Subunit interface selectivity of the alpha-neurotoxins for the nicotinic acetylcholine receptor

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate resid ues located in the two binding sites and for determining probable orientati ons of the bound peptide. We report here on a short alpha-neurotoxin from N aja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alpha gamma and alpha delta subunit interfaces (K-D similar to 100 pM) but binds with markedly reduced affinity to the alp ha epsilon interface (K-D similar to 100 nM). By constructing chimeras comp osed of portions of the gamma and epsilon subunits and coexpressing them wi th wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity . Within this region, gamma Pro-175 and gamma Glu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gamma Glu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin a nd measured binding of mutant toxin-receptor combinations. The data show st rong pairwise interactions or coupling between gamma Glu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop I I of this three-loop toxin, Thus, loop II of NmmI, and in particular the fa ce of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.