α‐Bungarotoxin Binds to Human Acetylcholine Receptor α‐Subunit Peptide 185–199 in Solution and Solid Phase but Not to Peptides 125–147 and 389–409

Guy E. Griesmann, Daniel J. McCormick, Henry J. De Aizpurua, Vanda A. Lennon

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

Abstract: The nicotinic acetylcholine receptor (AChR) of human skeletal muscle has a reducible disulfide bond near the neurotransmitter binding site in each of its α‐subunits. By testing a panel of overlapping synthetic peptides encompassing the α‐subunit segment 177–208 (containing cysteines 192 and 193) we found that specific binding of 125I‐labelled α‐bungarotoxin (α‐BTx) was maximal in the region 185–199. Binding was inhibited by unlabelled α‐BTx d‐tubocurarine > atropine > carbamylcholine. Peptide 193–208 did not bind α‐BTx, whereas 177–192 retained 40% binding activity. Peptides corresponding to regions 125–147 (containing cysteines 128 and 142) and 389–409, or peptides unrelated to sequences of the AChR failed to bind α‐BTx. No peptide bound 125I‐α‐labelled parathyroid hormone. The apparent affinity (KD) of α‐BTx binding to immobilized peptides 181–199 and 185–199 was ∼25 μM and 80 μM, respectively, in comparison with α‐BTx binding to native Torpedo ACh receptor (apparent KD∼0.5 nM). In solution phase, both peptides effectively competed with solubilized native human AChR for binding of α‐BTx, and peptide 185–199 showed little evidence of dissociation after 24 h. Peptides that bound α‐BTx did so when sulfhydryls were reduced. Cysteine modification, by N‐ethylmaleimide or acetamidomethylation, abolished α‐BTx‐binding activity. The data implicate the region of cysteines 192 and 193 in the binding of neurotransmitter to the human receptor.

Original languageEnglish (US)
Pages (from-to)1541-1547
Number of pages7
JournalJournal of neurochemistry
Volume54
Issue number5
DOIs
StatePublished - May 1990

Keywords

  • Nicotinic acetylcholine receptor
  • Synthetic peptides
  • α‐Bungarotoxin

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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