Hone_2012_Mol.Pharmacol_82_972

Nicotinic acetylcholine receptors (nAChRs) containing alpha6 and beta2 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 beta2 and beta4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind alpha6beta2 nAChRs also potently bind the closely related alpha6beta4 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 alpha6/alpha3beta2beta3 and alpha6/alpha3beta4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for alpha6/alpha3beta2beta3 nAChRs expressed in Xenopus oocytes (alpha6/alpha3 is a subunit chimera that contains the N-terminal ligand-binding domain of the alpha6 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 alpha6/alpha3beta2beta3 and alpha6/alpha3beta4 nAChRs with IC(50) values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to alpha6/alpha3beta2beta3 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 alpha6beta2 and alpha6beta4 nAChRs.