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.     

Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids.

The effects of negatively charged and neutral lipids on the function of the reconstituted nicotinic acetylcholine receptor from Torpedo californica were determined with two assays using acetylcholine receptor-containing vesicles: the ion flux response and the affinity-state transition. The receptor was reconstituted into three different lipid environments, with and without neutral lipids: (1) phosphatidylcholine/phosphatidylserine; (2) phosphatidylcholine/phosphatidic acid; and (3) phosphatidylcholine/cardiolipin. Analysis of the ion flux responses showed that: (1) all three negatively charged lipid environments gave fully functional acetylcholine receptor ion channels, provided neutral lipids were added; (2) in each lipid environment, the neutral lipids tested were functionally equivalent to cholesterol; and (3) the rate of receptor desensitization depends upon the type of neutral lipid and negatively charged phospholipid reconstituted with the receptor. The functional effects of neutral and negatively charged lipids on the acetylcholine receptor are discussed in terms of protein-lipid interactions and stabilization of protein structure by lipids.     

Correlation of phospholipid structure with functional effects on the nicotinic acetylcholine receptor. A modulatory role for phosphatidic acid.

Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy.     

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.     

Annular and nonannular binding sites for cholesterol associated with the nicotinic acetylcholine receptor

Interactions between lipids and the nicotinic acetylcholine receptor from Torpedo californica have been measured in reconstituted membranes containing purified receptor and defined lipids. The ability of brominated lipids to partially quench the intrinsic fluorescence of the acetylcholine receptor has been exploited to monitor contacts between the protein and the surrounding lipid. Relative binding constants for lipid binding to the protein have been quantitatively determined by measuring quenching observed in mixtures of brominated and nonbrominated lipids by use of equilibrium exchange equations developed by London and Feigenson [London, E., & Feigenson, G. W. (1981) Biochemistry 20, 1939-1948] and by Simmonds et al. [Simmonds, A. C., Rooney, E. K., & Lee, A. G. (1984) Biochemistry 23, 1432-1441]. Dioleoylphosphatidylcholine and its dibromo derivative are the two principal lipids used in the reconstituted membranes to establish the quenching parameters. Competition studies between cholesterol and phosphatidylcholine indicate that cholesterol does not compete effectively for the phospholipid sites presumed to surround the membrane-embedded portions of the receptor (annular lipids). However, dibromocholesterol partially quenches the receptor and leads to additional quenching of receptor in pure dibromophosphatidylcholine membranes. The results are consistent with the presence of additional binding sites for cholesterol that are not accessible to phospholipids (nonannular sites). Similar results are obtained by using cholesterol hemisuccinate and its dibromo analogue, both of which can be introduced into membranes more easily than cholesterol because of their greater solubility in water. Fatty acids appear to compete for both annular and nonannular sites, and analysis of the quenching data suggests that there are 5-10 nonannular sites associated with the receptor. Cholesterol has been shown to play a critical role in both acetylcholine receptor structural stabilization and ion channel activity, and the results presented here provide additional information about cholesterol-receptor interactions.     

The cholesterol dependence of activation and fast desensitization of the nicotinic acetylcholine receptor.

When nicotinic acetylcholine receptors are reconstituted into lipid bilayers lacking cholesterol, agonists no longer stimulate cation flux. The kinetics of this process are difficult to study because variations in vesicle morphology cause errors in flux measurements. We developed a new stopped-flow fluorescence assay to study activation independently of vesicle morphology. When receptors were rapidly mixed with agonist plus ethidium, the earliest fluorescence increase reported the fraction of channels that opened and their apparent rate of fast desensitization. These processes were absent when the receptor was reconstituted into dioleoylphosphatidylcholine or into a mixture of that lipid with dioleoylphosphatidic acid (12 mol%), even though a fluorescent agonist reported that resting-state receptors were still present. The agonist-induced channel opening probability increased with bilayer cholesterol, with a midpoint value of 9 +/- 1.7 mol% and a Hill coefficient of 1.9 +/- 0.69, reaching a plateau above 20-30 mol% cholesterol that was equal to the native value. On the other hand, the observed fast desensitization rate was comparable to that for native membranes from the lowest cholesterol concentration examined (5 mol%). Thus the ability to reach the open state after activation varies with the cholesterol concentration in the bilayer, whereas the rate of the open state to fast desensitized state transition is unaffected. The structural basis for this is unknown, but an interesting corollary is that the channels of newly synthesized receptors are not fully primed by cholesterol until they are inserted into the plasma membrane--a novel form of posttranslational processing.     

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.     

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.     

A minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptor

The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.     

