NEW SODIUM CHANNEL-BLOCKING CONOTOXINS ALSO AFFECT CALCIUM CURRENTS IN LYMNAEA NEURONS

Two new conotoxins that affect both sodium and calcium currents have been characterized from the venom of Conus marmoreus, using direct assays on voltage-gated currents in caudodorsal neurons (CDC) of the freshwater snail Lymnaea stagnalis. The designations and amino acid sequences of the new toxins are MrVIA, ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV, and MrVIB, ACSKKWEYCIVPILGFVYCCPGLICGPFVCV. Both toxins block voltage dependent sodium currents in snail neurons with ED(50)'s of 0.1-0.2 mu M. Effects are also observed on the fast-inactivating calcium current subtype in the CDC at greater than or equal to 1 mu M. At concentrations of 1-5 mu M, MrVIA acts as a calcium current agonist whereas MrVIB acts as a blocker. At higher doses both toxins block the fast-inactivating calcium current. Almost no effects of MrVIB are seen on the second (sustained kinetics) CDC calcium current subtype, while MrVIA also slightly blocks the sustained current. The calcium current block is rapidly reversible, whereas in contrast recovery of the sodium current requires extensive wash. MrVIA/B have the same cysteine framework as the omega- and delta-conotoxins and a high content of hydrophobic residues, in common with the delta-conotoxins. There is only one localized concentration of charged residues in MrVIA/B, in the first intercysteine loop. These two conotoxins provide unique probes for structure and function studies on voltage-gated sodium and L-type calcium channels. Their unusual cross-channel activity suggests they may represent an ''intermediate'' variant of conotoxin, in the diversification of one conotoxin structural family that selectively targets either sodium or calcium channels.