenziger, ELIC structure would instead reflect the distinct uncoupled state. 2009), similar to the mechanism of coupling/uncoupling asso- If this interpretation is correct, then the GLIC/GluCl and the 4.0 A˚ ciated with the PIP2-activated potassium channel (Hansen nAChR structures may still reflect the open state and closed et al., 2011). Lipids and detergents could influence interactions states, respectively. In this scenario, these structures would sug- between the ABD and TMD via the C terminus of the lipid- gest that only subtle changes in pore diameter occur upon chan- exposed M4 a helix, which in the nAChR structure interacts nel gating, consistent with the cryo-EM studies of nAChR gating, directly with the b6–b7 loop (Figure 4B). Although the broader functional studies, and MD simulations (Beckstein and functional significance of this putative interaction has yet to Sansom, 2004; Cymes and Grosman, 2008; Cymes et al., be demonstrated, the importance of the C terminus of M4 in 2005; Song and Corry, 2009; Unwin and Fujiyoshi, 2012; Wang nAChR function is well documented (Hosie et al., 2006; Jin et al., 2008, 2009). Strictly speaking, a definitive interpretation and Steinbach, 2011; Paradiso et al., 2001; Pons et al., of each crystal structure in terms of a specific conformation still 2004; Tobimatsu et al., 1987). requires experimental characterization of the conformation As noted, the coupling interface of the Torpedo nAChR ex- adopted in the crystalline state. hibits tight interactions between the ABD and TMD, whereas in ELIC, GLIC, and GluCl these contacts are weaker. Given that Summary and Future Directions the structures of ELIC, GLIC, and GluCl were solved in the deter- Our current understanding of pLGIC structure stems from gent-solubilized state, it is conceivable that contact between several major breakthroughs over the past 50 years, including the ABD and TMD is influenced by detergent-solubilization. the identification of the Torpedo nAChR as an abundant model One possible interpretation is that the weakened interactions for biochemical studies, the discovery and structural character- observed between the two domains could be similar to those ization of molluscan AChBPs, continued technical improve- thought to underlie the increased solvent accessibility detected ments in the EM imaging of Torpedo nAChRs, the discovery with the uncoupled Torpedo nAChR in unfavorable lipid environ- and structural characterization of prokaryotic pLGICs, and the ments. In this vein, it is interesting that the M4 a helix in the ELIC first X-ray structure of a eukaryotic pLGIC, GluCl. Each of these structures is partially unwound and tilted away from the remain- advancements generated new lines of investigation that collec- ing TMD with several C-terminal M4 residues not modeled tively led to detailed new insight into both structure and function, (Figure 5B). Contrasted with the nAChR structure (and that of but each line of investigation has its challenges and limitations. GLIC and GluCl), the ELIC structure does not exhibit contacts The Torpedo nAChR is abundant and amenable to biochemical between the C terminus of M4 and the b6–b7 loop, precisely studies but has proven refractory to crystallization. The AChBP the type of structural rearrangements thought to underlie lipid- and other water-soluble ABDs are amenable to crystallization dependent uncoupling (Figure 6B). without the complicating issues of detergent-solubilization, but

