Synthetic peptides corresponding to sequences contained within residues 173-204 of the alpha-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist alpha-bungarotoxin (BGTX) with relative high affinity. Since the synthetic peptide fragments of the receptor and BGTX each contain a small number of aromatic residues, intrinsic fluorescence studies were used to investigate their interaction. We examined a number of receptor-derived peptide fragments of increasing length (4-32 amino acids). Changes in the lambda max and quantum yield with increasing polypeptide chain length suggest an increase in the hydrophobicity of the tryptophan environment. When selective excitation and subtraction were used to reveal the tyrosine fluorescence of the peptides, a significant red shift in emission was observed and was found to be due to an excited-state tyrosinate. The binding of BGTX to the receptor-derived peptide fragments resulted in a large increase in fluorescence. In addition, at equilibrium, the lambda max of tryptophan fluorescence was shifted to shorter wavelengths. The. fluorescence enhancement, which was saturable with either peptide or BGTX, was used to determine the dissociation constants for the complexes. At pH 7.4, the apparent Kd for a dodecameric peptide (alpha 185-196), consisting of residues 185-196 in the alpha-subunit of the nAChR from Torpedo californica, was 1.4 microM. The Kd for an 18-mer (alpha 181-198), consisting of residues 181-198 of the Torpedo alpha-subunit, was 0.3 microM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the critical residues in a receptor's ligand-binding site provides valuable structural information important for understanding the basis for ligand recognition. The design of specific ligands targeted for receptor action will depend to a great extent on detailed structural knowledge of this kind. Although the nicotinic acetylcholine receptor (nAChR) is perhaps the best characterized of all receptors, the detailed configuration of the ligand-binding site remains unknown. Structural comparisons of nicotinic agonists and antagonists have long predicted a negative subsite on the receptor to interact with the positively charged alkyl-ammonium moiety common to nearly all nicotinic agents. We have used intrinsic fluorescence spectroscopic analyses together with binding studies of selectively modified peptide fragments of the nAChR to suggest that one or two invariant tyrosine residues at positions 190 and 198 on the alpha-subunit provide the critical negative subsite required for ligand binding. Tyrosines may similarly be part of the negative subsite of muscarinic receptors and other neurotransmitter receptors that bind cationic ligands.

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