A δ-Conotoxin from Conus ermineus Venom Inhibits Inactivation in Vertebrate Neuronal Na+ Channels but Not in Skeletal and Cardiac Muscles

Abstract

We have isolated δ-conotoxin EVIA (δ-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 ω-, δ-, μO-, and κ-conotoxins. However, it displays low sequence homology with the latter conotoxins. δ-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 δ-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. δ-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 δ-EVIA was confirmed by showing that a fluorescent derivative of δ-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, δ-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. δ-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. We have isolated δ-conotoxin EVIA (δ-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 ω-, δ-, μO-, and κ-conotoxins. However, it displays low sequence homology with the latter conotoxins. δ-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 δ-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. δ-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 δ-EVIA was confirmed by showing that a fluorescent derivative of δ-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, δ-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. δ-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. Defective nerve conduction is frequently associated with neurological diseases, particularly with those causing axonal demyelinization (1Kaji R. Muscle Nerve. 2003; 27: 285-296Crossref PubMed Scopus (129) Google Scholar, 2Hattori N. Yamamoto M. Yoshihara T. Koike H. Nakagawa M. Yoshikawa H. Ohnishi A. Hayasaka K. Onodera O. Baba M. Yasuda H. Saito T. Nakashima K. Kira J. Kaji R. Oka N. Sobue G. Brain. 2003; 126: 134-151Crossref PubMed Scopus (182) Google Scholar). Nerve conduction may be facilitated by blocking voltage-gated K+ channels and by inhibiting inactivation of voltage-gated Na+ channels. Ligands that act on such channels are plentiful, but most of them lack selectivity, i.e. they affect channels present in various tissues, including nerve tissue and skeletal, smooth, and cardiac muscles. Voltage-dependent Na+ channels play a fundamental role in cell membrane excitability, and they are targets for a large number of animal and plant toxins (3Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1669) Google Scholar, 4Cestèle S. Catterall W.A. Biochimie (Paris). 2000; 82: 883-892Crossref PubMed Scopus (595) Google Scholar). The α-subunit of each channel is a large membrane-spanning glycoprotein containing four homologous domains associated with smaller accessory β-subunits (3Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1669) Google Scholar). Mammals possess at least 10 Na+ channel genes, each encoding a distinct channel subtype (5Goldin A.L. Annu. Rev. Physiol. 2001; 63: 871-894Crossref PubMed Scopus (580) Google Scholar), and various Na+ channel repertoires endow various types of neurons and muscles with distinct transduction and encoding properties. During recent decades, many peptides isolated from cone snail venoms have been reported to target specific subtypes of voltage-sensitive Na+ channels (6Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar, 7Fainzilber M. Lodder J.C. Kits K.S. Kofman O. Vinnitsky I. Van Rietschoten J. Zlotkin E. Gordon D. J. Biol. Chem. 1995; 270: 1123-1129Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 8Olivera B.M. Mol. Biol. Cell. 1997; 8: 2101-2109Crossref PubMed Scopus (323) Google Scholar, 9Favreau P. Le Gall F. Benoit E. Molgó J. Acta Physiol. Pharmacol. Ther. Latinoam. 1999; 49: 257-267PubMed Google Scholar, 10French R.J. Dudley Jr., S.C. Methods Enzymol. 