Identification of equivalent residues in the γ, δ, and ε subunits of the nicotinic receptor that contribute to α-bungarotoxin binding

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Abstract

Cysteine was introduced from residues 116 to 121 of the γ 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 γL119C treated with the quaternary ammonium reagent MTET (trimethylammonium-ethyl methanethiosulfonate) show a decreased number of α- bungarotoxin (α-btx) sites. The decrease of 50% suggests that α-btx binding to the site harboring γL119C is blocked. Analysis of binding of the site- selective ligands dimethyl-d-tubocurarine (DMT) and α-conotoxin M1 (CTX) confirm specificity of modification for the site harboring γL119C. Cysteines placed at equivalent positions of the δ and ε subunits also lead to selective loss of α-btx binding following MTSET treatment. γL119C receptors treated with the primary amine reagent MTSEA (aminoethyl methanethiosulfonate) retain α-btx binding to both sites but show reduced affinity for DMT and CTX at the modified site. Lysine mutagenesis of Leu(γ119), Leu(δ121), and Leu(ε119) mimics MTSEA treatment, whereas mutagenesis of Thr(α119) and Gin(β119) is without effect, demonstrating subunit and residue specificity of MTSEA modification. MTSET modification of nearby γY117C does not block α-btx binding but markedly diminishes affinity for DMT and CTX. The overall findings indicate a localized point of interaction between α-btx and the modified γL119C, δL121C, and L119C.

Original languageEnglish (US)
Pages (from-to)23521-23527
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number38
DOIs
StatePublished - Sep 19 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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