kappa-Conotoxin PVIIA from the venom of Conus purpurascens is the first cone snail toxin that was described to block potassium channels. We synthesized chemically this toxin and showed that its disulfide bridge pattern is similar to those of omega- and delta-conotoxins. kappa-conotoxin competes with radioactive alpha-dendrotoxin for binding to rat brain synaptosomes, confirming its capacity to bind to potassium channels; however, it behaves as a weak competitor. The three-dimensional structure of kappa-conotoxin PVIIA, as elucidated by NMR spectroscopy and molecular modeling, comprises two large parallel loops stabilized by a triple-stranded antiparallel beta-sheet and three disulfide bridges. The overall fold of kappa-conotoxin is similar to that of calcium channel-blocking omega-conotoxins but differs from those of potassium channel-blocking toxins from sea anemones, scorpions, and snakes. Local topographies of kappa-conotoxin PVIIA that might account for its capacity to recognize Kv1-type potassium channels are discussed.

␦-Conotoxin EVIA, from Conus ermineus, is a 32-residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. ␦-Conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNa v 1.2a, rNa v 1.3, and rNa v 1.6), without affecting rat skeletal muscle (subtype rNa v 1.4) and human cardiac muscle (subtype hNa v 1.5) sodium channel (Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Mé nez, A., and Molgó , J. (2004) J. Biol. Chem. 279, 4680 -4685). Its structure was solved by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu 12 -Pro 13 peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be calculated separately. The isomerism occurs within a specific long disordered loop 2, including residues 11-19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the "inhibitor cystine-knot motif" of conotoxins from the "O superfamily" with a high structural order. To probe a possible functional role of the Leu 12 -Pro 13 cis/trans isomerism, a Pro 13 3 Ala ␦-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A ␦-conotoxin EVIA was estimated only two times less active than the wild-type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice.The new ␦-conotoxin EVIA (␦-EVIA), 1 a 32-amino acid conopeptide isolated from the venom of Conus ermineus, is the first conotoxin demonstrated to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNa v 1.2a, rNa v 1.3, and rNa v 1.6) without affecting rat skeletal muscle (subtype rNa v 1.4) and human cardiac muscle (subtype hNa v 1.5) sodium channel subtypes (1). This important recent discovery makes ␦-EVIA a unique tool to study the modulation mechanisms of neuronal Na ϩ channels. As a consequence, ␦-EVIA may also serve as a new lead molecule for the design of new drugs to treat neurological diseases characterized by defective nerve conduction, especially those causing an axonal demyelinization (2, 3). Nerve conduction could be facilitated by specific inhibition of Na ϩ channel inactivation. The knowledge of the detailed three-dimensional structure is therefore the first step necessary to understand the structureactivity relationships of this new lead conotoxin.Despite a low sequence identity with the -, -, and ␦-conotoxins, ␦-EVIA clearly belongs to the four-loop family of conotoxins characterized by a similar cysteine pairing giving a conserved 3-disulfide framework as shown in Fig. 1. Until now, the three-dimensional structure of 10 conotoxins belonging to this family was already determined as follows: the conotoxin -PVIIA (20) targeting potassium channels; conotoxins -MVIIA (21), -MVIIC (22), -MVIID (23), -GVIA (24), -SVIB (25), and -CVID (26) targeting calc...

We have isolated ␦-conotoxin EVIA (␦-EVIA) , a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described -, ␦-, ⌷-, and -conotoxins. However, it displays low sequence homology with the latter conotoxins. ␦-EVIA inhibits Na ؉ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na ؉ channels. Using amphibian myelinated axons and spinal neurons, we showed that ␦-EVIA increases the duration of action potentials by inhibiting Na ؉ channel inactivation. ␦-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of ␦-EVIA was confirmed by showing that a fluorescent derivative of ␦-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, ␦-EVIA inhibited inactivation of rat neuronal Na ؉ channel subtypes (rNa V 1.2a, rNa V 1.3, and rNa V 1.6) but did not affect rat skeletal (rNa V 1.4) and human cardiac muscle (hNa V 1.5) Na ؉ channel subtypes. ␦-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na ؉ channels without acting on Na ؉ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na ؉ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na ؉ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.

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