RESUMEN
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.
Asunto(s)
Lípidos de la Membrana/química , Receptores Nicotínicos/química , 2-Naftilamina/análogos & derivados , Animales , Fenómenos Biofísicos , Biofisica , Transferencia de Energía , Polarización de Fluorescencia , Colorantes Fluorescentes , Técnicas In Vitro , Cinética , Lauratos , Liposomas , Proteínas de la Membrana/química , TorpedoRESUMEN
The distribution of the two fluorescent phospholipid analogs across acetylcholine receptor (AChR)-rich membranes from Torpedo marmorata has been studied by a combination of nonradiative fluorescence resonance energy transfer using fluorescent lipid probes and quenching of their fluorescence with Co2+ and 2,4,6-trinitrobenzenesulfonic acid. The fluorescent lipid analogs were supplied to the AChR-rich membrane or liposome suspension by simply injecting ethanol solutions of the probes into the medium. The efficiency of the fluorescence energy transfer between NBD-labeled phosphatidylcholine and rhodamine-labeled ethanolamine glycerophospholipids was measured in model membranes prepared in such a way that the probes could be targeted at the same or opposite halves of the bilayer, and the results were compared with those obtained for native AChR-rich membranes. It is shown that NBD-PC and Rho-PE can be efficiently (95%) incorporated into AChR-rich membranes and liposomes. On the basis of the comparison with model liposomes, the energy transfer experiments suggest a preferential exofacial location of the parental phospholipids in the native AChR-rich membrane. Fluorescence quenching with Co2+ and TNBS showed these two phospholipid analogs to be located predominantly in the outer leaflet of the bilayer in AChR-rich membranes. From the Co2+ quenching of the lipid analogs, it was also possible to calculate the surface potential of the outer leaflet of the membrane as being on the order of -15 mV.