Zhangsun_2015_J.Biol.Chem_290_9855

alpha-Conotoxin LvIA (alpha-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of alpha3beta2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the alpha3beta2 nAChR subtype from the alpha6beta2* (* indicates the other subunit) and alpha3beta4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the beta2 subunit versus those of the beta4 subunit on the binding of alpha-CTx LvIA. Although two beta2 mutations, alpha3beta2[F119Q] and alpha3beta2[T59K], strongly enhanced the affinity of LvIA, the beta2 mutation alpha3beta2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the beta2-T59L mutant was combined with the alpha3 subunit. There were no significant difference in inhibition of alpha3beta2[T59I], alpha3beta2[Q34A], and alpha3beta2[K79A] nAChRs when compared with wild-type alpha3beta2 nAChR. alpha-CTx LvIA displayed slower off-rate kinetics at alpha3beta2[F119Q] and alpha3beta2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the beta2 subunit contributes to alpha-CTx LvIA binding and selectivity. The results demonstrate that Val(111) is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework alpha-conotoxins. Thr(59) and Phe(119) of the beta2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the beta4 subunit leads to increased affinity.