Key Structural Determinants in the Agonist Binding Loops of Human ?2 and ?4 Nicotinic Acetylcholine Receptor Subunits Contribute to ?3?4 Subtype Selectivity of ?-Conotoxins.

ABSTRACT: ?-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The ?3?4 nAChR, a predominant subtype in the peripheral nervous system, has been implicated in various pathophysiological conditions. As many ?-conotoxins have multiple pharmacological targets, compounds specifically targeting individual nAChR subtypes are needed. In this study, we performed mutational analyses to evaluate the key structural components of human ?2 and ?4 nAChR subunits that determine ?-conotoxin selectivity for ?3?4 nAChR. ?-Conotoxin RegIIA was used to evaluate the impact of non-conserved human ?2 and ?4 residues on peptide affinity. Two mutations, ?3?2[T59K] and ?3?2[S113R], strongly enhanced RegIIA affinity compared with wild-type ?3?2, as seen by substantially increased inhibitory potency and slower off-rate kinetics. Opposite point mutations in ?3?4 had the contrary effect, emphasizing the importance of loop D residue 59 and loop E residue 113 as determinants for RegIIA affinity. Molecular dynamics simulation revealed the side chains of ?4 Lys59 and ?4 Arg113 formed hydrogen bonds with RegIIA loop 2 atoms, whereas the ?2 Thr59 and ?2 Ser113 side chains were not long enough to form such interactions. Residue ?4 Arg113 has been identified for the first time as a crucial component facilitating antagonist binding. Another ?-conotoxin, AuIB, exhibited low activity at human ?3?2 and ?3?4 nAChRs. Molecular dynamics simulation indicated the key interactions with the ? subunit are different to RegIIA. Taken together, these data elucidate the interactions with specific individual ? subunit residues that critically determine affinity and pharmacological activity of ?-conotoxins RegIIA and AuIB at human nAChRs.

SUBMITTER: Cuny H 

PROVIDER: S-EPMC5095430 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature