ACKNOWLEDGEMENTSThis work was supported by the Deutsche Forschungsgemeinschaft (HE 2993/5-2 to Stefan H. Heinemann), FONACIT CNPq (PI 20040000385 to Adolfo Borges), and NIH (GM48677 to Baldomero M. Olivera). We thank Dr. J. Trimmer for providing the clone for rNa V 1.4 and the MzB SW seminar group for helpful discussions. ABSTRACTThe µO-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 (Na V 1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which µO-conotoxins affect Na V channels. Rat Na V 1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily µ-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the Na V 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 b-toxins. In fact, MrVIA functionally competed with the scorpion b-toxin Ts1 from Tityus serrulatus, while it did not show competition with µ-GIIIA. Ts1 and µ-GIIIA did not compete either. Thus, similar to scorpion b-toxins, µO-conotoxins are voltage-sensor toxins targeting receptor site-4 on Na V channels. They "block" Na + flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.