they cannot be used to study gating as they lack a TMD pore. by fluorescence and Fourier transform infrared difference spectroscopy. Cryo-EM studies of the Torpedo nAChR have yielded critical Biophys. J. 61, 983–992. insight into the structures of both the closed and open states Bali, M., and Akabas, M.H. (2007). The location of a closed channel gate in the and are unaffected by potential effects of detergent solubiliza- GABAA receptor channel. J. Gen. Physiol. 129, 145–159. tion, but the interpretation of these structures is limited by cur- Barrantes, F.J. (2002). Lipid matters: nicotinic acetylcholine receptor-lipid rent resolution. Prokaryotic pLGICs are relatively easy to express interactions (review). Mol. Membr. Biol. 19, 277–284. and crystallize, but little is known about their functional proper- Beckstein, O., and Sansom, M.S. (2004). The influence of geometry, surface ties in the detergent-solubilized state. character, and flexibility on the permeation of ions and water through biolog- Thus, despite exciting progress, a number of open questions ical pores. Phys. Biol. 1, 42–52. remain surrounding the interpretation of different conformations Beckstein, O., and Sansom, M.S. (2006). A hydrophobic gate in an ion channel: captured for different channels by different techniques. The the closed state of the nicotinic acetylcholine receptor. Phys. Biol. 3, 147–159. conformational ambiguities between the Torpedo nAChR struc- Billen, B., Spurny, R., Brams, M., van Elk, R., Valera-Kummer, S., Yakel, J.L., ture and those of ELIC, GLIC, and GluCl could reflect many fac- Voets, T., Bertrand, D., Smit, A.B., and Ulens, C. (2012). Molecular actions of tors, including the effects of crystal packing forces, differences in smoking cessation drugs at a4b2 nicotinic receptors defined in crystal struc- the resolution of the determined structures, intrinsic differences tures of a homologous binding protein. Proc. Natl. Acad. Sci. USA 109, 9173–9178. in the activation mechanisms of each pLGIC, and the possible transient nature of the open conformation in the presence of acti- Blanton, M.P., and Cohen, J.B. (1992). Mapping the lipid-exposed regions in the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 31, vating ligands. We contend that detergent-solubilization has the 3738–3750. potential to exert significant influence on the conformations of the crystallized pLGICs and that some of the crystallized struc- Blanton, M.P., and Cohen, J.B. (1994). Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implica- tures exhibit features consistent with the uncoupled nAChR tions. Biochemistry 33, 2859–2872. conformation observed in unfavorable membranes. Further work is required to develop probes that can be used to examine Bocquet, N., Prado de Carvalho, L., Cartaud, J., Neyton, J., Le Poupon, C., Taly, A., Grutter, T., Changeux, J.P., and Corringer, P.J. (2007). A prokaryotic the functional properties of pLGICs in the detergent solubilized proton-gated ion channel from the nicotinic acetylcholine receptor family. state and thus to unequivocally assess conformations adopted Nature 445, 116–119. during crystallization. An understanding of how lipids and deter- Bocquet, N., Nury, H., Baaden, M., Le Poupon, C., Changeux, J.P., Delarue, gents influence pLGIC structure may also assist in defining M., and Corringer, P.J. (2009). X-ray structure of a pentameric ligand-gated conditions for crystallization in unambiguous conformations. ion channel in an apparently open conformation. Nature 457, 111–114. Ultimately, definitive conformational insight is essential for a Bondarenko, V., Mowrey, D., Tillman, T., Cui, T., Liu, L.T., Xu, Y., and Tang, P. structural understanding of pLGIC function. Conformational (2012). NMR structures of the transmembrane domains of the a4b2 nAChR. insight is also essential for understanding the nature of drug Biochim. Biophys. Acta 1818, 1261–1268. action at these important therapeutic targets. Bondarenko, V., Mowrey, D., Liu, L.T., Xu, Y., and Tang, P. (2013). NMR resolved multiple anesthetic binding sites in the TM domains of the a4b2 nAChR. Biochim. Biophys. Acta 1828, 398–404. ACKNOWLEDGMENTS Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., and Marchot, P. (2005). Crystal structure of a Cbtx-AChBP complex reveals essential interactions This research was funded by a research grant to J.E.B. from the Canadian In- between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24, stitutes of Health Research (CIHR) as well as a CIHR Fellowship to C.J.B.D. 1512–1522. The role of CIHR in this research was to provide financial support. We thank Daniel Therien for help in the preparation of figures. Bouzat, C. (2012). New insights into the structural bases of activation of Cys- loop receptors. J. Physiol. Paris 106, 23–33.

REFERENCES Bouzat, C., Gumilar, F., Spitzmaul, G., Wang, H.L., Rayes, D., Hansen, S.B., Taylor, P., and Sine, S.M. (2004). Coupling of agonist binding to channel gating Akabas, M.H., Stauffer, D.A., Xu, M., and Karlin, A. (1992). Acetylcholine re- in an ACh-binding protein linked to an ion channel. Nature 430, 896–900. ceptor channel structure probed in cysteine-substitution mutants. Science 258, 307–310. Bouzat, C., Bartos, M., Corradi, J., and Sine, S.M. (2008). The interface between extracellular and transmembrane domains of homomeric Cys- Akabas, M.H., Kaufmann, C., Archdeacon, P., and Karlin, A. (1994). Identifica- loop receptors governs open-channel lifetime and rate of desensitization. tion of acetylcholine receptor channel-lining residues in the entire M2 segment J. Neurosci. 28, 7808–7819. of the alpha subunit. Neuron 13, 919–927. Brejc, K., van Dijk, W.J., Klaassen, R.V., Schuurmans, M., van Der Oost, J., Andersen, N., Corradi, J., Bartos, M., Sine, S.M., and Bouzat, C. (2011). Func- Smit, A.B., and Sixma, T.K. (2001). Crystal structure of an ACh-binding protein tional relationships between agonist binding sites and coupling regions of reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269–276. homomeric Cys-loop receptors. J. Neurosci. 31, 3662–3669. Bro¨ mstrup, T., Howard, R.J., Trudell, J.R., Harris, R.A., and Lindahl, E. (2013). Asmar-Rovira, G.A., Asseo-Garcıá, A.M., Quesada, O., Hanson, M.A., Cheng, Inhibition versus potentiation of ligand-gated ion channels can be altered by a A., Nogueras, C., Lasalde-Dominicci, J.A., and Stevens, R.C. (2008). Biophys- single mutation that moves ligands between intra- and inter-subunit sites. ical and ion channel functional characterization of the Torpedo californica Structure 21, this issue, 1307–1316. nicotinic acetylcholine receptor in varying detergent-lipid environments. J. Membr. Biol. 223, 13–26. Campos-Caro, A., Sala, S., Ballesta, J.J., Vicente-Agullo´ , F., Criado, M., and Sala, F. (1996). A single residue in the M2-M3 loop is a major determinant of Baenziger, J.E., and daCosta, C.J.B. (2013). Molecular mechanisms of acetyl- coupling between binding and gating in neuronal nicotinic receptors. Proc. choline receptor-lipid interactions: from model membranes to human biology. Natl. Acad. Sci. USA 93, 6118–6123. Biophys. Rev. 5, 1–9. Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., and Baenziger, J.E., Miller, K.W., and Rothschild, K.J. (1992). Incorporation of the Sixma, T.K. (2004). Nicotine and carbamylcholine binding to nicotinic acetyl- nicotinic acetylcholine receptor into planar multilamellar films: characterization choline receptors as studied in AChBP crystal structures. Neuron 41, 907–914.

