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Anionic Lipids Allosterically Modulate Multiple Nicotinic Acetylcholine Receptor Conformational Equilibria*

  • Corrie J.B. daCosta
    Footnotes
    Affiliations
    Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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  • Sarah A. Medaglia
    Affiliations
    Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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  • Nadine Lavigne
    Affiliations
    Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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  • Shuzhi Wang
    Affiliations
    Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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  • Casey L. Carswell
    Affiliations
    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 should be addressed: Dept. of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada. Tel.: 613-562-5800 (Ext. 8222); Fax: 613-562-5251
    Affiliations
    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 a grant from the Canadian Institutes of Health Research (to J. E. B.) and a Canadian Institutes of Health Research Canadian Graduate Scholarship (to C. J. B. D.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6, Table S1, and “Discussion.”
    1 Present address: Receptor Biology Laboratory, Dept. of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905.
Open AccessPublished:October 08, 2009DOI:https://doi.org/10.1074/jbc.M109.048280
      Anionic lipids influence the ability of the nicotinic acetylcholine receptor to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. We show here that anionic lipids with relatively small headgroups, and thus the greatest ability to influence lipid packing/bilayer physical properties, are the most effective at stabilizing an agonist-activatable receptor. The differing abilities of anionic lipids to stabilize an activatable receptor stem from differing abilities to preferentially favor resting over both uncoupled and desensitized conformations. Anionic lipids thus modulate multiple acetylcholine receptor conformational equilibria. Our data suggest that both lipids and membrane physical properties act as classic allosteric modulators influencing function by interacting with and thus preferentially stabilizing different native acetylcholine receptor conformational states.

      Introduction

      Cys loop receptors are a superfamily of membrane proteins that mediate synaptic transmission in both the central and peripheral nervous systems (
      • Sine S.M.
      • Engel A.G.
      ). They respond to the binding of the neurotransmitter by transiently opening an ion channel across the cell membrane. The resultant influx of ions into the cell alters the membrane potential leading to either the generation or the inhibition of an action potential. Any factor that influences the duration or magnitude of the neurotransmitter-induced response will alter the efficiency of synaptic transmission with important biological and potentially pathological consequences (
      • Shen X.M.
      • Deymeer F.
      • Sine S.M.
      • Engel A.G.
      ,
      • Engel A.G.
      • Ohno K.
      • Sine S.M.
      ).
      Lipids are potent modulators of Cys loop receptor function (
      • Barrantes F.J.
      ). The lipid sensitivity of one Cys loop receptor, the nicotinic acetylcholine receptor (nAChR)
      The abbreviations used are: nAChR
      nicotinic acetylcholine receptor
      PA
      1,2-diacyl-sn-glycero-3-phosphate
      PC
      1,2-diacyl-sn-glycero-3-phosphocholine
      PG
      1,2-diacyl-sn-glycero-3-phosphoglycerol
      PI
      1,2-diacyl-sn-glycero-3-phosphoinositol
      PS
      1,2-diacyl-sn-glycero-3-phospho-l-serine
      Chol
      cholesterol
      Carb
      carbamylcholine chloride.
      from Torpedo, has been particularly well studied. The ability of the nAChR to undergo allosteric transitions, and thus conduct cations across the membrane in response to agonist binding, is highly dependent upon the lipid composition of the membrane in which it is embedded (
      • Criado M.
      • Eibl H.
      • Barrantes F.J.
      ,
      • Fong T.M.
      • McNamee M.G.
      ,
      • Ryan S.E.
      • Demers C.N.
      • Chew J.P.
      • Baenziger J.E.
      ,
      • Rankin S.E.
      • Addona G.H.
      • Kloczewiak M.A.
      • Bugge B.
      • Miller K.W.
      ,
      • Baenziger J.E.
      • Morris M.L.
      • Darsaut T.E.
      • Ryan S.E.
      ,
      • Hamouda A.K.
      • Sanghvi M.
      • Sauls D.
      • Machu T.K.
      • Blanton M.P.
      ,
      • Sunshine C.
      • McNamee M.G.
      ,
      • Sunshine C.
