Nine residues influence the binding of alpha-bungarotoxin in alpha-subunit region 185-200 of human muscle acetylcholine receptor.
Identification of residues in the skeletal muscle nicotinic acetylcholine receptor (AChR) that bind snake venom alpha-neurotoxin antagonists of acetylcholine [e.g., alpha-bungarotoxin (alpha-BTx)] provides structural information about the neurotransmitter binding region of the receptor. Using synthetic peptides of the human AChR alpha-subunit region 177-208, we previously localized a pharmacologically specific binding site for alpha-BTx in segment 185-199. To define in more detail the residues that influence the binding of alpha-BTx to this region, we prepared 16 peptide analogues of the alpha-subunit segment 185-200, with the amino acid L-alanine sequentially replacing each native amino acid. Circular dichroism spectroscopy did not reveal changes in the secondary structure of the peptides except for the analogue in which Pro194 was substituted with alanine. This implies that any change in alpha-BTx binding could be attributed to replacement of the native residue's side chain by alanine's methyl group, rather than to a change in the structure of the peptide. The influence of each substitution with alanine was determined by comparing the analogue to the parental sequence alpha 185-200 in solution-phase competition with native human AChR for binding of 125I-labeled alpha-BTx. The binding of alpha-BTx by analogue peptides with alanine substituted for Tyr190, Cys192, or Cys193 was greatly diminished. Binding of alpha-BTx to peptides containing alanine replacements at Val188, Thr189, Pro194, Asp195, or Tyr198 was also reduced significantly (p < 0.003). An unanticipated finding was that substitution of alanine for Ser191 significantly increased alpha-BTx binding (p < 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)
McCormick
D J
DJ
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
Liebenow
J A
JA
Griesmann
G E
GE
Lennon
V A
VA
D000595:Amino Acid Sequence; D002038:Bungarotoxins / Q000378:metabolism*; D002942:Circular Dichroism; D004350:Drug Residues / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000378:metabolism*
D002038:Bungarotoxins; D010455:Peptides; D011950:Receptors, Cholinergic