title: Topological disposition of Cys 222 in the alpha-subunit of nicotinic acetylcholine receptor analyzed by fluorescence-quenching and electron paramagnetic resonance measurements.
abstract: The structure of the nicotinic acetylcholine receptor (AChR) has been studied using a combination of fluorescence quenching and electron paramagnetic resonance (EPR) collision gradient methods. The AChR from Torpedo californica was labeled with a fluorescent probe, N-(1-pyrenyl)maleimide, specific for sulfhydryls in a hydrophobic environment, under conditions of selective labeling of Cys222 in the alpha-subunit. alphaCys222 is located in the postulated M1 transmembrane domain and predicted to be at the center of an alpha-helical secondary structure. The spatial disposition of the acetylcholine receptor-bound pyrene with respect to the membrane bilayer was assessed by fluorescence quenching measurements. Quenching of pyrene fluorescence by spin-labeled fatty acids with the doxyl group at positions C-5 and C-12 revealed that the former was more effective, suggesting that the fluorophore is located closer to the membrane-water interface than to the hydrophobic interior. Power saturation EPR spectroscopy was also used to examine the effect of molecular oxygen and water-soluble paramagnetic reagents on the saturation behavior of a nitroxide spin label, which was specifically attached to the same alphaCys222 residue. Using the gradients of these paramagnetic reagents through the membrane-solution interface, the distance for the nitroxide derivative from the membrane-solution interface was measured to be approximately 7 A from the headgroup region of the phospholipid bilayer, in agreement with fluorescence quenching results. These results suggest that the M1 transmembrane domain of the AChR probably forms an irregular structure, a beta-strand, or an alpha-helical structure that may span the membrane in a way different from a linear alpha-helix.
authors.affiliation: Section of Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
authors.forename: J
authors.initials: J
authors.lastname: Kim
authors.forename: M G
authors.initials: MG
authors.lastname: McNamee
mesh_terms: D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes; D008301:Maleimides; D008566:Membranes; D008698:Mesylates / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D013113:Spin Labels; D014101:Torpedo
chemical_list: D002038:Bungarotoxins; D003497:Cyclic N-Oxides; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008301:Maleimides; D008698:Mesylates; D010713:Phosphatidylcholines; D011978:Receptors, Nicotinic; D013113:Spin Labels; C027724:iodo-alpha-bungarotoxin; D009532:Nickel; C034248:(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate; D004492:Edetic Acid; C003944:N-(3-pyrene)maleimide; D003545:Cysteine; D010100:Oxygen