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 acety]choline receptor (AChR)-ri ch membrane and compare them with those in reconstituted liposomes pre pared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 6 0 nm were observed as a function of temperature in the spectral emissi on maximum of laurdan embedded in these membranes. The so-called gener alized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by similar to 85% as temperatu re increased, but no phase transitions with a clear T-m 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 molecul es could be observed upon excitation at 290 nm. The efficiency of this process was similar to 55% for 1 mu M laurdan. A minimum donor-accept or distance r of 14 +/- 1 Angstrom could be calculated considering a d istance 0 < H < 10 Angstrom for the separation of the planes containin g donor and acceptor molecules, respectively, This value of r correspo nds closely to the diameter of the first-shell protein-associated lipi d, A value of similar to 1 was calculated for K-r, the apparent dissoc iation constant of laurdan, indicating no preferential affinity for th e 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 wate r penetration in the protein-vicinal and bulk bilayer lipid regions ca n 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 lipi ds reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic i nterface 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 t he native AChR membrane; 3) laurdan molecules within energy transfer d istance of the protein sense protein-associated lipid, which differs s tructurally and dynamically from the bulk bilayer lipid in terms of po larity and molecular motion and is associated with a lower degree of w ater penetration.