Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Mechanical properties of bilayers containing sperm sphingomyelins and ceramides with very long-chain polyunsaturated fatty acids.

Sphingomyelins (SM) and ceramides (Cer) with very long chain polyunsaturated fatty acids (V) are important components of spermatozoa membranes. In this study, the mechanical properties of bilayers of SM and Cer with nonhydroxy (n-V) and 2-hydroxy (h-V) fatty acid (30:5) were studied by molecular dynamics simulation at different temperatures and in the presence and the absence of salt. From our results, it was evidenced how n-V SM and h-V SM bilayers showed similar behavior. When n-V Cer was added to a h-V SM bilayer, the Gaussian curvature modulus and Ecurve of binary bilayers decreased. This variation in the mechanical properties of the bilayer can be associated with an incipient step during the fecundation process.

Boundary lipids in the nicotinic acetylcholine receptor microenvironment.

The structural and functional properties of the nicotinic acetylcholine receptor (AChR), the archetype molecule in the superfamily of Cys-looped ligand-gated ion channels, are strongly dependent on the lipids in the vicinal microenvironment. The influence on receptor properties is mainly exerted by the AChR-vicinal ("shell" or "annular") lipids, which occur in the liquid-ordered phase as opposed to the more disordered and "fluid" bulk membrane lipids. Fluorescence studies from our laboratory have identified discrete sites for fatty acids, phospholipids, and cholesterol on the AChR protein, and electron-spin resonance spectroscopy has enabled the establishment of the stoichiometry and selectivity of the shell lipid for the AChR and the disclosure of lipid sites in the AChR transmembrane region. Experimental evidence supports the notion that the interface between the protein moiety and the adjacent lipid shell is the locus of a variety of pharmacologically relevant processes, including the action of steroids and other lipids. I surmise that the outermost ring of M4 helices constitutes the boundary interface, most suitable to convey the signals from the lipid microenvironment to the rest of the transmembrane region, and to the channel inner ring in particular.

Topography of nicotinic acetylcholine receptor membrane-embedded domains.

The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in alphaM1, alphaM4, gammaM1, and gammaM4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear alpha-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.

Nongenomic effects of steroids on the nicotinic acetylcholine receptor.

A fast signaling mode of natural and synthetic steroids is exerted on some ion channels and cell-surface receptors. This activity contrasts with their classic mode of action, via intracellular receptors. Early studies from our laboratory demonstrated that spin-labeled androstanol and cholestane interact with the nicotinic acetylcholine receptor (AChR) and that lipid mobility at the lipid belt surrounding the AChR is reduced relative to that of the bulk membrane lipid. The occurrence of discrete and independent sites for phospholipids and sterols, both accessible to fatty acids, was subsequently disclosed in the native membrane. Synthetic and natural glucocorticoids were found to act as noncompetitive inhibitors of AChR function. The influence of different substituent groups in the cyclepentane perhydrophenanthrene ring on the channel-shortening potency of various steroids has also been assayed in muscle-type AChR, and we found a certain selectivity of this effect. Some organochlorine pesticides are xenoestrogens, that is, environmental agents capable of disrupting endocrine system signaling. We determined their effects on the AChR membrane using novel fluorescence techniques.

Effect of organochlorine insecticides on nicotinic acetylcholine receptor-rich membranes.

The so-called generalized polarization (GP) of the fluorescent probe Laurdan and the steady-state fluorescence anisotropy of the probe diphenylhexatriene (DPH) and its phenylpropionic derivative (PA-DPH) were used to study the effects of several organochlorine insecticides of the chlorophenylethane, chlorinated cyclohexane and chlorinated cyclodiene families on the Torpedo nicotinic acetylcholine receptor (AChR)-rich native membrane. All insecticides, with the exception of Lindane, augmented Laurdan GP both in the native membrane and in model lipid systems. Most organochlorine compounds produced a concentration-dependent decrease of DPH and PA-DPH anisotropy in the AChR-rich membrane. These compounds exhibited a dual behavior vis-à-vis the native AChR-rich membrane, exerting disordering effects at the bilayer core while ordering and/or excluding water molecules from the lipid-protein interface region, as sensed by DPH anisotropy and Laurdan GP, respectively. Furthermore, all insecticides decreased the efficiency of fluorescence resonance energy transfer between the intrinsic protein and Laurdan, albeit to different extents. On the basis of all these observations, the existence of potential target sites for insecticides in the protein-lipid interface region is postulated.

