Biological membranes show lateral and transverse asymmetric lipid distribution. Cholesterol (Chol) localizes in both hemilayers, but in the external one it is mostly condensed in lipid-ordered microdomains (raft domains), together with saturated phosphatidyl lipids and sphingolipids (including sphingomyelin and glycosphingolipids). Membrane asymmetries induce special membrane biophysical properties and behave as signals for several physiological and/or pathological processes. Alzheimer’s disease (AD) is associated with a perturbation in different membrane properties. Amyloid-β (Aβ) plaques and neurofibrillary tangles of tau protein together with neuroinflammation and neurodegeneration are the most characteristic cellular changes observed in this disease. The extracellular presence of Aβ peptides forming senile plaques, together with soluble oligomeric species of Aβ, are considered the major cause of the synaptic dysfunction of AD. The association between Aβ peptide and membrane lipids has been extensively studied. It has been postulated that Chol content and Chol distribution condition Aβ production and posterior accumulation in membranes and, hence, cell dysfunction. Several lines of evidence suggest that Aβ partitions in the cell membrane accumulate mostly in raft domains, the site where the cleavage of the precursor AβPP by β- and γ- secretase is also thought to occur. The main consequence of the pathogenesis of AD is the disruption of the cholinergic pathways in the cerebral cortex and in the basal forebrain. In parallel, the nicotinic acetylcholine receptor has been extensively linked to membrane properties. Since its transmembrane domain exhibits extensive contacts with the surrounding lipids, the acetylcholine receptor function is conditioned by its lipid microenvironment. The nicotinic acetylcholine receptor is present in high-density clusters in the cell membrane where it localizes mainly in lipid-ordered domains. Perturbations of sphingomyelin or cholesterol composition alter acetylcholine receptor location. Therefore, Aβ processing, Aβ partitioning, and acetylcholine receptor location and function can be manipulated by changes in membrane lipid biophysics. Understanding these mechanisms should provide insights into new therapeutic strategies for prevention and/or treatment of AD. Here, we discuss the implications of lipid-protein interactions at the cell membrane level in AD.

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.

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups (ϳ70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4°C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the spermcapacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.In a number of mammals including humans a series of very long-chain polyunsaturated fatty acids (VLCPUFA), 2 i.e. n-6 and n-3 tetraenoic, pentaenoic and hexaenoic fatty acids with up to 32 or 34 carbon atoms, depending on the species, was characterized in the sphingomyelin (SM) from testis and spermatozoa (1, 2). In the testis of various mammals, we focused on the fatty acids of the ceramide (Cer), a lipid molecule with which SM bears a close precursor-product relationship, showing that SM and Cer species containing VLCPUFA are a specific feature of cells of the spermatogenic lineage (3). Because these testicular cells are predecessors of spermatozoa, the question arose as to the quantitative importance of these molecules in spermatozoa, where they could play a role in sperm functions related to fertilization. Transit through the epididymis is a crucial phase in sperm maturation. Spermatozoa exiting the testis are immotile, unable to bind to eggs and to undergo the acrosomal reaction in vitro in response to commonly used stimuli. By the time they reach the region of cauda epididymis, sperm cells have acquired their progressive motility and their ability to bind, penetrate, and fertilize eggs (4). One of the questions we addressed was whether epididymal maturation gives rise to spermatozoa with a larger or a smaller proportion of SM and Cer containing these VLCPUFA as opposed to other fatty acids.Spermatozoa are functionally regionalized cells. Sperm-oocyte interactions are head-relat...

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.

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 ␣M1, ␣M4, ␥M1, and ␥M4. 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 ␣-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.The muscle and electric organ nicotinic acetylcholine receptor (AChR) 1 is a pentameric integral transmembrane protein of homologous ␣ 2 ␤␥␦ subunits. The AChR belongs to a superfamily of ligand-gated ion channels, together with the glycine receptor, a subtype of the serotonin receptor (5-HT 3 ), and the GABA A receptor (1-4). Each AChR subunit contains a relatively large amino-terminal extracellular domain of ϳ200 amino acids followed by four hydrophobic domains of 20 -30 amino acids in length (M1-M4) connected by hydrophilic loops of varying length and ending with a very short extracellular carboxyl terminus (reviewed in Ref. 5).Although the exact topology of the AChR relative to the membrane has not yet been determined unambiguously, it is usually accepted that the four hydrophobic segments M1-M4 correspond to transmembrane (TM) domains (6 -7). There is still contradictory evidence on their secondary structure. The original postulation of a four-helix bundle with an all-helical secondary structure (8) has been challenged by the results of cryoelectron microscopy of frozen AChR tubules (9 -10) and computer-aided molecular modeling indicating that the dimensions of the AChR TM region are not compatible with a pentameric four-helix bundle (11). Site-directed mutagenesis data combined with patch clamp electrophysiology, and results from photoaffinity labeling with noncompetitive channel blockers, support the notion that the M2 domain lines the walls of the ion channel proper and are indicative of ␣-helical periodicity in the residues exposed ...

