Molecular determinants by which a long chain toxin from snake venom interacts with the neuronal alpha 7-nicotinic acetylcholine receptor

Long chain curarimimetic toxins from snake venom bind with high affinities to both muscular type nicotinic acetylcholine receptors (AChRs) (K-d in the pM range) and neuronal alpha 7-AChRs (K-d in the nM range). To understand the molecular basis of this dual function, we submitted alpha-cobratoxin (a lpha-Cbtx), a typical long chain curarimimetic toxin, to an extensive mutat ional analysis. By exploring 36 toxin mutants, we found that Trp-25, Asp-27 , Phe-29, Arg-33, Arg-36, and Phe-65 are involved in binding to both neuron al and Torpedo (Antil, S., Servent, D., and Menez, A. (1999) J. Biol. Chem. 274, 34851-34858) AChRs and that some of them (Trp-25, Asp-27, and Arg-33) have similar binding energy contributions for the two receptors. In contra st, Ala-28, Lys-35, and Cys-26-Cys-30 selectively bind to the alpha 7-AChR, whereas Lys-23 and Lys-49 bind solely to the Torpedo AChR. Therefore, alph a-Cbtx binds to two AChR subtypes using both common and specific residues. Double mutant cycle analyses suggested that Arg-33 in alpha-Cbtx is close t o Tyr-187 and Pro-193 in the alpha 7 receptor. Since Arg-33 of another cura rimimetic toxin is close to the homologous alpha Tyr-190 of the muscular re ceptor (Ackermann, E. J., Ang, E. T. H., Kanter, J. R., Tsigelny, I., and T aylor, P. (1998) J. Biol Chem. 273, 10958-10964), toxin binding probably oc curs in homologous regions of neuronal and muscular AChRs. However, no coup ling was seen between alpha-Cbtx Arg-33 and alpha 7 receptor Trp-54, Leu-11 8, and Asp-163, in contrast to what was observed in a homologous situation involving another toxin and a muscular receptor (Osaka, H., Malany, S., Mol les, B. E., Sine, S. M., and Taylor, P. (2000) J. Biol Chem. 275, 5478-5484 ). Therefore, although occurring in homologous regions, the detailed modes of toxin binding to alpha 7 and muscular receptors are likely to be differe nt, These data offer a molecular basis for the design of toxins with predet ermined specificities for various members of the AChR family.