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

αO-Conotoxin GeXIVA is a selective α9α10
nicotinic
acetylcholine receptor (nAChR) inhibitor displaying two disulfide
bonds that can form three isomers. The bead (GeXIVA[1,2]) and ribbon
(GeXIVA[1,4]) isomers possess the highest activity on rat and human
α9α10 nAChRs. However, the molecular mechanism by which
they inhibit the α9α10 nAChR is unknown. Here, an alanine
scan of GeXIVA was used to elucidate key interactions between the
peptides and the α9α10 nAChR. The majority of GeXIVA[1,2]
analogues preserved affinity at α9α10 nAChR, but [R17A]GeXIVA[1,2]
enhanced selectivity on the α9α10 nAChR. The I23A replacement
of GeXIVA[1,4] increased activity at both rat and human α9α10
nAChRs by 10-fold. Surprisingly, these results do not support the
molecular model of an interaction in the orthosteric binding site
proposed previously, but rather may involve allosteric coupling with
the voltage-sensitive domain of the α9α10 nAChR. These
results could help to guide further development of GeXIVA analogues
as analgesics.