Structure-Activity Relationships of Alanine Scan Mutants O-Conotoxins GeXIVA[1,2] and GeXIVA[1,4]

& alpha;O-Conotoxin GeXIVA is a selective & alpha;9 & alpha;10nicotinicacetylcholine receptor (nAChR) inhibitor displaying two disulfidebonds that can form three isomers. The bead (GeXIVA[1,2]) and ribbon(GeXIVA[1,4]) isomers possess the highest activity on rat and human & alpha;9 & alpha;10 nAChRs. However, the molecular mechanism by whichthey inhibit the & alpha;9 & alpha;10 nAChR is unknown. Here, an alaninescan of GeXIVA was used to elucidate key interactions between thepeptides and the & alpha;9 & alpha;10 nAChR. The majority of GeXIVA[1,2]analogues preserved affinity at & alpha;9 & alpha;10 nAChR, but [R17A]GeXIVA[1,2]enhanced selectivity on the & alpha;9 & alpha;10 nAChR. The I23A replacementof GeXIVA[1,4] increased activity at both rat and human & alpha;9 & alpha;10nAChRs by 10-fold. Surprisingly, these results do not support themolecular model of an interaction in the orthosteric binding siteproposed previously, but rather may involve allosteric coupling withthe voltage-sensitive domain of the & alpha;9 & alpha;10 nAChR. Theseresults could help to guide further development of GeXIVA analoguesas analgesics.