Molecular mechanisms of acetylcholine receptor–lipid interactions: from model membranes to human biology

Lipids are potent modulators of the Torpedo nicotinic acetylcholine receptor. Lipids influence nicotinic receptor function by allosteric mechanisms, stabilizing varying proportions of pre-existing resting, open, desensitized, and uncoupled conformations. Recent structures reveal that lipids could alter function by modulating transmembrane α-helix/α-helix packing, which in turn could alter the conformation of the allosteric interface that links the agonist-binding and transmembrane pore domains—this interface is essential in the coupling of agonist binding to channel gating. We discuss potential mechanisms by which lipids stabilize different conformational states in the context of the hypothesis that lipid–nicotinic receptor interactions modulate receptor function at biological synapses.     

Lipid-protein interactions at the nicotinic acetylcholine receptor. A functional coupling between nicotinic receptors and phosphatidic acid-containing lipid bilayers.

The structural and functional properties of reconstituted nicotinic acetylcholine receptor membranes composed of phosphatidyl choline either with or without cholesterol and/or phosphatidic acid have been examined to test the hypothesis that receptor conformational equilibria are modulated by the physical properties of the surrounding lipid environment. Spectroscopic and chemical labeling data indicate that the receptor in phosphatidylcholine alone is stabilized in a desensitized-like state, whereas the presence of either cholesterol or phosphatidic acid favors a resting-like conformation. Membranes that effectively stabilize a resting-like state exhibit a relatively large proportion of non-hydrogen-bonded lipid ester carbonyls, suggesting a relatively tight packing of the lipid head groups and thus a well ordered membrane. Functional reconstituted membranes also exhibit gel-to-liquid crystal phase transition temperatures that are higher than those of nonfunctional reconstituted membranes composed of phosphatidylcholine alone. Significantly, incorporation of the receptor into phosphatidic acid-containing membranes leads to a dramatic increase in both the lateral packing densities and the gel-to-liquid crystal phase transition temperatures of the reconstituted lipid bilayers. These results suggest a functional link between the nicotinic acetylcholine receptor and the physical properties of phosphatidic acid-containing membranes that could underlie the mechanism by which this lipid preferentially enhances receptor function.     

Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor

The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590-24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.     

Nicotinic acetylcholine receptor–lipid interactions: Mechanistic insight and biological function

Membrane lipids are potent modulators of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable versus non-activatable conformations, as well as influencing the transitions between these conformational states. Of note, some membranes stabilize an electrically silent uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions. The uncoupled nAChR, however, does transition to activatable conformations in relatively thick lipid bilayers, such as those found in lipid rafts. In this review, we discuss current understanding of lipid–nAChR interactions in the context of increasingly available high resolution structural and functional data. These data highlight different sites of lipid action, including the lipid-exposed M4 transmembrane α-helix. Current evidence suggests that lipids alter nAChR function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These interactions have also been implicated in both the folding and trafficking of nAChRs to the cell surface. We review current mechanistic understanding of lipid–nAChR interactions, and highlight potential biological roles for lipid–nAChR interactions in modulating the synaptic response. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg⁹⁹⁵) contacts with β3(Asp⁷²³) but paradoxically do not diminish the contribution of αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.     

Phosphatidic acid and phosphatidylserine have distinct structural and functional interactions with the nicotinic acetylcholine receptor

Bilayers containing phosphatidylcholine (PC) and the anionic lipid phosphatidic acid (PA) are particularly effective at stabilizing the nicotinic acetylcholine receptor (nAChR) in a functional conformation that undergoes agonist-induced conformational change. The physical properties of PC membranes containing PA are also substantially altered upon incorporation of the nAChR. To test whether or not the negative charge of PA is responsible for this "bi-directional coupling," the nAChR was reconstituted into membranes composed of PC with varying levels of the net negatively charged lipid phosphatidylserine (PS). In contrast to PA, increasing levels of PS in PC membranes do not stabilize an increasing proportion of nAChRs in a functional resting conformation, nor do they slow nAChR peptide hydrogen exchange kinetics. Incorporation of the nAChR had little effect on the physical properties of the PC/PS membranes, as monitored by the gel-to-liquid crystal phase transition temperatures of the bilayers. These results show that a net negative charge alone is not sufficient to account for the unique interactions that occur between the nAChR and PC/PA membranes. Incorporation of the receptor into PC/PS membranes, however, did lead to an altered head group conformation of PS possibly by recruiting divalent cations to the membrane surface. The results show that the nAChR has complex and unique interactions with both PA and PS. The interactions between the nAChR and PS may be bridged by divalent cations, such as calcium.     

