Amino acid residues that confer high selectivity of the α6 nicotinic acetylcholine receptor subunit to α-conotoxin MII[S4A, E11A, L15A]

Nicotinic acetylcholine receptors (nAChRs) containing alpha 3 and beta 2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha 6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog of alpha-conotoxin MII, MII[ S4A, E11A, L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha 6/alpha 3 beta 2 beta 3 nAChRs with an IC50 of 1.2 nM; in contrast, its IC50 on the closely related alpha 3 beta 2 as well as non-alpha 6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha 3 subunit inserted into the homologous positions of the alpha 6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha 6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha 3 subunit increased the IC(5)0 of the peptide by> 1000-fold. Conversely, when these key residues in alpha 3 were replaced with those from alpha 6, the IC50 decreased by almost 150-fold. Similar effects were seen with other alpha 6- selective conotoxins, suggesting the general importance of these alpha 6 residues in conferring selective binding.