α-Conotoxin PeIA[S9H,V10A,E14N] Potently and Selectively Blocks α6β2β3 versus α6β4 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) containing alpha 6 and beta 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 beta 2 and beta 4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind alpha 6 beta 2 nAChRs also potently bind the closely related alpha 6 beta 4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described alpha-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including alpha 6/alpha 3 beta 2 beta 3 and alpha 6/alpha 3 beta 4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for alpha 6/alpha 3 beta 2 beta 3 nAChRs expressed in Xenopus oocytes (alpha 6/alpha 3 is a subunit chimera that contains the N-terminal ligand-binding domain of the alpha 6 subunit). On the basis of these results, second-generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by alpha 6/alpha 3 beta 2 beta 3 and alpha 6/alpha 3 beta 4 nAChRs with IC50 values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to alpha 6/alpha 3 beta 2 beta 3 nAChRs yielded a k(off) of 0.096 +/- 0.001 min(-1) and a k on of 0.23 +/- 0.019 min(-1) M-9. The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between alpha 6 beta 2 and alpha 6 beta 4 nAChRs.