Celie, P.H., Klaassen, R.V., van Rossum-Fikkert, S.E., van Elk, R., van Nierop, Fong, T.M., and McNamee, M.G. (1986). Correlation between acetylcholine re- P., Smit, A.B., and Sixma, T.K. (2005). Crystal structure of acetylcholine-bind- ceptor function and structural properties of membranes. Biochemistry 25, ing protein from Bulinus truncatus reveals the conserved structural scaffold 830–840. and sites of variation in nicotinic acetylcholine receptors. J. Biol. Chem. 280, 26457–26466. Fu, D.X., and Sine, S.M. (1994). Competitive antagonists bridge the alpha- gamma subunit interface of the acetylcholine receptor through quaternary Chakrapani, S., Bailey, T.D., and Auerbach, A. (2004). Gating dynamics of the ammonium-aromatic interactions. J. Biol. Chem. 269, 26152–26157. acetylcholine receptor extracellular domain. J. Gen. Physiol. 123, 341–356. Galzi, J.L., Revah, F., Black, D., Goeldner, M., Hirth, C., and Changeux, J.P. Changeux, J.P. (2012). The nicotinic acetylcholine receptor: the founding (1990). Identification of a novel amino acid alpha-tyrosine 93 within the father of the pentameric ligand-gated ion channel superfamily. J. Biol. cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity Chem. 287, 40207–40215. labeling. Additional evidence for a three-loop model of the cholinergic ligands- binding sites. J. Biol. Chem. 265, 10430–10437. Changeux, J.P., Kasai, M., and Lee, C.Y. (1970). Use of a snake venom toxin to characterize the cholinergic receptor protein. Proc. Natl. Acad. Sci. USA 67, Gao, F., Bren, N., Burghardt, T.P., Hansen, S., Henchman, R.H., Taylor, P., 1241–1247. McCammon, J.A., and Sine, S.M. (2005). Agonist-mediated conformational changes in acetylcholine-binding protein revealed by simulation and intrinsic Corringer, P.J., Le Nove` re, N., and Changeux, J.P. (2000). Nicotinic receptors tryptophan fluorescence. J. Biol. Chem. 280, 8443–8451. at the amino acid level. Annu. Rev. Pharmacol. Toxicol. 40, 431–458. Gonzalez-Gutierrez, G., and Grosman, C. (2010). Bridging the gap between Corringer, P.J., Galzi, J.L., Eisele´ , J.L., Bertrand, S., Changeux, J.P., and structural models of nicotinic receptor superfamily ion channels and their cor- Bertrand, D. (1995). Identification of a new component of the agonist binding responding functional states. J. Mol. Biol. 403, 693–705. site of the nicotinic alpha 7 homooligomeric receptor. J. Biol. Chem. 270, 11749–11752. Gonzalez-Gutierrez, G., Lukk, T., Agarwal, V., Papke, D., Nair, S.K., and Gros- man, C. (2012). Mutations that stabilize the open state of the Erwinia chrisan- Corringer, P.J., Poitevin, F., Prevost, M.S., Sauguet, L., Delarue, M., and themi ligand-gated ion channel fail to change the conformation of the pore Changeux, J.P. (2012). Structure and pharmacology of pentameric receptor domain in crystals. Proc. Natl. Acad. Sci. USA 109, 6331–6336. channels: from bacteria to brain. Structure 20, 941–956. Grosman, C., Salamone, F.N., Sine, S.M., and Auerbach, A. (2000). The extra- Criado, M., Eibl, H., and Barrantes, F.J. (1984). Functional properties of the cellular linker of muscle acetylcholine receptor channels is a gating control acetylcholine receptor incorporated in model lipid membranes. Differential element. J. Gen. Physiol. 116, 327–340. effects of chain length and head group of phospholipids on receptor affinity states and receptor-mediated ion translocation. J. Biol. Chem. 259, 9188– Grutter, T., and Changeux, J.P. (2001). Nicotinic receptors in wonderland. 9198. Trends Biochem. Sci. 26, 459–463.