On Some Structural Analogies between Acetylcholinesterase and the Macromolecular Receptor of Acetylcholine

Several properties of the enzyme acetylcholinesterase (AChE) isolated in vitro are compared with those of the membrane receptor(s) of acetylcholine expressed by the in vivo electrical response of the electroplax membrane. AChE strongly binds in vitro effectors of the electroplax: agonists e.g., decamethonium or antagonists, e.g., d-tubocurarine and flaxedil. It also reacts covalently with an affinity labeling reagent of the acetylcholine receptor site(s) in vivo (TDF). Two classes of sites on AChE molecule account for the binding of these quaternary nitrogen containing compounds: (1) the anionic site of the active center and (2) noncatalytic "peripheral anionic centers" located outside the active center. A disulfide bond breaking agent, dithiothreitol (DTT) alters in a parallel manner the reaction of AChE and the excitable membrane of the electroplax to TDF. The irreversibility of TDF action is lost in both cases, after exposure to DTT. Both AChE and the acetylcholine receptor thus contain disulfide bonds—they are closely related but not necessarily identical proteins.     

Two distinct anionic phospholipid-dependent events involved in SecA-mediated protein translocation

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed by the Sec translocase, which in its minimal form consists of the protein-conducting channel SecYEG, and the motor ATPase SecA. SecA binds via its positively charged N-terminus to membranes containing anionic phospholipids, leading to a lipid-bound intermediate. This interaction induces a conformational change in SecA, resulting in a high-affinity association with SecYEG, which initiates protein translocation. Here, we examined the effect of anionic lipids on the SecA-SecYEG interaction in more detail, and discovered a second, yet unknown, anionic lipid-dependent event that stimulates protein translocation. Based on molecular dynamics simulations we identified an anionic lipid-enriched region in vicinity of the lateral gate of SecY. Here, the anionic lipid headgroup accesses the lateral gate, thereby stabilizing the pre-open state of the channel. The simulations suggest flip-flop movement of phospholipid along the lateral gate. Electrostatic contribution of the anionic phospholipids at the lateral gate may directly stabilize positively charged residues of the signal sequence of an incoming preprotein. Such a mechanism allows for the correct positioning of the entrant peptide, thereby providing a long-sought explanation for the role of anionic lipids in signal sequence folding during protein translocation.     

Membrane-active peptides and the clustering of anionic lipids

There is some overlap in the biological activities of cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs). We compared nine AMPs, seven CPPs, and a fusion peptide with regard to their ability to cluster anionic lipids in a mixture mimicking the cytoplasmic membrane of Gram-negative bacteria, as measured by differential scanning calorimetry. We also studied their bacteriostatic effect on several bacterial strains, and examined their conformational changes upon membrane binding using circular dichroism. A remarkable correlation was found between the net positive charge of the peptides and their capacity to induce anionic lipid clustering, which was independent of their secondary structure. Among the peptides studied, six AMPs and four CPPs were found to have strong anionic lipid clustering activity. These peptides also had bacteriostatic activity against several strains (particularly Gram-negative Escherichia coli) that are sensitive to lipid clustering agents. AMPs and CPPs that did not cluster anionic lipids were not toxic to E. coli. As shown previously for several types of AMPs, anionic lipid clustering likely contributes to the mechanism of antibacterial action of highly cationic CPPs. The same mechanism could explain the escape of CPPs from intracellular endosomes that are enriched with anionic lipids.     

A Lipid-dependent Uncoupled Conformation of the Acetylcholine Receptor

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.     

Cations Mediate Interactions between the Nicotinic Acetylcholine Receptor and Anionic Lipids

Interactions between the nicotinic acetylcholine receptor (nAChR) and phosphatidic acid (PA) are bidirectional in that membranes containing PA are effective at stabilizing an agonist-responsive nAChR, whereas incorporation of the nAChR into the same membranes leads to a substantial increase in lipid lateral packing density. A previous study suggested that the ability of PA to adopt a dianionic ionization state is key. We monitored the ionization state of PA in both reconstituted and protein-free membranes. In model membranes composed of PA and 3:2 (mol/mol) phosphatidylcholine (PC)/PA, the monoanionic-to-dianionic transition of PA was detected with a pKa of 8.7 and 6.5, respectively. In the reconstituted 3:2 PC/PA membranes, however, PA was stabilized in a monoanionic state at pH values up to 10. Although dianionic PA does not play a role in nAChR function, we found that both the stabilization of monoanionic PA and the concentration of other cations at the bilayer surface can account for changes in bilayer physical properties that are observed upon incorporation of the nAChR into 3:2 PC/PA membranes. A nAChR-induced concentration of cations at the bilayer surface likely mediates interactions between the nAChR and the anionic lipids in its membrane environment.     

Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.     

Anionic lipids in Ca(2+)-triggered fusion

Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca(2+)-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca(2+)-sensing and even bind Ca(2+) directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca(2+) sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.     

Acetylcholine receptor binding properties and ion permeability response after covalent attachment of the local anaesthetic quinacrine

Membrane vesicles rich in nicotinic acetylcholine receptor prepared from Torpedo californica electric tissue have been irreversibly modified with quinacrine mustard, an alkylating derivative of the local anaesthetic quinacrine. The reaction blocked the ion channel regulated by the acetylcholine receptor. Acetylcholine still bound to the modified membrane vesicles with K_D approx. 10^?8. The number of binding sites was reduced by up to 50%. Stopped-flow experiments showed that in contrast to what had been found with reversibly binding quinacrine no fluorescence changes caused by energy transfer from the irradiated protein to the fluorescent local anaesthetic occurred after addition of agonist. This indicates that the conformational changes associated with the activation of the ion channel are blocked by the covalent reaction with quinacrine mustard. Analysis of the membrane vesicles by SDS-polyacrylamide gel electrophoresis showed that all polypeptide chains assumed to be part of the receptor complex had reacted with the mustard. Even small components, probably lipids, migrating with the dye front, showed fluorescence.     

Loss of Acetylcholine Signaling Reduces Cell Clearance Deficiencies in Caenorhabditis elegans

The ability to eliminate undesired cells by apoptosis is a key mechanism to maintain organismal health and homeostasis. Failure to clear apoptotic cells efficiently can cause autoimmune diseases in mammals. Genetic studies in Caenorhabditis elegans have greatly helped to decipher the regulation of apoptotic cell clearance. In this study, we show that the loss of levamisole-sensitive acetylcholine receptor, but not of a typical neuronal acetylcholine receptor causes a reduction in the number of persistent cell corpses in worms suffering from an engulfment deficiency. This reduction is not caused by impaired or delayed cell death but rather by a partial restoration of the cell clearance capacity. Mutants in acetylcholine turn-over elicit a similar phenotype, implying that acetylcholine signaling is the process responsible for these observations. Surprisingly, tissue specific RNAi suggests that UNC-38, a major component of the levamisole-sensitive receptor, functions in the dying germ cell to influence engulfment efficiency. Animals with loss of acetylcholine receptor exhibit a higher fraction of cell corpses positive for the “eat-me” signal phosphatidylserine. Our results suggest that modulation by ion channels of ion flow across plasma membrane in dying cells can influence the dynamics of phosphatidylserine exposure and thus clearance efficiency.     

Synthesis and properties of novel tetraalkyl cationic lipids

The synthesis, physical properties, and transfection potencies of two representives of a new class of divalent, tetraalkyl cationic lipids is described. These cationic lipids are dimers of N,N-Dioleyl-N,N-dimethylammonium chloride (DODAC) joined by a hydrocarbon tether three or six carbons in length (TODMAC3 and TODMAC6, respectively). It is shown that TODMAC6 can display improved transfection properties in comparison to DODAC when formulated into plasmid DNA-cationic lipid complexes. These improved transfection potencies are observed at cationic lipid to DNA charge ratios of two or higher. It is also shown that TODMAC6 exhibits equivalent or improved ability (as compared to DODAC) to induce nonbilayer structure in mixtures with anionic lipid. This is consistent with the hypothesis that the ability of cationic lipids to induce nonbilayer structures when mixed with anionic lipids is correlated to their transfection potency. Complexes containing TODMAC3 on the other hand exhibit lower transfection potencies than achieved with DODAC, behavior that is consistent with steric effects limiting the formation of ion pairs with anionic lipids. It is concluded that TODMAC6 exhibits potential as a transfection agent for in vitro and in vivo use and that the design of cationic lipids according to their ability to induce nonbilayer structure provides a useful guide for synthesis of new cationic lipids.     

