Alpha-bungarotoxin binds to human acetylcholine receptor alpha-subunit peptide 185-199 in solution and solid phase but not to peptides 125-147 and 389-409.
The nicotinic acetylcholine receptor (AChR) of human skeletal muscle has a reducible disulfide bond near the neurotransmitter binding site in each of its alpha-subunits. By testing a panel of overlapping synthetic peptides encompassing the alpha-subunit segment 177-208 (containing cysteines 192 and 193) we found that specific binding of 125I-labelled alpha-bungarotoxin (alpha-BTx) was maximal in the region 185-199. Binding was inhibited by unlabelled alpha-BTx greater than d-tubocurarine greater than atropine greater than carbamylcholine. Peptide 193-208 did not bind alpha-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 alpha-BTx. No peptide bound 125I-alpha-labelled parathyroid hormone. The apparent affinity (KD) of alpha-BTx binding to immobilized peptides 181-199 and 185-199 was approximately 25 microM and 80 microM, respectively, in comparison with alpha-BTx binding to native Torpedo ACh receptor (apparent KD approximately 0.5 nM). In solution phase, both peptides effectively competed with solubilized native human AChR for binding of alpha-BTx, and peptide 185-199 showed little evidence of dissociation after 24 h. Peptides that bound alpha-BTx did so when sulfhydryls were reduced. Cysteine modification, by N-ethylmaleimide or acetamidomethylation, abolished alpha-BTx-binding activity. The data implicate the region of cysteines 192 and 193 in the binding of neurotransmitter to the human receptor.
Griesmann
G E
GE
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
McCormick
D J
DJ
De Aizpurua
H J
HJ
Lennon
V A
VA
D002038:Bungarotoxins / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012996:Solutions
D002038:Bungarotoxins; D010446:Peptide Fragments; D010455:Peptides; D011950:Receptors, Cholinergic; D012996:Solutions