Cromer, B.A., Morton, C.J., and Parker, M.W. (2002). Anxiety over GABA(A) Grutter, T., de Carvalho, L.P., Dufresne, V., Taly, A., Edelstein, S.J., and receptor structure relieved by AChBP. Trends Biochem. Sci. 27, 280–287. Changeux, J.P. (2005). Molecular tuning of fast gating in pentameric ligand- gated ion channels. Proc. Natl. Acad. Sci. USA 102, 18207–18212. Cymes, G.D., and Grosman, C. (2008). Pore-opening mechanism of the nico- Hamouda, A.K., Sanghvi, M., Sauls, D., Machu, T.K., and Blanton, M.P. (2006). tinic acetylcholine receptor evinced by proton transfer. Nat. Struct. Mol. Biol. Assessing the lipid requirements of the Torpedo californica nicotinic acetyl- 15, 389–396. choline receptor. Biochemistry 45, 4327–4337. Cymes, G.D., Ni, Y., and Grosman, C. (2005). Probing ion-channel pores one Hansen, S.B., Radic’, Z., Talley, T.T., Molles, B.E., Deerinck, T., Tsigelny, I., proton at a time. Nature 438, 975–980. and Taylor, P. (2002). Tryptophan ﬂuorescence reveals conformational changes in the acetylcholine binding protein. J. Biol. Chem. 277, 41299– Czajkowski, C., Kaufmann, C., and Karlin, A. (1993). Negatively charged amino 41302. acid residues in the nicotinic receptor delta subunit that contribute to the binding of acetylcholine. Proc. Natl. Acad. Sci. USA 90, 6285–6289. Hansen, S.B., Talley, T.T., Radic, Z., and Taylor, P. (2004). Structural and ligand recognition characteristics of an acetylcholine-binding protein from daCosta, C.J. (2006). A Structural Role for Lipids in Coupling Ligand Binding to Aplysia californica. J. Biol. Chem. 279, 24197–24202. Channel Gating in a Neurotransmitter Receptor (Ottawa: University of Ottawa). Hansen, S.B., Sulzenbacher, G., Huxford, T., Marchot, P., Taylor, P., and daCosta, C.J., and Baenziger, J.E. (2009). A lipid-dependent uncoupled Bourne, Y. (2005). Structures of Aplysia AChBP complexes with nicotinic ago- conformation of the acetylcholine receptor. J. Biol. Chem. 284, 17819–17825. nists and antagonists reveal distinctive binding interfaces and conformations. EMBO J. 24, 3635–3646. daCosta, C.J., Medaglia, S.A., Lavigne, N., Wang, S., Carswell, C.L., and Baenziger, J.E. (2009). Anionic lipids allosterically modulate multiple nicotinic Hansen, S.B., Tao, X., and MacKinnon, R. (2011). Structural basis of PIP2 acti- acetylcholine receptor conformational equilibria. J. Biol. Chem. 284, 33841– vation of the classical inward rectiﬁer K+ channel Kir2.2. Nature 477, 495–498. 33849. Heidmann, T., Sobel, A., Popot, J.L., and Changeux, J.P. (1980). Reconstitu- Dellisanti, C.D., Yao, Y., Stroud, J.C., Wang, Z.Z., and Chen, L. (2007). Crystal tion of a functional acetylcholine receptor. Conservation of the conformational structure of the extracellular domain of nAChR alpha1 bound to alpha-bungar- and allosteric transitions and recovery of the permeability response; role of otoxin at 1.94 A resolution. Nat. Neurosci. 10, 953–962. lipids. Eur. J. Biochem. 110, 35–55.

Dennis, M., Giraudat, J., Kotzyba-Hibert, F., Goeldner, M., Hirth, C., Chang, Hertling-Jaweed, S., Bandini, G., Mu¨ ller-Fahrnow, A., Dommes, V., and J.Y., Lazure, C., Chre´ tien, M., and Changeux, J.P. (1988). Amino acids of the Hucho, F. (1988). Rapid preparation of the nicotinic acetylcholine receptor Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photo- for crystallization in detergent solution. FEBS Lett. 241, 29–32. afﬁnity ligand for the acetylcholine binding site. Biochemistry 27, 2346–2357. Herz, J.M., Johnson, D.A., and Taylor, P. (1987). Interaction of noncompetitive Dougherty, D.A. (1996). Cation-pi interactions in chemistry and biology: a new inhibitors with the acetylcholine receptor. The site speciﬁcity and spectro- view of benzene, Phe, Tyr, and Trp. Science 271, 163–168. scopic properties of ethidium binding. J. Biol. Chem. 262, 7238–7247.