Disclosure of discrete sites for phospholipid and sterols at the protein-lipid interface in native acetylcholine receptor-rich membrane.

There is an increasing body of evidence to support the notion that the function of the nicotinic acetylcholine receptor (AChR) is influenced by its lipid microenvironment [see Barrantes, F. J. (1993) FASEB J. 7, 1460-1467]. We have recently made use of the so-called generalized polarization (GP) of the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) to learn about the physical state of the lipids in Torpedo marmorata AChR native membrane [Antollini, S. S., Soto, M. A., Bonini de Romanelli, I., Gutiérrez Merino, C., Sotomayor, P., and Barrantes, F. J. (1996) Biophys. J. 70, 1275-1284] and cells expressing endogenous or heterologous AChR [Zanello, L. P., Aztiria, E., Antollini, S., and Barrantes, F. J. (1996) Biophys. J. 70, 2155-2164]. In the present work, Laurdan GP was measured in T. marmorata native AChR membrane by direct excitation or under energy transfer conditions in the presence of exogenous lipids. GP was found to diminish in these two regions upon addition of oleic acid and dioleoylphosphatidylcholine and not to vary significantly upon addition of cholesterol hemisuccinate, indicating an increase in the polarity of the single, ordered-liquid lipid phase in the two former cases. Complementary information about the bulk lipid order was obtained from measurements of fluorescence anisotropy of DPH and two of its derivatives. The membrane order diminished in the presence of oleic acid and dioleoylphosphatidylcholine. The location of Laurdan was determined using the parallax method. Laurdan lies at approximately 10 A from the center of the bilayer, i.e., at depth of approximately 5 A from the lipid-water interface. Exogenous lipids modified the energy transfer efficiency from the intrinsic fluorescence to Laurdan. This strategy is introduced as a new analytic tool that discloses for the first time the occurrence of discrete and independent sites for phospholipids and sterols, respectively, both accessible to fatty acids, and presumably located at a shallow depth close to the phospholipid polar head region in the native AChR membrane.

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer.

The spectral properties of the fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) were exploited to learn about the physical state of the lipids in the nicotinic acetylcholine receptor (AChR)-rich membrane and compare them with those in reconstituted liposomes prepared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 60 nm were observed as a function of temperature in the spectral emission maximum of laurdan embedded in these membranes. The so-called generalized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by approximately 85% as temperature increased, but no phase transitions with a clear Tm were observed. A still unexploited property of laurdan, namely its ability to act as a fluorescence energy transfer acceptor from tryptophan emission, has been used to measure properties of the protein-vicinal lipid. Energy transfer from the protein in the AChR-rich membrane to laurdan molecules could be observed upon excitation at 290 nm. The efficiency of this process was approximately 55% for 1 microM laurdan. A minimum donor-acceptor distance r of 14 +/- 1 A could be calculated considering a distance 0 < H < 10 A for the separation of the planes containing donor and acceptor molecules, respectively. This value of r corresponds closely to the diameter of the first-shell protein-associated lipid. A value of approximately 1 was calculated for Kr, the apparent dissociation constant of laurdan, indicating no preferential affinity for the protein-associated probe, i.e., random distribution in the membrane. From the spectral characteristics of laurdan in the native AChR-rich membrane, differences in the structural and dynamic properties of water penetration in the protein-vicinal and bulk bilayer lipid regions can be deduced. We conclude that 1) the physical state of the bulk lipid in the native AChR-rich membrane is similar to that of the total lipids reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic interface upon increasing the temperature; 2) the wavelength dependence of laurdan generalized polarization spectra indicates the presence of a single, ordered (from the point of view of molecular axis rotation)-liquid (from the point of view of lateral diffusion) lipid phase in the native AChR membrane; 3) laurdan molecules within energy transfer distance of the protein sense protein-associated lipid, which differs structurally and dynamically from the bulk bilayer lipid in terms of polarity and molecular motion and is associated with a lower degree of water penetration.