      • McNamee M.G.
      ). In native Torpedo membranes, a complex mixture of lipids works synergistically to stabilize predominantly the agonist-activatable resting nAChR (
      • Baenziger J.E.
      • Morris M.L.
      • Darsaut T.E.
      • Ryan S.E.
      ). In contrast, the nAChR reconstituted into phosphatidylcholine membranes (PC-nAChR) does not undergo agonist-induced allosteric transitions unless neutral and/or anionic lipids are present (
      • Criado M.
      • Eibl H.
      • Barrantes F.J.
      ,
      • Fong T.M.
      • McNamee M.G.
      ,
      • Ryan S.E.
      • Demers C.N.
      • Chew J.P.
      • Baenziger J.E.
      ,
      • Rankin S.E.
      • Addona G.H.
      • Kloczewiak M.A.
      • Bugge B.
      • Miller K.W.
      ,
      • Baenziger J.E.
      • Morris M.L.
      • Darsaut T.E.
      • Ryan S.E.
      ,
      • Hamouda A.K.
      • Sanghvi M.
      • Sauls D.
      • Machu T.K.
      • Blanton M.P.
      ,
      • Sunshine C.
      • McNamee M.G.
      ,
      • Sunshine C.
      • McNamee M.G.
      ,
      • McCarthy M.P.
      • Moore M.A.
      ).
      PC-nAChR is unresponsive to agonist because it adopts an uncoupled conformation where allosteric communication between the agonist-binding and transmembrane pore domains is lost, even though both domains adopt structures suggestive of the activatable resting state (
      • daCosta C.J.
      • Baenziger J.E.
      ). Lipids are thought to influence coupling by interacting with the highly lipid-exposed M4 transmembrane α-helix. M4 extends beyond the lipid bilayer toward the eponymous Cys loop, which is central for coupling agonist binding to channel gating (
      • Bouzat C.
      • Gumilar F.
      • Spitzmaul G.
      • Wang H.L.
      • Rayes D.
      • Hansen S.B.
      • Taylor P.
      • Sine S.M.
      ,
      • Shen X.M.
      • Ohno K.
      • Tsujino A.
      • Brengman J.M.
      • Gingold M.
      • Sine S.M.
      • Engel A.G.
      ). M4 may act as a “lipid sensor” relaying membrane properties to the coupling interface between the agonist binding and transmembrane pore domains. By interacting preferentially with M4 in the coupled resting conformation, lipids may stabilize the resting state (
      • daCosta C.J.
      • Baenziger J.E.
      ).
      Most studies suggest that the nAChR requires both anionic and neutral lipids in a PC membrane to adopt an agonist-activatable conformation (
      • Fong T.M.
      • McNamee M.G.
      ,
      • Sunshine C.
      • McNamee M.G.
      ,
      • Sunshine C.
      • McNamee M.G.
      ). Of the many natural anionic lipids, phosphatidic acid (PA) is particularly effective. High levels of PA in a PC membrane are sufficient to stabilize an agonist-activatable nAChR (
      • Baenziger J.E.
      • Morris M.L.
      • Darsaut T.E.
      • Ryan S.E.
      ,
      • daCosta C.J.
      • Ogrel A.A.
      • McCardy E.A.
      • Blanton M.P.
      • Baenziger J.E.
      ). In contrast, the nAChR in a PC membrane containing the anionic lipid, phosphatidylserine (PS), is not responsive to agonist unless cholesterol (Chol) is present (
      • daCosta C.J.
      • Wagg I.D.
      • McKay M.E.
      • Baenziger J.E.
      ). The strikingly different efficacies of PA and PS suggest that PA imparts a unique chemical and/or physical property onto the reconstituted membranes that is required to stabilize the resting conformation. One obvious chemical difference is the charge distribution within the two lipid headgroups (Fig. 1), which could lead to essential interactions between PA and polar side chains in the resting conformation. Alternatively, the PA headgroup is much smaller than the headgroup of PS and will have a larger influence on lipid packing/bilayer physical properties (
      • Kooijman E.E.
      • Chupin V.
      • Fuller N.L.
      • Kozlov M.M.