Duret, G., Van Renterghem, C., Weng, Y., Prevost, M., Moraga-Cid, G., Huon, Hibbs, R.E., and Gouaux, E. (2011). Principles of activation and permeation in C., Sonner, J.M., and Corringer, P.J. (2011). Functional prokaryotic-eukaryotic an anion-selective Cys-loop receptor. Nature 474, 54–60. chimera from the pentameric ligand-gated ion channel family. Proc. Natl. Acad. Sci. USA 108, 12143–12148. Hilf, R.J., and Dutzler, R. (2008). X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature 452, 375–379. Finer-Moore, J., and Stroud, R.M. (1984). Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc. Natl. Acad. Hilf, R.J., and Dutzler, R. (2009). Structure of a potentially open state of a pro- Sci. USA 81, 155–159. ton-activated pentameric ligand-gated ion channel. Nature 457, 115–118.

Hilf, R.J., Bertozzi, C., Zimmermann, I., Reiter, A., Trauner, D., and Dutzler, R. Miyazawa, A., Fujiyoshi, Y., and Unwin, N. (2003). Structure and gating mech- (2010). Structural basis of open channel block in a prokaryotic pentameric anism of the acetylcholine receptor pore. Nature 423, 949–955. ligand-gated ion channel. Nat. Struct. Mol. Biol. 17, 1330–1336. Moore, M.A., and McCarthy, M.P. (1995). Snake venom toxins, unlike smaller Hosie, A.M., Wilkins, M.E., da Silva, H.M., and Smart, T.G. (2006). Endogenous antagonists, appear to stabilize a resting state conformation of the nicotinic neurosteroids regulate GABAA receptors through two discrete transmem- acetylcholine receptor. Biochim. Biophys. Acta 1235, 336–342. brane sites. Nature 444, 486–489. Nury, H., Bocquet, N., Le Poupon, C., Raynal, B., Haouz, A., Corringer, P.J., Imoto, K., Methfessel, C., Sakmann, B., Mishina, M., Mori, Y., Konno, T., and Delarue, M. (2010). Crystal structure of the extracellular domain of a bac- Fukuda, K., Kurasaki, M., Bujo, H., Fujita, Y., et al. (1986). Location of a terial ligand-gated ion channel. J. Mol. Biol. 395, 1114–1127. delta-subunit region determining ion transport through the acetylcholine receptor channel. Nature 324, 670–674. Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J.P., Sonner, J.M., Delarue, M., and Corringer, P.J. (2011). X-ray Ivanov, I., Cheng, X., Sine, S.M., and McCammon, J.A. (2007). Barriers to ion structures of general anaesthetics bound to a pentameric ligand-gated ion translocation in cationic and anionic receptors from the Cys-loop family. J. Am. channel. Nature 469, 428–431. Chem. Soc. 129, 8217–8224. O’Leary, M.E., Filatov, G.N., and White, M.M. (1994). Characterization of Jha, A., Cadugan, D.J., Purohit, P., and Auerbach, A. (2007). Acetylcholine re- d-tubocurarine binding site of Torpedo acetylcholine receptor. Am. J. Physiol. ceptor gating at extracellular transmembrane domain interface: the cys-loop 266, C648–C653. and M2-M3 linker. J. Gen. Physiol. 130, 547–558. Paas, Y., Cartaud, J., Recouvreur, M., Grailhe, R., Dufresne, V., Pebay- Jin, X., and Steinbach, J.H. (2011). A portable site: a binding element for Peyroula, E., Landau, E.M., and Changeux, J.P. (2003). Electron microscopic 17b-estradiol can be placed on any subunit of a nicotinic a4b2 receptor. evidence for nucleation and growth of 3D acetylcholine receptor microcrystals J. Neurosci. 31, 5045–5054. in structured lipid-detergent matrices. Proc. Natl. Acad. Sci. USA 100, 11309– Kao, P.N., and Karlin, A. (1986). Acetylcholine receptor binding site contains a 11314. disulﬁde cross-link between adjacent half-cystinyl residues. J. Biol. Chem. 261, 8085–8088. Paas, Y., Gibor, G., Grailhe, R., Savatier-Duclert, N., Dufresne, V., Sunesen, M., de Carvalho, L.P., Changeux, J.P., and Attali, B. (2005). Pore conforma- Kash, T.L., Jenkins, A., Kelley, J.C., Trudell, J.R., and Harrison, N.L. (2003). tions and gating mechanism of a Cys-loop receptor. Proc. Natl. Acad. Sci. Coupling of agonist binding to channel gating in the GABA(A) receptor. Nature USA 102, 15877–15882. 421, 272–275. Padilla-Morales, L.F., Morales-Pe´ rez, C.L., De La Cruz-Rivera, P.C., Asmar- Kollman, J.M., Pandi, L., Sawaya, M.R., Riley, M., and Doolittle, R.F. (2009). Rovira, G., Ba´ ez-Paga´ n, C.A., Quesada, O., and Lasalde-Dominicci, J.A. Crystal structure of human ﬁbrinogen. Biochemistry 48, 3877–3886. (2011). Effects of lipid-analog detergent solubilization on the functionality and lipidic cubic phase mobility of the Torpedo californica nicotinic acetylcho- Labriola, J.M., Pandhare, A., Jansen, M., Blanton, M.P., Corringer, P.J., and line receptor. J. Membr. Biol. 243, 47–58. Baenziger, J.E. (2013). Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment. J. Biol. Chem. 288, Pan, J., Chen, Q., Willenbring, D., Mowrey, D., Kong, X.P., Cohen, A., Divito, 11294–11303. C.B., Xu, Y., and Tang, P. (2012a). Structure of the pentameric ligand-gated ion channel GLIC bound with anesthetic ketamine. Structure 20, 1463–1469. Law, R.J., Henchman, R.H., and McCammon, J.A. (2005). A gating mechanism proposed from a simulation of a human alpha7 nicotinic acetylcholine recep- Pan, J., Chen, Q., Willenbring, D., Yoshida, K., Tillman, T., Kashlan, O.B., tor. Proc. Natl. Acad. Sci. USA 102, 6813–6818. Cohen, A., Kong, X.P., Xu, Y., and Tang, P. (2012b). Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist Lee, W.Y., and Sine, S.M. (2005). Principal pathway coupling agonist binding acetylcholine. Nat. Commun. 3, 714. to channel gating in nicotinic receptors. Nature 438, 243–247. Paradiso, K., Zhang, J., and Steinbach, J.H. (2001). The C terminus of the Lee, W.Y., Free, C.R., and Sine, S.M. (2009). Binding to gating transduction in human nicotinic alpha4beta2 receptor forms a binding site required for poten- nicotinic receptors: Cys-loop energetically couples to pre-M1 and M2-M3 re- tiation by an estrogenic steroid. J. Neurosci. 21, 6561–6568. gions. J. Neurosci. 29, 3189–3199. Parikh, R.B., Bali, M., and Akabas, M.H. (2011). Structure of the M2 transmem- Li, S.X., Huang, S., Bren, N., Noridomi, K., Dellisanti, C.D., Sine, S.M., and brane segment of GLIC, a prokaryotic Cys loop receptor homologue from Chen, L. (2011). Ligand-binding domain of an a7-nicotinic receptor chimera Gloeobacter violaceus, probed by substituted cysteine accessibility. J. Biol. and its complex with agonist. Nat. Neurosci. 14, 1253–1259. Chem. 286, 14098–14109.

