PROTEIN-LIPID INTERACTIONS AND TORPEDO-CALIFORNICA NICOTINIC ACETYLCHOLINE-RECEPTOR FUNCTION .1. SPATIAL DISPOSITION OF CYSTEINE RESIDUES IN THE GAMMA-SUBUNIT ANALYZED BY FLUORESCENCE-QUENCHING AND ENERGY-TRANSFER MEASUREMENTS

The nicotinic acetylcholine receptor from Torpedo californica was labe led with a fluorescent, lipophilic probe, N-(1-pyrenyl)maleimide, spec ific for sulfhydryls in a hydrophobic environment, and was found to al kylate Cys 416, Cys 420 and Cys 451 in the gamma subunit [Li, L., Schu chard, M., Palma, A., Pradier, L., & McNamee, M. G. (1990) Biochemistr y 29, 5428-5436]. The spatial disposition of the acetylcholine recepto r-bound pyrene with respect to the membrane bilayer was assessed by a combination of fluorescence-quenching and resonance energy transfer me asurements, under conditions of selective labeling of the gamma subuni t. Quenching of pyrene fluorescence by spin-labeled fatty acids with t he doxyl group at positions C-5 and C-12 revealed that the former was more effective, with a Stern-Volmer quenching constant of 0.187 compar ed to 0.072 for the latter, suggesting that the fluorophore(s) are loc ated closer to the membrane-water interface rather than the hydrophobi c interior. Energy transfer was found to occur from tryptophan in the acetylcholine receptor to cysteine-bound pyrene with a distance of sep aration of approximately 18 angstrom. However, there was no energy tra nsfer when pyrene-labeled AChR was reconstituted into membranes contai ning brominated phospholipids and cholesterol, suggesting that the flu orophore(s) responsible for energy transfer are located in the membran e domain. Thus, the N-(1-pyrenyl)maleimide can be used to monitor lipi d-protein interactions of the AChR.