Key structural determinants in the agonist binding loops of human beta 2 and beta 4 nicotinic acetylcholine receptor subunits contribute toalpha 3 beta 4 subtype selectivity of alpha-conotoxins

alpha-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The 34 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 aR. -Conotoxin RegIIA was used to evaluate the impact of non-conserved hund 4 nAChR subunits that determine -conotoxin selectivity for 34 nAChman 2 and 4 residues on peptide affinity. Two mutations, 32[T59K] and 32[S113R], strongly enhanced RegIIA affinity compared with wild-type 32, as seen by substantially increased inhibitory potency and slower off-rate kinetics. Opposite point mutations in 34 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 Lys(59) and 4 Arg(113) formed hydrogen bonds with RegIIA loop 2 atoms, whereas the 2 Thr(59) and 2 Ser(113) side chains were not long enough to form such interactions. Residue 4 Arg(113) has been identified for the first time as a crucial component facilitating antagonist binding. Another -conotoxin, AuIB, exhibited low activity at human 32 and 34 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.