Lummis, S.C. (2012). 5-HT(3) receptors. J. Biol. Chem. 287, 40239–40245. Pons, S., Sallette, J., Bourgeois, J.P., Taly, A., Changeux, J.P., and Devillers- Thie´ ry, A. (2004). Critical role of the C-terminal segment in the maturation and Lummis, S.C., Beene, D.L., Lee, L.W., Lester, H.A., Broadhurst, R.W., and export to the cell surface of the homopentameric alpha 7-5HT3A receptor. Eur. Dougherty, D.A. (2005). Cis-trans isomerization at a proline opens the pore J. Neurosci. 20, 2022–2030. of a neurotransmitter-gated ion channel. Nature 438, 248–252. Prevost, M.S., Sauguet, L., Nury, H., Van Renterghem, C., Huon, C., Poitevin, Lynch, J.W., Rajendra, S., Pierce, K.D., Handford, C.A., Barry, P.H., and Scho- ﬁeld, P.R. (1997). Identiﬁcation of intracellular and extracellular domains medi- F., Baaden, M., Delarue, M., and Corringer, P.J. (2012). A locally closed conformation of a bacterial pentameric proton-gated ion channel. Nat. Struct. Mol. ating signal transduction in the inhibitory glycine receptor chloride channel. Biol. 19, 642–649. EMBO J. 16, 110–120.

