Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues.
A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.
D000109:Acetylcholine / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D003553:Cystine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D005033:Ethylmaleimide / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo
D000345:Affinity Labels; D004220:Disulfides; D000644:Quaternary Ammonium Compounds; D011950:Receptors, Cholinergic; C008313:4-(N-maleimido)benzyltrimethylammonium; D003553:Cystine; D000109:Acetylcholine; D005033:Ethylmaleimide; D003488:Cyanogen Bromide