The 9-Arginine Residue of α-Conotoxin GI Is Responsible for Its Selective High Affinity for the αγ Agonist Site on the Electric Organ Acetylcholine Receptor

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Abstract
The two agonist-binding domains of the electric organ nicotinic acetylcholine receptor are located at the αγ and αδ subunit interfaces. α-Conotoxins GI and MI are competitive antagonists of this receptor and, like d-tubocurarine, bind to the αγ site with much higher affinity than to the αδ site. In the present study, α-conotoxin SIA also displayed strong affinity for the αγ site but no measurable affinity for the αδ site, thus showing even greater site-selectivity. In contrast, α-conotoxin SI does not distinguish between the two agonist sites, although its sequence differs from that of GI at only three positions: GI, ECCNPACGRHYSC; SI, ICCNPACGPKYSC. Analogues of SI and GI modified at these three positions were studied to identify the determinants of GI's αγ selectivity. Substituting arginine for proline at position 9 produced peptides which displayed “GI-like” selectivity for the αγ site. Conversely, substituting proline for arginine at position 9 resulted in “SI-like” nonselective inhibitors. An SI analogue having alanine in place of proline 9 did not distinguish between the two agonist sites and displayed about the same affinity as SI, indicating the importance of the arginyl cation. Interchanging the residues at position 1 or at position 10 influenced the affinity for the receptor but did not measurably change peptide selectivity. Therefore, of the three sequence differences in SI and GI, the variation at position 9, proline and arginine, respectively, is sufficient to account for GI's selective high-affinity binding to the αγ site on the electric organ acetylcholine receptor.