      • de Kruijff B.
      • Burger K.N.
      • Rand P.R.
      ). Membrane physical properties could influence transmembrane helix:helix associations to favor the resting nAChR.
      Figure thumbnail gr1
      FIGURE 1Chemical structures of anionic phospholipids. A, phospholipid molecules studied here contain palmitoyl (R1) and oleoyl (R2) acyl chains attached to a phosphoglycerol backbone. The headgroup (X) connects to the phosphate moiety. For PI, both R1 and R2 are oleoyl chains. B, structures of various headgroups (X). Cardiolipin (Card) is composed of two phospholipid moieties (Y in A) linked through a single glycerol moiety.
      The initial goal of this work was to probe the mechanism(s) by which PC/PA membranes stabilize an agonist-activatable nAChR. We examined a variety of reconstituted PC/anionic lipid membranes and found that anionic lipids with relatively small headgroup cross-sectional areas, and thus the greatest ability to influence lipid packing/bilayer physical properties, are the most effective at stabilizing an agonist-activatable resting conformation. This suggests that membrane physical properties play a role in stabilizing the resting nAChR.
      More importantly, we also found that the differing abilities of PC/anionic lipid membranes to stabilize an activatable nAChR stem from differing abilities to preferentially stabilize resting over both uncoupled and desensitized conformations. In other words, anionic lipids modulate multiple nAChR conformational equilibria. Our findings suggest that both lipids and membrane physical properties act as classic allosteric modulators influencing nAChR function by preferentially interacting with and thus stabilizing native conformational states.

      DISCUSSION

      Cation flux through the nAChR is usually interpreted in the context of a conformational model involving resting, open, and desensitized states. In this model, the magnitude of an agonist-induced macroscopic flux depends on the relative proportions of pre-existing activatable/resting versus nonactivatable desensitized conformations, as well the ability of an agonist to transition the nAChR to an open state (Fig. 7). The observation that PC-nAChR adopts a distinct uncoupled conformation adds complexity to this conformational scheme (
      • daCosta C.J.
      • Baenziger J.E.
      ). The macroscopic response of the nAChR in a reconstituted membrane must be interpreted in terms of a model that includes resting, desensitized, open, and uncoupled conformations (Fig. 7) (
      • daCosta C.J.
      • Baenziger J.E.
      ).
      Figure thumbnail gr7
      FIGURE 7Conformational scheme for the nAChR in reconstituted membranes. The effects of lipids on nAChR function have been interpreted previously using a conformational scheme involving resting (R), open (O), and desensitized (D) conformations (scheme 2). The nAChR in reconstituted membranes can be stabilized in an uncoupled conformation (
      • daCosta C.J.
      • Baenziger J.E.
      ). Lipid effects on nAChR function in reconstituted membranes must therefore include equilibria between uncoupled (U) and coupled (R, O, and D) states (scheme 1).
      Previous studies have shown that the addition of both PA and Chol to a reconstituted PC membrane stabilizes predominantly a resting nAChR (
      • Fong T.M.
      • McNamee M.G.
      ,
      • Hamouda A.K.
      • Sanghvi M.
      • Sauls D.
      • Machu T.K.
      • Blanton M.P.
      ,
      • McCarthy M.P.
      • Moore M.A.
      ,
      • daCosta C.J.
      • Baenziger J.E.
      ). This shows that Chol and PA together modulate the equilibrium between uncoupled and resting conformations (
      • daCosta C.J.
      • Baenziger J.E.
      ). We show here that anionic lipids influence the equilibria between uncoupled, resting, and desensitized conformations. Different PC/anionic lipid membranes have different abilities to stabilize the nAChR in an agonist-activatable resting conformation because they have different abilities to stabilize resting over both uncoupled and desensitized states. For example, PC/PA membranes stabilize a large proportion of resting nAChRs because they limit the numbers of both uncoupled and desensitized receptors. PC/PG membranes are less effective at stabilizing an agonist-activatable nAChR because they stabilize a large proportion of both uncoupled and desensitized nAChRs. In contrast, PC/PS and PC/PI membranes are relatively ineffective because both membranes favor the uncoupled state.
