Background: Ligands that selectively target ␣6␤2* nAChRs are needed. Results: An analog of ␣-conotoxin PeIA was synthesized that was Ͼ15,000-fold more potent at inhibiting ␣6/␣3␤2␤3 receptors than the closely related ␣3␤2 subtype.

Nicotinic acetylcholine receptors (nAChRs) containing ␣6 and ␤2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinson's disease and nicotine dependence. However, the expression profile of ␤2 and ␤4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind ␣6␤2 nAChRs also potently bind the closely related ␣6␤4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described ␣-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including ␣6/ ␣3␤2␤3 and ␣6/␣3␤4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for ␣6/␣3␤2␤3 nAChRs expressed in Xenopus oocytes (␣6/␣3 is a subunit chimera that contains the N-terminal ligand-binding domain of the ␣6 subunit). On the basis of these results, second-generation analogs were then synthesized.