Topological Disposition of Cys 222 in the α-Subunit of Nicotinic Acetylcholine Receptor Analyzed by Fluorescence-Quenching and Electron Paramagnetic Resonance Measurements

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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 α-subunit. αCys222 is located in the postulated M1 transmembrane domain and predicted to be at the center of an α-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 αCys222 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 Å 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 β-strand, or an α-helical structure that may span the membrane in a way different from a linear α-helix.