1999; 294: 575-605Crossref PubMed Scopus (27) Google Scholar). Thus, the μ-conotoxins GIIIA and GIIIB selectively block tetrodotoxin-sensitive Na+ channels in skeletal muscles (6Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar, 10French R.J. Dudley Jr., S.C. Methods Enzymol. 1999; 294: 575-605Crossref PubMed Scopus (27) Google Scholar, 11Li R.A. Ennis I.L. French R.J. Dudley Jr., S.C. Tomaselli G.F. Marban E. J. Biol. Chem. 2001; 276: 11072-11077Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The μ-conotoxin PIIIA also differentiates Na+ channel subtypes, i.e. toxin concentrations that block rNaV1.2 (rat brain type II/IIA) and rNaV1.4 (rat skeletal muscle) channels only slightly inhibit rNaV1.7 (rat peripheral nerve PN1) channels (12Shon K.J. Olivera B.M. Watkins M. Jacobsen R.B. Gray W.R. Floresca C.Z. Cruz L.J. Hillyard D.R. Brink A. Terlau H. Yoshikami D. J. Neurosci. 1998; 18: 4473-4481Crossref PubMed Google Scholar, 13Safo P. Rosenbaum T. Shcherbatko A. Choi D.Y. Han E. Toledo-Aral J.J. Olivera B.M. Brehm P. Mandel G. J. Neurosci. 2000; 20: 76-80Crossref PubMed Google Scholar). The δ-conotoxins represent a novel category of Na+ channel probes because their receptor/binding site differs from those of other neurotoxins affecting Na+ channel inactivation (7Fainzilber M. Lodder J.C. Kits K.S. Kofman O. Vinnitsky I. Van Rietschoten J. Zlotkin E. Gordon D. J. Biol. Chem. 1995; 270: 1123-1129Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 14Hillyard D.R. Olivera B.M. Woodward S. Corpuz G.P. Gray W.R. Ramilo C.A. Cruz L.J. Biochemistry. 1989; 28: 358-361Crossref PubMed Scopus (88) Google Scholar, 15Fainzilber M. Gordon D. Hasson A. Spira M.E. Zlotkin E. Eur. J. Biochem. 1991; 202: 589-595Crossref PubMed Scopus (89) Google Scholar, 16Shon K.J. Grilley M.M. Marsh M. Yoshikami D. Hall A.R. Kurz B. Gray W.R. Imperial J.S. Hillyard D.R. Olivera B.M. Biochemistry. 1995; 34: 4913-4918Crossref PubMed Scopus (92) Google Scholar, 17Shichor I. Fainzilber M. Pelhate M. Malecot C.O. Zlotkin E. Gordon D. J. Neurochem. 1996; 67: 2451-2460Crossref PubMed Scopus (20) Google Scholar, 18Bulaj G. DeLaCruz R. Azimi-Zonooz A. West P. Watkins M. Yoshikami D. Olivera B.M. Biochemistry. 2001; 40: 13201-13208Crossref PubMed Scopus (68) Google Scholar). The goal of our studies was to discover δ-conopeptides that exclusively inhibit inactivation of Na+ channel subtypes in the nervous systems of vertebrates. We report here the isolation and characterization of δ-conotoxin EVIA (δ-EVIA), 1The abbreviations used are: δ-EVIAδ-conotoxin EVIAα-BTXα-bungarotoxinHPLChigh pressure liquid chromatographyδ-REVIAα-rhodaminyl B-labeled δ-EVIAEPPend plate potential.1The abbreviations used are: δ-EVIAδ-conotoxin EVIAα-BTXα-bungarotoxinHPLChigh pressure liquid chromatographyδ-REVIAα-rhodaminyl B-labeled δ-EVIAEPPend plate potential. a conopeptide in Conus ermineus venom. The purified and synthesized toxin inhibits Na+ channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNav1.2a, rNav1.3, and rNav1.6), without affecting rat skeletal muscle (subtype rNav1.4) and human cardiac muscle (subtype hNav1.5) Na+ channels. Thus, δ-EVIA may be a valuable new tool for discriminating Na+ channel subtypes, for studying the mechanisms involved in neuronal Na+ channel modulation, and for designing new drugs to treat neurological diseases characterized by defective nerve conduction. δ-conotoxin EVIA α-bungarotoxin high pressure liquid chromatography α-rhodaminyl B-labeled δ-EVIA end plate potential. δ-conotoxin EVIA α-bungarotoxin high pressure liquid chromatography α-rhodaminyl B-labeled δ-EVIA end plate potential. Reagents—Specimens of C. ermineus were collected from the Atlantic Ocean along the Senegalese coast of West Africa and maintained in an aquarium. Venom stripped from freshly dissected ducts, was stored at –80 °C after lyophilization. δ-Conotoxin TxVIA was isolated from the venom