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A Lipid-dependent Uncoupled Conformation of the Acetylcholine Receptor*

  • Corrie J.B. daCosta
    Correspondence
    To whom correspondence may be addressed
    Affiliations
    From the Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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  • John E. Baenziger
    Correspondence
    To whom correspondence may be addressed. Tel.: 613-562-5800 (ext.: 8222); Fax: 613-562-5251;
    Affiliations
    From the Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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  • Author Footnotes
    * This work was supported by the Canadian Institutes of Health Research and a Canadian Graduate Scholarship (to C. J. B. D.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Experimental Procedures, references, Figs. S1–S6, and Tables S1–S3.
Open AccessPublished:April 08, 2009DOI:https://doi.org/10.1074/jbc.M900030200
      Lipids influence the ability of Cys-loop receptors to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. With the nicotinic acetylcholine receptor (nAChR) from Torpedo, current models suggest that lipids modulate the natural equilibrium between resting and desensitized conformations. We show that the lipid-inactivated nAChR is not desensitized, instead it adopts a novel conformation where the allosteric coupling between its neurotransmitter-binding sites and transmembrane pore is lost. The uncoupling is accompanied by an unmasking of previously buried residues, suggesting weakened association between structurally intact agonist-binding and transmembrane domains. These data combined with the extensive literature on Cys-loop receptor-lipid interactions suggest that the M4 transmembrane helix plays a key role as a lipid-sensor, translating bilayer properties into altered nAChR function.
      Neurotransmission at chemical synapses is fundamental to the propagation of electrical signals within the nervous system. Central to this process is the ability of Cys-loop receptors to convert a chemical input into an electrical output by conducting ions across the synaptic membrane in response to neurotransmitter binding (
      • Sine S.M.
      • Engel A.G.
      ). At the molecular level, this not only requires the ability to bind agonist and conduct ions, but also the ability to effectively translate agonist binding into ion channel opening/gating. The agonist-binding sites, which are located on the extra-membranous surface of the receptor, are thus allosterically coupled to the distant transmembrane ion pore (Fig. 1). Factors that affect the ability of Cys-loop receptors to bind agonist, conduct ions, and/or couple agonist binding to ion channel gating have the potential to modulate the synaptic response, and thus influence the transmission of electrical signals.
      Figure thumbnail gr1
      FIGURE 1Structure of the nAChR from Torpedo (Protein Data Bank code 2BG9), and a minimal model of nAChR conformational equilibria. A, the entire nAChR pentamer, with labeled extracellular agonist-binding (ABD), transmembrane pore (TMD), and cytoplasmic (CD) domains. Side chains of residues forming part of the agonist-binding site (1; αTrp-149) and the ion pore gate (2; αLeu-251, as well as analogous β-, γ-, and δ-subunit leucines) are shown in orange and purple. Views shown are from the synaptic space of B, the agonist-binding domain, and C, the transmembrane pore. D, our data show that lipid composition influences the activatable pool of receptors by controlling the proportion of nAChRs in uncoupled (U) versus coupled/resting (R) conformations (Scheme 1, boxed). Because gating appears to involve structural rearrangements at the lipid-protein interface (
      • Mitra A.
      • Bailey T.D.
      • Auerbach A.L.
      ), lipids may also influence the equilibrium between R, O (open), and D (desensitized) states (Scheme 2).
      One factor that is known to affect the activity of several Cys-loop receptors is the lipid composition of the membrane in which they are embedded (
      • Barrantes F.J.
      ). Lipid sensitivity of the nicotinic acetylcholine receptor (nAChR)
      The abbreviations used are: nAChR
      nicotinic acetylcholine receptor
      PA
      phosphatidic acid
      PC
      phosphatidylcholine
      Chol
      cholesterol
      Carb
      carbamylcholine
      TCP
      tenocylclidine.