Amyloid β peptide (Aβ) is a key player in the development of Alzheimer’s disease (AD). It is the primary component of senile plaques in AD patients and is also found in soluble forms. Cholinergic activity mediated by α7 nicotinic receptors has been shown to be affected by Aβ soluble forms. To shed light into the molecular mechanism of this effect, we explored the direct actions of oligomeric Aβ1–40 and Aβ1–42 on human α7 by fluorescence spectroscopy and single-channel recordings. Fluorescence measurements using the conformational sensitive probe crystal violet (CrV) revealed that in the presence of Aβ α7 undergoes concentration-dependent conformational changes. Exposure of α7 to 100 pM Aβ changes CrV KD towards that of the desensitized state. However, α7 is still reactive to high carbamylcholine (Carb) concentrations. These observations are compatible with the induction of active/desensitized states as well as of a novel conformational state in the presence of both Aβ and Carb. At 100 nM Aβ, α7 adopts a resting-state-like structure which does not respond to Carb, suggesting stabilization of α7 in a blocked state. In real time, we found that Aβ is capable of eliciting α7 channel activity either in the absence or presence of the positive allosteric modulator (PAM) PNU-120596. Activation by Aβ is favored at picomolar or low nanomolar concentrations and is not detected at micromolar concentrations. At high Aβ concentrations, the mean duration of activation episodes elicited by ACh in the presence of PNU-120596 is significantly reduced, an effect compatible with slow open-channel block. We conclude that Aβ directly affects α7 function by acting as an agonist and a negative modulator. Whereas the capability of low concentrations of Aβ to activate α7 could be beneficial, the reduced α7 activity in the presence of higher Aβ concentrations or its long exposure may contribute to the cholinergic signaling deficit and may be involved in the initiation and development of AD.

Free fatty acids (FFA) are essential components of the cell, where they play a key role in lipid and carbohydrate metabolism, and most particularly in cell membranes, where they are central actors in shaping the physicochemical properties of the lipid bilayer and the cellular adaptation to the environment. FFA are continuously being produced and degraded, and a feedback regulatory function has been attributed to their turnover. The massive increase observed under some pathological conditions, especially in brain, has been interpreted as a protective mechanism possibly operative on ion channels, which in some cases is of stimulatory nature and in other cases inhibitory. Here we discuss the correlation between the structure of FFA and their ability to modulate protein function, evaluating the influence of saturation/unsaturation, number of double bonds, and cis vs. trans isomerism. We further focus on the mechanisms of FFA modulation operating on voltage-gated and ligand-gated ion channel function, contrasting the still conflicting evidence on direct vs. indirect mechanisms of action.

We investigated the effect of the physical state of the cell membrane on the activity of the nicotinic acetylcholine receptor (AChR) in various clonal cell lines transfected with the cDNAs of embryonic or adult AChR by measuring single-channel properties and some membrane physicochemical properties as a function of temperature. Unitary conductance and channel closing rate, alpha, had Q(10) values of 1.2 and 2.2, respectively. Using Eyring's transition state theory, it was calculated that both embryonic and adult-type AChR had relatively low thermal sensitivity of ionic conductance and activation energy (E(a) of 3.0-5.0 kcal-mol(-1) at 20 degrees C), indicating that once the AChR channel opens, ion movement is dominated by diffusional processes. Channel closure exhibited higher energy requirements, with E(a) values of about 13 kcal-mol(-1). This process appears to be more endothermic (higher delta H(a) values) than ion permeation, and it is plausible that the energy acquired by the system can be used in the maintenance of its degree of order, as revealed by the delta S(a) 0 calculated for channel closure. The influence of the membrane environment on AChR function is reinforced by the observation that the conductance of the same, embryonic-type AChR protein, expressed in qualitatively different cellular lipid environments, appeared to have different energetic requirements. A correlation between the electrophysiological and thermodynamic parameters of the AChR and physicochemical properties of the membrane bilayer in which the protein is embedded could be established using measurements of the so-called generalized polarization (GP) of the lipophilic probe laurdan. Both embryonic and adult AChR exhibited a higher GP and a higher sensitivity to temperature-dependent changes in GP when heterologously expressed in stable form in Chinese hamster ovary (CHO)-derived cells than did the native embryonic AChR in BC3H-1 cells, indicating that these two properties are determined by the host membrane and are not inherent properties of the AChR type. In addition, the differences in the macroscopic physical states of the lipids and membrane-associated solvent (water) dipolar relaxation between BC3H-1 and CHO-derived cells indicated by the spectroscopic properties of laurdan suggest that both lipid and associated water may influence the microscopic activity of individual AChR molecules embedded in the lipid bilayer. Finally, the different dependence of AChR channel conductance and mean open time as a function of GP observed between the different AChR subtypes in clonal cell lines suggests the importance of specific lipid-protein interactions in addition to bulk membrane properties.