Structural analysis and amphiphilic properties of a chemically synthesized mitochondrial signal peptide

Anionic phospholipids induce a marked conformational change in a synthetic peptide corresponding to residues 1-27 of pre-ornithine carbamyltransferase. The peptide designated, pO-(1-27)-peptide amide, becomes more alpha-helical in the presence of cardiolipin or dimyristoylphosphatidylglycerol but not in the presence of dimyristoylphosphatidylcholine. The greater helix-promoting action of anionic versus zwitterionic lipids is predicted by helix-coil transition theory. This statistical mechanical theory also predicts that a shorter peptide, N-acetyl-pO-(16-27)-peptide amide, has less helix-forming tendency, even in the presence of sodium dodecyl sulfate, despite the fact that it has a comparable number of positive charges. The N-acetyl-pO-(16-27)-peptide amide has no helical structure in buffer with or without dimyristoylphosphatidylglycerol but it has a small (5%) helical content in methanol. Thus, the ability of anionic lipids to promote helix formation requires more than the presence of cationic groups on the peptide. The angular dependence of the hydrophobic moment of the putative helical segment of pO-(1-27)-peptide amide demonstrates that any helical structure which is formed would have some amphiphilic character. The pO-(1-27)-peptide amide disrupts large lipid aggregates to form discoid micelles about 30 to 50 nm in diameter. The ability to lyse membranes into disc-shaped micelles is characteristic of peptides containing an amphiphathic helix. In the case of the mitochondrial signal peptide, this membrane-lytic behavior may contribute to the translocation of the protein into the organelle.     

Structure,phase behaviour and membrane interactions of N-acylethanolamines and N-acylphosphatidylethanolamines

N-Acylethanolamines (NAEs) and N-acylphosphatidylethanolamines (NAPEs) are naturally occurring membrane lipids, whose content increases dramatically in a variety of organisms when subjected to stress, suggesting that they may play a role in the stress-combating mechanisms of organisms. In the light of this, it is of great interest to characterize the structure, physical properties, phase transitions and membrane interactions of these two classes of lipids. This review will present the current status of our understanding of the structure and phase behaviour of NAEs and NAPEs and their interaction with major membrane lipids, namely phosphatidylcholine, phosphatidylethanolamine and cholesterol. The relevance of such interactions to the putative stress-combating and membrane stabilizing properties of these lipids will also be discussed.     

Change in the intrinsic fluorescence of acetylcholine receptor purified from Narke japonica upon binding with cholinergic ligands

Effects of various cholinergic ligands on the intrinsic fluorescence of acetylcholine receptor purified from the electric organ of Narke japonica were investigated. Binding with acetylcholine decreased the fluorescence by 7–8%, and that with carbamylcholine by 4–5% at 20 °C. Decamethonium and d-tubocurarine did not affect significantly the fluorescence intensity, while hexamethonium enhanced it. These changes were completely inhibited by preincubation of the receptor with α-bungarotoxin, which indicated that the observed intrinsic fluorescence change was due to the specific binding of each ligand. Data of the quenching experiment using iodide ion as an extrinsic quencher suggested the occurrence of the conformational change in the receptor upon binding with various cholinergic ligands. Considering these results together with those on intrinsic fluorescence change, conformational change provoked by binding with acetylcholine or carbamylcholine seems to differ from that provoked by binding with other cholinergic ligands examined.     

Zwitterionic phospholipids and sterols modulate antimicrobial peptide-induced membrane destabilization

Cationic amphipathic α-helical peptides preferentially disrupt anionic lipids in mixed model membranes, potentially causing a catastrophic release of the cell contents or attenuation of the membrane potential. The effective role of such peptides requires considerable discrimination between target and host cells, which is likely to occur at the level of the cell membrane. Here, we explore the roles of a variety of common membrane constituents in mediating the interaction between the antimicrobial peptide pleurocidin and model membranes. We employ intrinsic tryptophan fluorescence and circular dichroism to observe the effect of increasing concentrations of sterol in the membrane on peptide binding, using ²H solid-state NMR of chain deuterated lipids simultaneously to probe the effective chain disruption of the anionic phospholipid component of the membrane. We show that the degree of ordering of the lipid acyl chains in the membrane is dependent on the nature of the zwitterionic phospholipid headgroup in mixed anionic membranes. Furthermore, the presence of cholesterol and ergosterol increases acyl chain order in the liquid crystalline model membranes, but to differing degrees. Our results show how sterols can protect even negatively charged membranes from the disruptive effects of antimicrobial peptides, thereby providing a molecular view of the differences in sensitivity of various target membranes to linear cationic antibiotic peptides where bacteria (no sterols) are most susceptible, lower eukaryotes including fungi (containing ergosterol) exhibit an intermediate degree of sensitivity, and higher organisms (containing cholesterol) are largely resistant to antimicrobial peptides.