      3The abbreviations used are: nAChR
      nicotinic acetylcholine receptor
      PA
      phosphatidic acid
      PC
      phosphatidylcholine
      Chol
      cholesterol
      Carb
      carbamylcholine
      TCP
      tenocylclidine.
      from Torpedo, the prototypical Cys-loop receptor, has been known since the earliest attempts to isolate the nAChR from receptor-rich Torpedo membranes (
      • Epstein M.
      • Racker E.
      ,
      • Heidmann T.
      • Sobel A.
      • Popot J.L.
      • Changeux J.P.
      ). Initial studies showed that to preserve a fully functional nAChR, the receptor must be purified in the presence of exogenous lipid and then reconstituted into a membrane with a particular lipid composition (
      • Fong T.M.
      • McNamee M.G.
      ).
      Subsequent studies have focused on defining the roles that individual lipid species play in supporting nAChR function (
      • Fong T.M.
      • McNamee M.G.
      ,
      • Criado M.
      • Eibl H.
      • Barrantes F.J.
      ,
      • Rankin S.E.
      • Addona G.H.
      • Kloczewiak M.A.
      • Bugge B.
      • Miller K.W.
      ,
      • daCosta C.J.
      • Ogrel A.A.
      • McCardy E.A.
      • Blanton M.P.
      • Baenziger J.E.
      ,
      • Hamouda A.K.
      • Sanghvi M.
      • Sauls D.
      • Machu T.K.
      • Blanton M.P.
      ). The consensus is that both cholesterol (Chol) and anionic lipids (such as phosphatidic acid; PA) are required in a reconstituted phosphatidylcholine (PC) membrane to provide an optimal environment. Chol and PA both increase the proportion of nAChRs stabilized in an agonist-activatable conformation (
      • Baenziger J.E.
      • Morris M.L.
      • Darsaut T.E.
      • Ryan S.E.
      ). As the chemical labeling pattern of the non-activatable PC-nAChR is similar to that of the desensitized nAChR, it has been suggested that lipids modulate the natural equilibrium between resting and desensitized receptors (
      • daCosta C.J.
      • Ogrel A.A.
      • McCardy E.A.
      • Blanton M.P.
      • Baenziger J.E.
      ,
      • Hamouda A.K.
      • Sanghvi M.
      • Sauls D.
      • Machu T.K.
      • Blanton M.P.
      ,
      • McCarthy M.P.
      • Moore M.A.
      ). Other studies, however, hint that PC-nAChR is not desensitized, suggesting a more complex mechanism of nAChR-lipid interactions (
      • Rankin S.E.
      • Addona G.H.
      • Kloczewiak M.A.
      • Bugge B.
      • Miller K.W.
      ,
      • daCosta C.J.
      • Kaiser D.E.
      • Baenziger J.E.
      ,
      • Baenziger J.E.
      • Ryan S.E.
      • Goodreid M.M.
      • Vuong N.Q.
      • Sturgeon R.M.
      • daCosta C.J.
      ). The lack of detailed characterization of the inactive PC-nAChR conformation has prevented the development of descriptive models of nAChR-lipid interactions. It is this lack of definitive insight into the lipid-dependent nAChR conformations that is addressed here.
      As a first step toward understanding how lipids influence function, we characterize here the activatable and non-activatable conformations of the Torpedo nAChR stabilized in PC/PA/Chol (PC/PA/Chol-nAChR) and PC (PC-nAChR) membranes. We show that lipid-dependent inactivation is not related to agonist-induced desensitization. Instead, PC-nAChR adopts a novel conformation in which allosteric coupling between the agonist-binding sites and transmembrane pore is lost (Fig. 1D). Furthermore, uncoupling leads to a substantial increase in solvent accessibility, with minimal effects on nAChR secondary structure and thermal stability. Together, our data show that the lipid environment surrounding the nAChR transmembrane domain influences communication between the intact agonist-binding and transmembrane pore domains. In the context of recent structural and mutagenesis data, our findings suggest that the lipid-exposed transmembrane M4 helix acts as a lipid-sensor modulating interactions at a coupling interface between the two domains. The existence of this novel uncoupled conformation could explain how membrane-soluble allosteric modulators (including lipids) influence Cys-loop receptor function.

