Francisco J. Barrantes

Cholesterol Modulates the Organization of the γM4 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 Förster 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.

Fluorescence and molecular dynamics studies of the acetylcholine receptor γM4 transmembrane peptide in reconstituted systems

A combination of fluorescence spectroscopy and molecular dynamics (MD) is applied to assess the conformational dynamics of a peptide making up the outermost ring of the nicotinic acetylcholine receptor (AChR) transmembrane region and the effect of membrane thickness and cholesterol on the hydrophobic matching of this peptide. The fluorescence studies exploit the intrinsic fluorescence of the only tryptophan residue in a synthetic peptide corresponding to the fourth transmembrane domain of the AChR γ subunit (γM4-Trp6) reconstituted in lipid bilayers of varying thickness, and combine this information with quenching studies using depth-sensitive phosphatidylcholine spin-labeled probes and acrylamide, polarization of fluorescence, and generalized polarization of Laurdan. A direct correlation was found between bilayer width and the depth of insertion of Trp6. We further extend our recent MD study of the conformational dynamics of the AChR channel to focus on the crosstalk between M4 and the lipid-belt region. The isolated γM4 peptide is shown to possess considerable orientational flexibility while maintaining a linear α-helical structure, and to vary its tilt depending on bilayer width and cholesterol (Chol) content. MD studies also show that γM4 also establishes contacts with the other TM peptides on its inner face, stabilizing a shorter TM length that is still highly sensitive to the lipid environment. In the native membrane the topology of the M4 ring is likely to exhibit a similar behavior, dynamically modifying its tilt to match the hydrophobic thickness of the bilayer.

Steroids differentially inhibit the nicotinic acetylcholine receptor

The effect of various natural and synthetic steroids on the function of the nicotinic acetylcholine receptor (AChR) was studied at the single-channel level. AChR channel kinetics was affected by some substitutions in the cyclopentaneperhydrophenantrene ring. Functionally relevant substitutions shortened channel open state duration, an effect that varied for different steroids. The presence of a polar group at C11 contributed to the inhibitory potency of the steroid. Among mono-hydroxy- lated steroids such as 11- and 17-OH progesterone, the highest potency was displayed by the former showing a level similar to that of the reference compound, hydrocortisone. When the effects were analyzed in terms of the octanol–water partition coefficient, a linear relationship was unexpectedly found between the hydrophilicity of the steroids and their inhibitory potency.

Sphingomyelin composition and physical asymmetries in native acetylcholine receptor-rich membranes

Abstract. Selective enzymatic hydrolysis, lipid compositional analyses, and fluorescence studies have been carried out on acetylcholine receptor (AChR)-rich membranes from Torpedinidae to investigate the topology of sphingomyelin (SM) in the native membrane and its relationship with the AChR protein. Controlled sphingomyelinase hydrolysis of native membranes showed that SM is predominantly (~60%) localized in the outer half of the lipid bilayer. Differences were also observed in the distribution of SM fatty acid molecular species in the two bilayer leaflets. A fluorescent SM derivative {N-[10-(1-pyrenyl)decanoyl]sphingomyelin; Py-SM} was used to study protein-lipid interactions in the AChR-rich membrane and in affinity-purified Torpedo AChR reconstituted in liposomes made from Torpedo electrocyte lipid extracts. The efficiency of Förster resonance energy transfer (FRET) from the protein to the pyrenyl-labeled lipid as a function of acceptor surface density was used to estimate distances and topography of the SM derivative relative to the protein. The dynamics of the lipid acyl chains were explored by measuring the thermal dependence of Py-SM excimer formation, sensitive to the fluidity of the membrane. Differences were observed in the concentration dependence of excimer/monomer pyrenyl fluorescence when measured by direct excitation of the probe as against under FRET conditions, indicating differences in the intermolecular collisional frequency of the fluorophores between bulk and protein-vicinal lipid environments, respectively. Py-SM exhibited a moderate selectivity for the protein-vicinal lipid domain, with a calculated relative affinity Kr≈0.55. Upon sphingomyelinase digestion of the membrane, FRET efficiency increased by about 50%, indicating that the resulting pyrenyl-ceramide species have higher affinity for the protein than the parental SM derivative.

Fluorescence studies of the nicotinic acetylcholine receptor in its membrane environment.

Since the discovery of the fraction of immobilized lipid in contact with the nicotinic acetylcholine receptor (AChR), the lipid-belt region around this protein has become the focus of a variety of biophysical studies aimed at defining its properties. Here we summarize recent spectroscopic studies from our laboratory using Laurdan fluorescence to characterize distinct sites for lipids and to describe their effect on the AChR microenvironment.