LOCALIZATION OF HISTIDINE-RESIDUES RELEVANT FOR THE BINDING OF ALPHA-BUNGAROTOXIN TO THE ACETYLCHOLINE-RECEPTOR ALPHA-SUBUNIT IN V8-PROTEOLYTIC FRAGMENTS

Histidine residues have been shown to be critical for alpha-BgTx bindi ng to the acetylcholine receptor (Lacorazza et al., 1992; Bouzat et al ., 1993; Lacorazza et al., 1995). Receptor subunits from Discopyge tsc hudii were modified with diethylpyrocarbonate (DEP). DEP treatment pro duces a concentration-dependent decrease of [I-125]alpha-BgTx binding to the alpha subunit. The neurotoxin binding capacity was fully restor ed by adding the nucleophile hydroxylamine. By proteolytic mapping of the alpha-subunit with V8-protease, we determined that the binding cap acity to the fragment alpha V8-19 decreased 80% by DEP treatment. In a ddition, the [I-125]alpha-BgTx binding to the same fragment decreased by 70% when the subunits were reduced and affinity-alkylated. We repor t the N-terminal sequence of both subunits and V8-fragments (alpha V8- 10, alpha V8-13, and alpha V8-18), which constitute a first contributi on to the knowledge of the primary structure of the Discopyge tschudii receptor. We propose that the fragment alpha V8-19 contains one or mo re of the histidine residues involved in the alpha-BgTx binding and pr obably-includes the Cys alpha 192-193 disulfide bond. Only two histidi ne residues are present in the extracellular sequence of Torpedo calif ornica for such fragments: His alpha 186 and alpha 204. Copyright (C) 1996 Elsevier Science Ltd.