      DISCUSSION

      The effects of agonists and allosteric modulators on the nAChR are usually interpreted in terms of a simplified conformational scheme involving three main inter-convertible conformations: the resting, open, and desensitized states (Fig. 1D, Scheme 2) (
      • Bertrand D.
      • Gopalakrishnan M.
      ). In the absence of agonist, an equilibrium exists primarily between the resting and desensitized conformations, with the equilibrium in native membranes strongly favoring the resting state (
      • Boyd N.D.
      • Cohen J.B.
      ). It has been suggested that many allosteric modulators, including lipids, influence nAChR function by modulating this natural equilibrium (
      • Baenziger J.E.
      • Morris M.L.
      • Darsaut T.E.
      • Ryan S.E.
      ,
      • Baenziger J.E.
      • Ryan S.E.
      • Goodreid M.M.
      • Vuong N.Q.
      • Sturgeon R.M.
      • daCosta C.J.
      ). The data presented here, however, show unequivocally that the lipid-dependent non-activatable nAChR adopts a novel conformation that is neither resting, nor desensitized. Lipid action at the nAChR therefore does not result from a modulation of the resting-to-desensitized equilibrium, instead another non-activatable conformation is involved. The identification of this novel conformation has repercussions for nAChR-lipid interactions, but may also have broader implications for our general understanding of nAChR function.
      We previously hypothesized that lipids play a role in coupling agonist binding to channel gating (
      • daCosta C.J.
      ). In support of this hypothesis, the functional hallmark of the lipid-dependent non-activatable nAChR is a loss of allosteric coupling between the agonist binding and ion channel functions of the receptor. Specifically, agonist binding does not induce the expected conformational change of the transmembrane pore. Reciprocally, TCP, a non-competitive inhibitor that binds to the nAChR pore, fails to trigger a conformational change in the extracellular agonist-binding domain. Effectively, the nAChR is no longer an agonist-activated ion channel because its agonist-binding site is functionally uncoupled from its transmembrane pore. For this reason we refer to this conformation as the lipid-dependent “uncoupled state.”
      The existence of this uncoupled conformation leads to two important, but related questions. First, how does membrane lipid composition influence coupling between the agonist-binding sites and the transmembrane pore? Second, does uncoupling play a role in nAChR (or Cys-loop receptor) function in vivo?
      With regards to the first question, our biochemical and biophysical data can now be interpreted in terms of the nAChR structural model. This model shows that the agonist-binding and ion channel functions reside in two distinct structural domains, demarked by an abrupt change in secondary structure (FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5A) (
      • Unwin N.
      ). The two domains meet at an interface located ∼10 Å above the bilayer surface. This interface consists of several loops from the agonist-binding (β1-β2, β6-β7 or Cys-loop, and β8-β9) and transmembrane pore (M2-M3L) domains, as well as the covalent link between them (β10-M1). Complex interactions between these interfacial loops are critical for effective communication between the two domains (
      • Sine S.M.
      • Engel A.G.
      ). Lipid-dependent uncoupling must result from structural rearrangements that ultimately disrupt these inter-domain interactions. Weakened associations between the two domains with increased exposure of previously buried interfacial residues could account for the increased solvent accessibility observed with the uncoupled state.
      Figure thumbnail gr5
      FIGURE 5Potential role of M4 as a lipid-sensor modulating allosteric coupling in the Torpedo nAChR. A, view of the α-subunit agonist-binding domain (ABD) and transmembrane pore domain (TMD), highlighting structures at their interface (Cys-loop, post-M4, β1-β2 loop, and the M2-M3 linker). Also labeled are the C-loop and Trp-149, which form part of the agonist-binding site, and Leu-251 thought to form part of the pore gate. B and C, close up views of the coupling interface highlighting possible residues important for: B, relaying M4 (orange) induced pressure on the Cys-loop (green) to the M2-M3 linker (red); and C, M4 residues in close contact with the Cys-loop. D, schematic depicting possible lipid-dependent structural rearrangements of the transmembrane helices resulting in loss of interactions between the C-terminal end of M4 and the Cys-loop.
