α-Conotoxin MII, a peptide toxin isolated from Conus magus, antagonizes a subset of neuronal nicotinic receptors. Rat α3β2 receptors, expressed in Xenopus oocytes, are blocked with an IC50 of 3.7 ± 0.3 nM. To identify structural features that determine toxin potency, a series of alanine-substituted toxins were synthesized and tested for the ability to block the function of α3β2 receptors. Circular dichroism and protein modeling were used to assess the structural integrity of the mutant toxins. Three residues were identified as major determinants of toxin potency. Replacement of asparagine 5, proline 6, or histidine 12 with alanine resulted in >2700-fold, 700-fold, and ∼2700-fold losses in toxin potency, respectively. A decrease in pH improved toxin potency, while an increase in pH eliminated toxin blockade, suggesting that, in the active form of the toxin, histidine 12 is charged. The imidazole ring of histidine 12 protrudes from one side, while asparagine 5 and proline 6 are located at the opposite end of the toxin structure. The side chains of these three residues are exposed on the surface of the toxin, suggesting that they directly interact with the α3β2 receptor.
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