      The finding that lipids influence multiple nAChR conformational equilibria leads to an important shift in how we both interpret and investigate nAChR-lipid interactions. Many studies have focused on elucidating how specific lipids influence “function,” typically as measured by a single assay that probes the ability of the nAChR to flux cations or undergo an allosteric transition from one conformation to another. This approach implies that there is a single mechanism by which each lipid interacts and thus influences nAChR function.
      The fact that lipids influence multiple conformational equilibria, however, raises the possibility that there are a number of mechanisms by which lipids alter function. Lipids may interact preferentially with, and thus preferentially stabilize, either resting, desensitized, or uncoupled conformations. Note that this does not necessarily require distinct lipid-binding sites on each conformation; it may simply suggest that lipids at a single site interact more strongly with one conformation over another. Also, the membrane-exposed surface of the nAChR transmembrane domain may itself act as an “allosteric site,” which is sensitive to bulk membrane mechanical properties, such as hydrophobic mismatch, intrinsic curvature, etc. To understand how lipids influence the proportions of activatable (resting) versus nonactivatable (uncoupled and desensitized) conformations, one must characterize how lipids and different membrane mechanical properties interact with each individual conformational state. In fact, some lipids may have complex interactions with the nAChR in that they preferentially stabilize one conformation by binding to a specific lipid-binding site, while preferentially stabilizing another via effects on bulk membrane mechanical properties.
      Although we have studied equilibrium conditions, our fluorescence data also show a rich complexity to the rates of ethidium binding, suggesting that lipids influence the rates of transitions between conformational states. To understand how lipids influence these rates, one must elucidate how lipids interact with transition states to alter the activation energies governing conformational transitions.
      The fact that lipids influence multiple conformations also impacts on our understanding of lipid specificity at the nAChR. Some studies have concluded that neutral and anionic lipids are both essential, with considerable research focusing on the role of Chol (
      • Fong T.M.
      • McNamee M.G.
      ,
      • Rankin S.E.
      • Addona G.H.
      • Kloczewiak M.A.
      • Bugge B.
      • Miller K.W.
      ,
      • Criado M.
      • Barrantes F.J.
      ). It has been suggested that Chol and anionic lipids influence nAChR secondary (
      • Fong T.M.
      • McNamee M.G.
      ,
      • Fernandez-Ballester G.
      • Castresana J.
      • Fernandez A.M.
      • Arrondo J.L.
      • Ferragut J.A.
      • Gonzalez-Ros J.M.
      ) or tertiary/quaternary structure (
      • Méthot N.
      • Demers C.N.
      • Baenziger J.E.
      ,
      • Brannigan G.
      • Hénin J.
      • Law R.
      • Eckenhoff R.
      • Klein M.L.
      ). The implication is that lipids are required for the nAChR to “fold” into a native conformation.
      The hypothesis that specific lipids are required to stabilize a native structure contrasts with an allosteric model, which implies that lipids interact with and preferentially stabilize pre-existing conformational states. There are many different nAChR conformations in reconstituted membranes. Each of these may interact differently with different lipids or lipid properties. Given the diversity of lipids found in biological membranes and their potentially complex effects on membrane physical properties, it is likely that many lipids influence nAChR conformational equilibria. Although some, such as Chol, may have a greater influence than others, no specific lipid may be absolutely essential for function. In agreement with this hypothesis, all PC/anionic lipid membranes studied here stabilize a proportion of agonist-activatable nAChRs, although the proportions vary substantially from one membrane to another.
      The relative efficacies of the PC/anionic lipid membranes for stabilizing an agonist-activatable resting state that undergoes both gating and desensitization are PC/PA > PC/PG > PC/PS > PC/PI ≅ PC/cardiolipin, with the latter two being relatively ineffective. A similar rank potency was found in the efficacy of PA, PS, and PI to stabilize a functional nAChR in reconstituted membranes containing PC and Chol (
      • Hamouda A.K.
      • Sanghvi M.
      • Sauls D.
      • Machu T.K.
      • Blanton M.P.
      ). Why do anionic lipids vary in their abilities to stabilize a functional nAChR?