Martin, M., Czajkowski, C., and Karlin, A. (1996). The contributions of aspartyl Prince, R.J., and Sine, S.M. (1996). Molecular dissection of subunit interfaces residues in the acetylcholine receptor gamma and delta subunits to the binding in the acetylcholine receptor. Identiﬁcation of residues that determine agonist of agonists and competitive antagonists. J. Biol. Chem. 271, 13497–13503. selectivity. J. Biol. Chem. 271, 25770–25777.

Me´ thot, N., Demers, C.N., and Baenziger, J.E. (1995). Structure of both the Rankin, S.E., Addona, G.H., Kloczewiak, M.A., Bugge, B., and Miller, K.W. ligand- and lipid-dependent channel-inactive states of the nicotinic acetylcho- (1997). The cholesterol dependence of activation and fast desensitization of line receptor probed by FTIR spectroscopy and hydrogen exchange. the nicotinic acetylcholine receptor. Biophys. J. 73, 2446–2455. Biochemistry 34, 15142–15149. Rayes, D., De Rosa, M.J., Sine, S.M., and Bouzat, C. (2009). Number and Middleton, R.E., and Cohen, J.B. (1991). Mapping of the acetylcholine binding locations of agonist binding sites required to activate homomeric Cys-loop site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaf- receptors. J. Neurosci. 29, 6022–6032. ﬁnity label. Biochemistry 30, 6987–6997. Reeves, D.C., and Lummis, S.C. (2002). The molecular basis of the structure Miledi, R., Molinoff, P., and Potter, L.T. (1971). Isolation of the cholinergic and function of the 5-HT3 receptor: a model ligand-gated ion channel (review). receptor protein of Torpedo electric tissue. Nature 229, 554–557. Mol. Membr. Biol. 19, 11–26.

Reeves, D.C., Jansen, M., Bali, M., Lemster, T., and Akabas, M.H. (2005). A Thompson, A.J., Alqazzaz, M., Ulens, C., and Lummis, S.C. (2012). The phar- role for the beta 1-beta 2 loop in the gating of 5-HT3 receptors. J. Neurosci. macological proﬁle of ELIC, a prokaryotic GABA-gated receptor. Neurophar- 25, 9358–9366. macology 63, 761–767.

Rovira, J.C., Ballesta, J.J., Vicente-Agullo´ , F., Campos-Caro, A., Criado, M., Tobimatsu, T., Fujita, Y., Fukuda, K., Tanaka, K., Mori, Y., Konno, T., Mishina, Sala, F., and Sala, S. (1998). A residue in the middle of the M2-M3 loop of M., and Numa, S. (1987). Effects of substitution of putative transmembrane the beta4 subunit specifically affects gating of neuronal nicotinic receptors. segments on nicotinic acetylcholine receptor function. FEBS Lett. 222, 56–62. FEBS Lett. 433, 89–92. Unwin, N. (1995). Acetylcholine receptor channel imaged in the open state. Rovira, J.C., Vicente-Agullo´ , F., Campos-Caro, A., Criado, M., Sala, F., Sala, Nature 373, 37–43. S., and Ballesta, J.J. (1999). Gating of alpha3beta4 neuronal nicotinic receptor Unwin, N. (2005). Refined structure of the nicotinic acetylcholine receptor at 4A can be controlled by the loop M2-M3 of both alpha3 and beta4 subunits. resolution. J. Mol. Biol. 346, 967–989. Pflugers Arch. 439, 86–92. Unwin, N., and Fujiyoshi, Y. (2012). Gating movement of acetylcholine receptor Rucktooa, P., Smit, A.B., and Sixma, T.K. (2009). Insight in nAChR subtype caught by plunge-freezing. J. Mol. Biol. 422 , 617–634. selectivity from AChBP crystal structures. Biochem. Pharmacol. 78, 777–787. Unwin, N., Miyazawa, A., Li, J., and Fujiyoshi, Y. (2002). Activation of the nico- Sauguet, L., Howard, R.J., Malherbe, L., Lee, U.S., Corringer, P.J., Harris, tinic acetylcholine receptor involves a switch in conformation of the alpha sub- R.A., and Delarue, M. (2013a). Structural basis for potentiation by alcohols units. J. Mol. Biol. 319, 1165–1176. and anaesthetics in a ligand-gated ion channel. Nat. Commun. 4, 1697. Velisetty, P., and Chakrapani, S. (2012). Desensitization mechanism in pro- Sauguet, L., Poitevin, F., Murail, S., Van Renterghem, C., Moraga-Cid, G., karyotic ligand-gated ion channel. J. Biol. Chem. 287, 18467–18477. Malherbe, L., Thompson, A.W., Koehl, P., Corringer, P.J., Baaden, M., and Delarue, M. (2013b). Structural basis for ion permeation mechanism in pen- Velisetty, P., Chalamalasetti, S.V., and Chakrapani, S. (2012). Conformational tameric ligand-gated ion channels. EMBO J. 32, 728–741. transitions underlying pore opening and desensitization in membrane- embedded Gloeobacter violaceus ligand-gated ion channel (GLIC). J. Biol. Schofield, P.R., Darlison, M.G., Fujita, N., Burt, D.R., Stephenson, F.A., Rodri- Chem. 287, 36834–36872. guez, H., Rhee, L.M., Ramachandran, J., Reale, V., Glencorse, T.A., et al. (1987). Sequence and functional expression of the GABA A receptor shows Wang, H.L., Milone, M., Ohno, K., Shen, X.M., Tsujino, A., Batocchi, A.P., a ligand-gated receptor super-family. Nature 328, 221–227. Tonali, P., Brengman, J., Engel, A.G., and Sine, S.M. (1999). Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel Shen, X.M., Ohno, K., Tsujino, A., Brengman, J.M., Gingold, M., Sine, S.M., gating. Nat. Neurosci. 2, 226–233. and Engel, A.G. (2003). Mutation causing severe myasthenia reveals functional Wang, H.L., Cheng, X., Taylor, P., McCammon, J.A., and Sine, S.M. (2008). asymmetry of AChR signature cystine loops in agonist binding and gating. Control of cation permeation through the nicotinic receptor channel. PLoS J. Clin. Invest. 111, 497–505. Comput. Biol. 4, e41.