      How could lipids alter inter-domain interactions that occur ∼10 Å above the bilayer surface? One possibility is that lipids disrupt these inter-domain interactions by altering the packing of transmembrane helices. Transmembrane helix packing could ultimately dictate the conformation of the M2-M3 and β10-M1 linkers, thereby influencing crucial interactions at the interface between the transmembrane pore and agonist-binding domains (
      • Brannigan G.
      • Hénin J.
      • Law R.
      • Eckenhoff R.
      • Klein M.L.
      ).
      Lipid-dependent uncoupling could also stem from more localized structural rearrangements at the lipid-nAChR interface. The five M4 helices (one from each subunit) are the most lipid-exposed transmembrane segments (Fig. 1C) (
      • Miyazawa A.
      • Fujiyoshi Y.
      • Unwin N.
      ). Mutagenesis of lipid-facing M4 residues leads to altered nAChR gating kinetics, suggesting that M4 plays a role in coupling agonist binding to channel gating (
      • Li L.
      • Lee Y.H.
      • Pappone P.
      • Palma A.
      • McNamee M.G.
      ,
      • Lee Y.H.
      • Li L.
      • Lasalde J.
      • Rojas L.
      • McNamee M.
      • Ortiz-Miranda S.I.
      • Pappone P.
      ,
      • Lasalde J.A.
      • Tamamizu S.
      • Butler D.H.
      • Vibat C.R.
      • Hung B.
      • McNamee M.G.
      ,
      • Bouzat C.
      • Roccamo A.M.
      • Garbus I.
      • Barrantes F.J.
      ,
      • Tamamizu S.
      • Guzmán G.R.
      • Santiago J.
      • Rojas L.V.
      • McNamee M.G.
      • Lasalde-Dominicci J.A.
      ,
      • Shen X.M.
      • Deymeer F.
      • Sine S.M.
      • Engel A.G.
      ). According to the current nAChR structural model, the C-terminal end of M4 extends beyond those of the other transmembrane helices and is located in close proximity to several highly conserved residues within the Cys-loop (Fig. 5, B and C). This is significant given that previous experiments have shown that the extended length of M4 is an absolute requirement for nAChR function (
      • Tobimatsu T.
      • Fujita Y.
      • Fukuda K.
      • Tanaka K.
      • Mori Y.
      • Konno T.
      • Mishina M.
      • Numa S.
      ). Furthermore, a C-terminal residue in the human α4-subunit, which is known to be critical for mediating steroid-induced potentiation (
      • Paradiso K.
      • Zhang J.
      • Steinbach J.H.
      ), conspicuously aligns with Gln-435 in M4 of the Torpedo α-subunit, a residue that is poised to interact with the highly conserved αPhe-137 within the Cys-loop (Fig. 5C). Clearly, the C-terminal end of M4 is an important, but unappreciated part of the interface between the agonist-binding and transmembrane domains. By interacting directly with both membrane lipids and residues in the Cys-loop, M4 is perfectly situated to relay bilayer properties to this coupling interface. We propose that lipid-dependent bilayer properties are important for positioning the C-terminal end of M4 so that it effectively contacts the Cys-loop. M4/Cys-loop interactions in turn affect the ability of the Cys-loop to communicate with the remainder of the transmembrane domain, particularly the all important M2-M3 linker (Fig. 5D). M4 essentially acts as a lipid-sensor relaying bilayer properties to the coupling interface.
      By binding to discrete transmembrane sites, lipids (and other allosteric modulators, including steroids) may stabilize the nAChR transmembrane domain in conformations that permit effective communication with the agonist-binding domain, perhaps by facilitating interactions between the C-terminal end of M4 and the Cys-loop. Indeed, the identification of a steroid-binding site between M4 and M1-M3 in the mouse GABAA receptor, as well as a similar modulatory site in the rat α7 nAChR, appear to support this (
      • Hosie A.M.
      • Wilkins M.E.
