α-Conotoxin Residues that Interact at Close Range with γ-Tyrosine-111 and Mutant δ-Tyrosine-113 on the Torpedo Nicotinic Acetylcholine Receptor

The alpha-conotoxins MI and GI display stronger affinities for the alphagamma agonist site on the Torpedo californica electrocyte nicotinic acetylcholine receptor (ACHR) than for the alphadelta agonist site, while alpha-conotoxin SI binds with the same affinity to both sites. Prior studies reported that the arginine at position 9 on GI and the tyrosine at position 111 on the receptor gamma subunit were responsible for the stronger alphagamma affinities of GI and MI, respectively. This study was undertaken to determine if the alpha-conotoxin midchain cationic residues interact with Torpedo gammaY111. The findings show that lysine 10 on MI is responsible for the alphagamma selectivity of MI and confirm the previously reported importance of R9 on GI and on the SI analogue, SIP9R. The results also show that gammaY111 contributes substantially to the selective alphagamma high affinity of all three peptides. Double-mutant cycle analyses reveal that, in the alphagamma site, K10 on MI and R9 on SIP9R interact with the aromatic ring of gammaY111 to stabilize the high-affinity complex, while in contrast, R9 on GI does not. The substitution of Y for R at position 113 on the delta subunit converts the alphadelta site into a high-affinity site for MI, GI, and SIP9R through the interacting of deltaY113 with K10 on MI and with R9 on both GI and SIP9R. The overall data show that the residues in the two sites with which MI interacts, other than at gamma111/delta113, are either the same or similar enough to exert equivalent effects on MI, indicating that MI binds in the same orientation at the alphagamma and alphadelta sites. Similar findings show that SIP9R probably also binds in the same orientation at the wild-type alphagamma and alphadelta sites. The finding that R9 on GI interacts closely with deltaR113Y but not with gammaY111 means that GI binds in different orientations at the alphagamma and alphadelta sites. This report also discusses the molecular basis of the difference in the MI high-affinity sites on Torpedo and embryonic mouse muscle ACHRs.