Silman, I., and Karlin, A. (1969). Acetylcholine receptor: covalent attachment of Wang, H.L., Toghraee, R., Papke, D., Cheng, X.L., McCammon, J.A., Ravaioli, depolarizing groups at the active site. Science 164, 1420–1421. U., and Sine, S.M. (2009). Single-channel current through nicotinic receptor produced by closure of binding site C-loop. Biophys. J. 96, 3582–3590. Sine, S.M. (2002). The nicotinic receptor ligand binding domain. J. Neurobiol. 53, 431–446. Wang, H.L., Cheng, X., and Sine, S.M. (2012). Intramembrane proton binding site linked to activation of bacterial pentameric ion channel. J. Biol. Chem. 287, Sine, S.M., and Engel, A.G. (2006). Recent advances in Cys-loop receptor 6482–6489. structure and function. Nature 440, 448–455. Weill, C.L., McNamee, M.G., and Karlin, A. (1974). Affinity-labeling of purified Sine, S.M., Kreienkamp, H.J., Bren, N., Maeda, R., and Taylor, P. (1995). acetylcholine receptor from Torpedo californica. Biochem. Biophys. Res. Molecular dissection of subunit interfaces in the acetylcholine receptor: iden- Commun. 61, 997–1003. tification of determinants of alpha-conotoxin M1 selectivity. Neuron 15, 205–211. Weng, Y., Yang, L., Corringer, P.J., and Sonner, J.M. (2010). Anesthetic sensitivity of the Gloeobacter violaceus proton-gated ion channel. Anesth. Analg. Smit, A.B., Syed, N.I., Schaap, D., van Minnen, J., Klumperman, J., Kits, K.S., 110, 59–63. Lodder, H., van der Schors, R.C., van Elk, R., Sorgedrager, B., et al. (2001). A Xiu, X., Hanek, A.P., Wang, J., Lester, H.A., and Dougherty, D.A. (2005). A uni- glia-derived acetylcholine-binding protein that modulates synaptic transmis- fied view of the role of electrostatic interactions in modulating the gating of Cys sion. Nature 411, 261–268. loop receptors. J. Biol. Chem. 280, 41655–41666.

Song, C., and Corry, B. (2009). Computational study of the transmembrane Xu, M., Covey, D.F., and Akabas, M.H. (1995). Interaction of picrotoxin with domain of the acetylcholine receptor. Eur. Biophys. J. 38, 961–970. GABAA receptor channel-lining residues probed in cysteine mutants. Biophys. J. 69, 1858–1867. Taly, A., and Changeux, J.P. (2008). Functional organization and conformational dynamics of the nicotinic receptor: a plausible structural interpretation Zimmermann, I., and Dutzler, R. (2011). Ligand activation of the prokaryotic of myasthenic mutations. Ann. N Y Acad. Sci. 1132, 42–52. pentameric ligand-gated ion channel ELIC. PLoS Biol. 9, e1001101.

Tasneem, A., Iyer, L.M., Jakobsson, E., and Aravind, L. (2005). Identiﬁcation of Zimmermann, I., Marabelli, A., Bertozzi, C., Sivilotti, L.G., and Dutzler, R. the prokaryotic ligand-gated ion channels and their implications for the mech- (2012). Inhibition of the prokaryotic pentameric ligand-gated ion channel anisms and origins of animal Cys-loop ion channels. Genome Biol. 6, R4. ELIC by divalent cations. PLoS Biol. 10, e1001429.