Identification of equivalent residues in the gamma, delta, and epsilon subunits of the nicotinic receptor that contribute to alpha-bungarotoxin binding.
Cysteine was introduced from residues 116 to 121 of the gamma subunit of the fetal mouse acetylcholine receptor, and the mutant receptors were treated with methanethiosulfonate reagents and examined for changes in ligand binding properties. Of the 18 combinations of mutant and reagent, only receptors harboring gammaL119C treated with the quaternary ammonium reagent MTSET (trimethylammonium-ethyl methanethiosulfonate) show a decreased number of alpha-bungarotoxin (alpha-btx) sites. The decrease of 50% suggests that alpha-btx binding to the site harboring gammaL119C is blocked. Analysis of binding of the site-selective ligands dimethyl-d-tubocurarine (DMT) and alpha-conotoxin M1 (CTX) confirm specificity of modification for the site harboring gammaL119C. Cysteines placed at equivalent positions of the delta and epsilon subunits also lead to selective loss of alpha-btx binding following MTSET treatment. gammaL119C receptors treated with the primary amine reagent MTSEA (aminoethyl methanethiosulfonate) retain alpha-btx binding to both sites but show reduced affinity for DMT and CTX at the modified site. Lysine mutagenesis of Leugamma119, Leudelta121, and Leuepsilon119 mimics MTSEA treatment, whereas mutagenesis of Thralpha119 and Glnbeta119 is without effect, demonstrating subunit and residue specificity of MTSEA modification. MTSET modification of nearby gammaY117C does not block alpha-btx binding but markedly diminishes affinity for DMT and CTX. The overall findings indicate a localized point of interaction between alpha-btx and the modified gammaL119C, deltaL121C, and epsilonL119C.
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Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA.
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism*; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D008239:Lysine; D003545:Cysteine