Changes in conformation upon agonist binding, and nonequivalent labeling, of the membrane-spanning regions of the nicotinic acetylcholine receptor subunits.
All four subunits of the acetylcholine receptor (AChR) are labeled by the lipid-soluble photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) with different stoichiometries and levels of saturable modification sites, dependent on the conformational state of the AChR. This probe is specific for hydrophobic targets such as the membrane-spanning regions of intrinsic proteins. In the resting state, the gamma subunit is labeled 4.5 times greater and the beta and delta subunits 1.65-1.69 greater than the alpha subunit. Carbamylcholine-induced desensitization of the AChR lowers the level and alters the stoichiometry of [125I]TID incorporation into each subunit. This effect is shown to be specific in two ways. First, it is eliminated by prior equilibration with excess alpha-bungarotoxin, which does not change the [125I]TID-labeling pattern of the AChR from that of the resting state. Second, bacteriorhodopsin is labeled by [125I]TID to the same extent both in the presence and absence of carbamylcholine. The noncompetitive blocker phencyclidine does not alter [125I]TID labeling of the AChR relative to the resting state. The 43-kDa protein, which is believed to cross-link the AChR to the cytoskeleton at the synapse, is not modified by [125I]TID, in agreement with earlier conclusions that the 43-kDa protein is not an intrinsic membrane protein.
McCarthy
M P
MP
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Stroud
R M
RM
D000818:Animals; D001389:Azirines / Q000378:metabolism; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D010622:Phencyclidine / Q000494:pharmacology; D010777:Photochemistry; D011485:Protein Binding; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D001389:Azirines; D002038:Bungarotoxins; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D002217:Carbachol; D010622:Phencyclidine