?O-Conotoxins inhibit NaV channels by interfering with their voltage sensors in domain-2

The ?O-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (NaV1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which ?O-conotoxins affect NaV channels. Rat NaV1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily ?-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the NaV channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion ?-toxins. In fact, MrVIA functionally competed with the scorpion ?-toxin Ts1 from Tityus serrulatus, while it did not show competition with ?-GIIIA. Ts1 and ?-GIIIA did not compete either. Thus, similar to scorpion ?-toxins, ?O-conotoxins are voltage-sensor toxins targeting receptor site-4 on NaV channels. They ?block? Na+ flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.