Differential scanning calorimetry and Fourier transform infrared analysis of lipid-protein interactions involving the nicotinic acetylcholine receptor

Lipid-protein interactions were studied using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into dielaidoylphosphatidylcholine (DEPC, 18:1 trans-9,10) membranes. The structural and thermodynamic behavior of lipids in the vicinity of the protein were studied by differential scanning calorimetry and Fourier transform infrared spectroscopy. The effects of AChR on the thermodynamic parameters associated with lipid phase transitions were to reduce the enthalpy change, lower the transition temperature and reduce the cooperative behavior of the lipid molecules. A stoichiometry of approx. 95 lipids per AChR molecule was found by simulating the decrease in enthalpy in terms of a simple model in which a fixed number of lipid molecules are prevented from undergoing a cooperative phase transition. In parallel, the vibrational spectra of pure DEPC and AChR reconstituted in DEPC membranes at various lipid to protein ratios were examined. Profiles of the 3000-2800 cm-1 C-H stretching region and 1350-950 cm-1 characteristic of the headgroup region of the lipid exhibit little sensitivity to protein/lipid ratio reflecting weak interaction of AChR with DEPC. The lipid carbonyl on the other hand appear to be increasingly hydrogen bonded in the presence of AChR. The results provide new information about the size and physical state of the motionally restricted lipid environment that surrounds the acetylcholine receptor. The results are discussed in the context of lipid-mediated alterations in acetylcholine receptor function.     

The effect of amantadine on nicotinic acetylcholine receptor (nAchR) in reconstituted membranes

The antiviral drug amantadine is also a potent neuromuscular blocking agent. When the nicotinic receptor from a Torpedinidae species is reconstituted into soybean liposomes, the binding of α-bungarotoxin is not altered although the carbamylcholine induced radioactive cation influx is blocked.By studying cation fluxes in amantadine preincubated membranes previously exposed to different concentrations of carbamylcholine for different periods of time, we have shown that the drug accelerates the conversion of the nicotinic acetylcholine receptor from a state of low affinity to a state of high affinity for carbamyalcholine, a phenomenon correlated with receptor desensitization. The drug did not induce such a shift by itself.The present data and those by Earnest et al. (Biochemistry22, 5523–5535, 1984) show that the nicotinic acetylcholine receptor reconstituted into liposomes is a good model for studying the effects of noncompetitive blockers of nicotinic acetylcholine receptor function.     

Nicotinic acetylcholine receptor induces lateral segregation of phosphatidic acid and phosphatidylcholine in reconstituted membranes

Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup and fatty acyl chain saturation but also on AChR protein-lipid interactions. Additional evidence suggests a possible competition between Chol and POPA for the same binding sites on the AChR protein.     

Cholesterol modulates the organization of the gammaM4 transmembrane domain of the muscle nicotinic acetylcholine receptor

A 28-mer gammaM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor gamma-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gammaM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gammaM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low F?rster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gammaM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gammaM4 peptide occupies approximately 30% of the patch area.     

Arg-Lys-Asp-Val-Tyr (thymopentin) accelerates the cholinergic-induced inactivation (desensitization) of reconstituted nicotinic receptor

1. The thymic hormone thymopoietin blocks neuromuscular transmission and was proposed (Goldstein, 1974) as a modulator of synaptic conductivity. 2. The cholinergic-induced inactivation of nicotinic receptor reconstituted into asolectin lipid vesicles was studied in the presence and in the absence of thymopentin, a synthetic pentapeptide corresponding to positions 32-36 of thymopoietin. 3. The present data show that thymopentin accelerates desensitization of the nicotinic acetylcholine receptor, supporting the aforementioned physiological role proposed for thymopoietin. 4. They also suggest that the hormone itself and/or a yet unidentified hormine-derived peptide fragment may act as an endogenous ligand for nicotinic acetylcholine receptor desensitization.     

Behavioral differences between phosphatidic acid and phosphatidylcholine in the presence of the nicotinic acetylcholine receptor

It has been found experimentally that negatively charged phosphatidic acid (PA) lipids and cholesterol molecules stabilize the nicotinic acetylcholine receptor (nAChR) in a functional resting state that can participate in an agonist-induced conformational change. In this study, we compare phosphatidylcholine (PC) and PA lipid behavior in the presence of the nAChR to determine why PC lipids do not support a functional nAChR. For lipids that are located within 1.0 nm of the protein, both PC and PA lipids have very similar order-parameter and bilayer-thickness values, which indicate that the annular lipid properties are protein-dependent. The most significant difference between the PC and PA bilayers is the formation of a lipid domain around the protein, which is visible in the PA bilayer but not the PC bilayer. This suggests that the PA domain may help stabilize the nAChR resting state. The PA lipids in the microdomain have a decreased order compared to a homogeneous PA bilayer and the lipids near the protein attempt to increase the free space in their vicinity by residing in multiple lateral planes.     

Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution

The nicotinic acetylcholine receptor (AChR) is in intimate contact with the lipids in its native membrane. Here we analyze the possibility that it is the intrinsic properties of the AChR that determine its partition into a given lipid domain. Torpedo AChR or a synthetic peptide corresponding to the AChR γM4 segment (the one in closer contact with lipids) was reconstituted into “raft”-containing model membranes. The distribution of the AChR was assessed by Triton X-100 extraction in combination with fluorescence studies, and lipid analyses were performed on each sample. The influence of rapsyn, a peripheral protein involved in AChR aggregation, was studied. Raft-like domain aggregation was also studied using membranes containing the ganglioside GM1 followed by GM1 crosslinking. The γM4 peptide displays a marked preference for raft-like domains. In contrast, AChR alone or in the presence of rapsyn or ganglioside aggregation exhibits no such preference for raft-like domains, but it does cause a significant reduction in the total amount of these domains. The results indicate that the distribution of the AChR in lipid domains cannot be due exclusively to the intrinsic physicochemical properties of the protein and that there must be an external signal in native cell membranes that directs the AChR to a specific membrane domain.     

Correlation between acetylcholine receptor function and structural properties of membranes

Protein-lipid interactions were studied by using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into membranes containing various synthetic lipids and native lipids. AChR function was determined by measuring two activities at 4 degrees C: (1) low to high agonist affinity-state transition of AChR in the presence of an agonist (carbamylcholine) in either membrane fragments or sealed vesicles and (2) ion-gating activity of AChR-containing vesicles in response to carbamylcholine. Sixteen samples were examined, each containing different lipid compositions including phosphatidylcholine, cholesterol, phosphatidic acid, phosphatidylethanolamine, asolectin, neutral lipid depleted asolectin, native lipids, and cholesterol-depleted native lipids. Phosphatidylcholines with different configurations of fatty acyl chains were used. The dynamic structures of these membranes were probed by incorporating spin-labeled fatty acid into AChR-containing vesicles and measuring the order parameters. It was found that both aspects of AChR function were highly dependent on the lipid environment even though carbamylcholine binding itself was not affected. An appropriate membrane fluidity was necessarily required to allow the interconversion between the low and high affinity states of AChR. An optimal fluidity hypothesis is proposed to account for the conformational transition properties of membrane proteins. In addition, the conformational change was only a necessary, but not sufficient, condition for the AChR-mediated ion flux activity. Among membranes in which AChR manifested the affinity-state transition, only those containing both cholesterol and negatively charged phospholipids (such as phosphatidic acid) retained the ion-gating activity.     

The nicotinic acetylcholine receptor from Discopyge tschudii: purification, characterization and reconstitution into liposomes

The nicotinic acetylcholine receptor from Discopyge tschudii electroplax was purified by affinity chromatography on Affi-Gel 401 using bromoacetylcholine as the ligand. Its specific activity was about 4000 pmol 125I-alpha-bungarotoxin/mg protein. SDS-polyacrylamide gel electrophoresis revealed four bands of apparent molecular weights: 41,200, 49,500, 60,000 and 66,300. The amino acid composition of each individual subunit was determined. Native membranes, rich in nicotinic receptor exhibited carbamylcholine-catalysed cation transport (blocked by curare and desensitized by prior incubation with the cholinergic agonist). The functional activity of the purified material could be reconstituted into soybean lecithin liposomes. Our data show that the Discopyge tschudii nicotinic receptor is similar to that from Torpedo californica.     

Postsynaptic membranes in the electric tissue of Narcine: I. Organization and innervation of electric cells. Fine structure of nicotinic receptor-channel molecules revealed by transmission microscopy

The ventral, postsynaptic membranes of the electroplaques of Narcine were examined for structures corresponding in location, numbers, position and form with nicotinic acetylcholine receptor-channel molecules. Electron micrographs of conventionally fixed material showed that the thickened outer lamina of these membranes contained about 6100 stained structures/μm² ; and calculations from this packing density and the estimated area of postsynaptic membranes/g tissue yielded a total number of structures within a factor of two of the number of receptor molecules/g determined from the binding of alpha-bungarotoxin. Examination of tissue prepared with a tannic acid/glutaraldehyde fixative revealed that the structures were uniform in character, cylindrical in shape, transmembrane in position, and that each contained a central core. Their diameter matched that of isolated receptor molecules, and their size can account for most of the receptor protein. We conclude that these receptor molecules are largely intramembranous, cylindrical and exhibit transmembrane pores.     

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.