      • da Silva H.M.
      • Smart T.G.
      ,
      • Young G.T.
      • Zwart R.
      • Walker A.S.
      • Sher E.
      • Millar N.S.
      ). Simulations have also hinted that a similar site might exist for cholesterol in the Torpedo nAChR (
      • Brannigan G.
      • Hénin J.
      • Law R.
      • Eckenhoff R.
      • Klein M.L.
      ).
      Alternatively, a physical (rather than a chemical) property of the bilayer may dictate the favorability of helix-helix versus helix-lipid interactions. Tryptophan substitutions in M4, which in model peptides stabilize transmembrane helix associations (
      • Ridder A.
      • Skupjen P.
      • Unterreitmeier S.
      • Langosch D.
      ), lead to increased open probability of both Torpedo and human nAChRs (
      • Li L.
      • Lee Y.H.
      • Pappone P.
      • Palma A.
      • McNamee M.G.
      ,
      • Lee Y.H.
      • Li L.
      • Lasalde J.
      • Rojas L.
      • McNamee M.
      • Ortiz-Miranda S.I.
      • Pappone P.
      ,
      • Lasalde J.A.
      • Tamamizu S.
      • Butler D.H.
      • Vibat C.R.
      • Hung B.
      • McNamee M.G.
      ,
      • Tamamizu S.
      • Guzmán G.R.
      • Santiago J.
      • Rojas L.V.
      • McNamee M.G.
      • Lasalde-Dominicci J.A.
      ). Presumably the M4 tryptophan substitutions drive increased association between M4 and M1-M3, which in turn facilitate interactions between the C-terminal extension of M4 and the Cys-loop.
      Undeniably M4-lipid interactions play a key role in Cys-loop receptor function in native (or native-like) membrane environments. M4-lipid interactions even affect the open probability of human nAChRs (
      • Shen X.M.
      • Deymeer F.
      • Sine S.M.
      • Engel A.G.
      ). So whereas, M4-mediated uncoupling explains our in vitro data, the existence of this uncoupled conformation may have broader implications for Cys-loop receptor function in vivo. Lipids (and potentially other allosteric modulators) could influence the pool of agonist-activatable receptors by modulating an equilibrium between functional coupled and non-functional uncoupled states (Fig. 1D, Scheme 1).
      From a pharmacological perspective, it is clear that membrane lipid composition can modulate the efficacy of nAChR agonists. Although we have concentrated on highly simplified and therefore non-physiological membranes, what we observe likely represents extremes at opposite ends of a spectrum. Subtle alterations in lipid composition may fine-tune the synaptic response in vivo. Indeed, the fact that endogenous lipids modulate agonist responses in other Cys-loop receptors directly supports this (
      • Paradiso K.
      • Zhang J.
      • Steinbach J.H.
      ,
      • Hosie A.M.
      • Wilkins M.E.
      • da Silva H.M.
      • Smart T.G.
      ). Even subtle changes in the proportion of coupled receptors could lead to appreciable changes in macroscopic agonist responses, thereby having significant effects on pathology.
      Interestingly, structures of nAChR prokaryotic homologues show that their M4/C-terminal tails are too short to reach the β6-β7/“Cys-loop” (
      • Hilf R.J.
      • Dutzler R.
      ,
      • Hilf R.J.
      • Dutzler R.
      ,
      • Bocquet N.
      • Nury H.
      • Baaden M.
      • Le Poupon C.
      • Changeux J.P.
      • Delarue M.
      • Corringer P.J.
      ). Although the lipid sensitivity of these bacterial channels is presently unknown, their shortened M4 segments preclude a role for the C-terminal end of M4 at the coupling interface between their extra-membranous and transmembrane domains. It is thus tempting to speculate that the emergence of lipid sensitivity in the Cys-loop receptor superfamily coincided with the increasing demands of an evolving nervous system.

      Acknowledgments

      We thank Drs. Z. Yao and M. Pelchat for use of their equipment and N. Lavigne, N. Vuong, and S. Wang for technical assistance.

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