      One possible explanation is that the charge distribution within the anionic lipid headgroups may dictate their ability to interact preferentially with the resting conformation. It has been suggested that the nAChR stabilizes PA in a dianionic state and that dianionic PA is particularly effective at stabilizing the nAChR in an agonist responsive conformation (
      • Bhushan A.
      • McNamee M.G.
      ). A recent study, however, showed the contrary.
      R. M. Sturgeon and J. E. Baenziger, submitted for publication.
      The nAChR stabilizes PA in a reconstituted membrane in the mono-anionic state, possibly by concentrating cations, including protons, at the membrane surface.
      Another possible explanation stems from the observation that the efficacy of an anionic lipid is related to the surface area of the headgroup. Those anionic lipids that have smaller headgroup cross-sectional areas (
      • Dickey A.
      • Faller R.
      ,
      • Elmore D.E.
      ) are more effective at stabilizing an agonist-activatable resting nAChR (i.e. PA and PG) than those with larger headgroups (PI and PS). For example, one study estimated the relative surface areas of PA, PG, and PC to be 5.20 ± 0.04, 5.48 ± 0.04, and 6.41 ± 0.06 Å2 (
      • Dickey A.
      • Faller R.
      ). Lipids with small headgroup surface areas, such as PA and diacylglycerol, exhibit a negative intrinsic curvature (see below), although lipids with larger headgroup, such as PC and PS, exhibit minimal intrinsic curvature (
      • Kooijman E.E.
      • Chupin V.
      • Fuller N.L.
      • Kozlov M.M.
      • de Kruijff B.
      • Burger K.N.
      • Rand P.R.
      ). The most effective anionic lipid, PA, thus has a substantially smaller headgroup than any other anionic lipid.
      The relative cross-sectional areas of the headgroup and acyl chain regions of a phospholipid influences the ability of a lipid to pack effectively into a bilayer (
      • Kooijman E.E.
      • Chupin V.
      • Fuller N.L.
      • Kozlov M.M.
      • de Kruijff B.
      • Burger K.N.
      • Rand P.R.
      ,
      • Lee A.G.
      ,
      • Alley S.H.
      • Ces O.
      • Barahona M.
      • Templer R.H.
      ,
      • Cullis P.R.
      • Fenske D.B.
      • Hope M.J.
      Biochemistry of Lipids, Lipoproteins and Membranes.
      ). Lipids with smaller headgroups, such as phosphatidylethanolamine, exhibit inverted cone-like shapes that tend to favor hexagonal phases. When forced into a bilayer, phosphatidylethanolamine leads to curvature stress, a form of potential energy essentially stored within the bilayer. Given that PA (and to a lesser extent PG) has a headgroup that is relatively small, one would expect incorporation of large amounts of PA (or PG) into a planar PC bilayer to result in curvature frustration. In contrast, anionic lipids with headgroups similar in size to that of the PC choline group (i.e. PS and PI) should pack relatively seamlessly into PC bilayers. Curvature frustration may drive transmembrane helix associations leading to effective nAChR coupling. Both anionic lipids and an appropriate physical environment may preferentially stabilize a resting conformation over uncoupled and desensitized nAChRs. Tightly associated lipids have been identified in the crystal structure of a prokaryotic homolog of the nAChR (
      • Bocquet N.
      • Nury H.
      • Baaden M.
      • Le Poupon C.
      • Changeux J.P.
      • Delarue M.
      • Corringer P.J.
      ). The possible links between membrane mechanical properties and nAChR function require further investigation.
      In conclusion, our data show that anionic lipids exhibit strikingly different abilities to stabilize the nAChR in an agonist-activatable resting conformation because they stabilize different proportions of resting, desensitized, and uncoupled states. Bulk membrane physical properties, related to headgroup size, appear to play a role in the efficacies of some anionic lipids. Lipids and membrane physical properties act as allosteric modulators influencing nAChR function by interacting with and preferentially stabilizing native nAChR conformations.

      Acknowledgments

      We thank Martin Pelchat for the extensive use of the fluorescence spectrometer.

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