Nine Residues Influence the Binding of α‐Bungarotoxin in α‐Subunit Region 185–200 of Human Muscle Acetylcholine Receptor

Abstract

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)

Mimotopes of the Nicotinic Receptor Binding Site Selected by a Combinatorial Peptide Library

“…The fact that peptide mimotopes can be more efficient binders than the corresponding native sequence is not too surprising considering that amino acid substitutions can result in a better mimicry of the conformational structure of a binding site, which could not be reproduced by the native sequence when this is cut "out" of the protein threedimensional structure. Some data indicating that modified peptides reproducing the nicotinic receptor binding site sequence could result in higher R-bgt binding have already been reported in the case of the human muscle R 185-200 sequence, where substitution of S191 with A produced a peptide which was more efficient than the corresponding native sequence in inhibiting R-bgt binding to the receptor in solution (40).…”

“…Biochemistry, Vol. 40 thus increasing the possibility to select mimotopes that could better reproduce the receptor recognition surface. In this view, we also chose to concentrate most of the totally variant positions between residues 193 and 199.…”

“…This choice was dictated by the necessity to avoid postsynthetic oxidation. The importance of the disulfide bridge between C192 and C193 in R-bgt binding synthetic peptides is debated since contrasting results have been described (19,(37)(38)(39)(40). Nonetheless, results obtained with peptides selected from a random phage library (27) indicate that peptides with two serines in positions 192 and 193 can efficiently bind R-bgt.…”

Mecamylamine but not the α7 receptor antagonist α‐bungarotoxin blocks sensitization to the locomotor stimulant effects of nicotine

“…Previous studies have also noted seizures following a-bgt administration to rodents. The seizures are thought to be mediated at the level of the CA3 ®eld of the hippocampus where a-bgt sites are located on GABA-containing interneurones (Miner & Collins, 1989;Marks et al, 1989;McCormick et al, 1993;Freedman et al, 1993).…”

Interaction of protein ligands with receptor fragments

“…More precise defimtion of the curaremimetic toxin bmdmg site has been attempted wtth fragments of a subunits generated as proteolytic [15-l 81, synthetic [19][20][21][22][23][24][25][26][27] or fusion peptides obtained by genetic means It is accepted that the region around the cysteme 192-193 is recognized by most AcChoR hgands. Systematic replacement of the residues by alanme suggest a special contribution of residues Tyr-190, Cys-192, Cys-193, Val-188, Thr-189, to formatton of toxin-peptide complexes [31]. Other a subunit fragments may also be recognized by the toxms [ 19,20,; however, these data require further confirmation since they have sometimes been questionned Among the other possible toxm bmdmg fragments 1s the loop between the disulfide 128-142 which 1s conserved m the family of hgand-gated channels, and has been predicted as interacting with chohnergtc hgands [35] and controversially proposed as a target for snake toxms [19,20,33,34,36,37] Curaremimettc toxms are small proteins from Elapidae and Hydrophndae venoms that bmd to AcChoR wrth htgh affinity (& = lo-" M) They are classified as long-chain or short-cham toxms when they respectively possess between 66 and 74 residues and five disulfides or between 60 and 62 residues and four drsulfides [38].…”