of Conus textile collected in New Caledonia. Tubocurarine chloride and tetrodotoxin were from Sigma-Aldrich, acetonitrile (UV grade) was from Merckeurolab (Fontenay sous Bois, France), fluorescein isothiocyanate-conjugated α-bungarotoxin (α-BTX) was from Molecular Probes Europe BV (Leiden, The Netherlands). Other solvents and chemicals were purchased from commercial sources and were of the highest purity commercially available. Purification of δ-EVIA—Aliquots (10 mg) of lyophilized venom were extracted (1 h, at 4 °C in a rotatory shaker) with 0.2 m ammonium acetate (pH 7.4). The extract was clarified twice by centrifugation (6,000 × g, 4 min), and the supernatant fluids from all extractions were pooled, applied to a column (1.8 × 76 cm) of Sephadex G-50 (Amersham Biosciences), and eluted (flow rate, ∼4 ml·h-1) with 0.2 m ammonium acetate (pH 7.4). δ-EVIA was further purified by three steps of reverse phase HPLC with Vydac C18 and Merck C18 columns (see Fig. 1). Amino Acid Sequencing—Aliquots of δ-EVIA were incubated for 1 h at room temperature in a solution containing 6 m guanidine hydrochloride, 20 mm dithiothreitol, 2 mm EDTA, and 0.5 m Tris-HCl (pH 7.5). They were then treated for 3 h at room temperature with 1.5 mm 4-vinylpyridine. The derivative was purified by reverse phase HPLC with a C18 Vydac column (4.6 mm x 25 cm; 5-μm particle size), and the amino acid sequence was determined by Edman degradation using an Applied Biosystems 477A microsequencer. Synthesis of δ-EVIA—δ-EVIA was synthesized by solid phase synthesis methods (Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry). Folding procedures, including oxidation of the three be described The of δ-EVIA with that determined by not B-labeled δ-EVIA was as described that was used as an was to amino acid and were with a an a France), and were with and The were and the were with a The was as the in of the 2 h frog nerve muscle were used to in venom muscle and to the of purified were by nerve muscle were dissected and in a solution containing mm mm mm and mm (pH In most was with 2 m as previously described Gall F. P. Benoit E. C. F. A. Molgó J. Eur. J. Neurosci. 1999; PubMed Scopus Google Scholar). and synaptic potentials were at with with 3 m using Gall F. P. Benoit E. C. F. A. Molgó J. Eur. J. Neurosci. 1999; PubMed Scopus Google Scholar). In a with the of an was used to and action potentials at a membrane of with of were at °C with myelinated axons isolated from the frog potentials and Na+ were using and as previously described E. Gordon D. 2001; PubMed Scopus Google Scholar). Na+ were after K+ with mm and 10 mm δ-EVIA was applied to the of the E. Gordon D. 2001; PubMed Scopus Google Scholar). were as previously described J.C. S. A. PubMed Scopus Google Scholar, N. M.M. G. K. Nerve Scholar). and the associated tissue of J. of were for in a and solution containing mm 2 mm 0.5 mm EDTA, and mm (pH The were in a containing and solution containing mm 2 mm mm and mm (pH The were with and used h after at neurons were at using the cell of the with an from and on a and were using and Na+ were in an solution containing mm 3 mm 2 mm 1 mm 20 mm 10 mm (pH 7.4). The solution mm 10 mm 2 mm 2 mm and 10 mm (pH and Using encoding the of rat skeletal muscle human cardiac muscle and rat brain and Na+ channels and the human were in and by a as described D. N. N. N. D. L. I. O. A. M. M. Eur. J. Biochem. 2003; 270: PubMed Scopus Google Scholar). The solution mm 2 mm 1 mm mm and mm (pH solution of δ-EVIA was in and was with solution containing (1 was used to the with solution 4 1 of solution was the in a at room to the did not affect the of to TxVIA (1 was incubated for 2 at room temperature with 0.5 of in containing (10 of a and from The was purified with a Vydac C18 was as previously described N. C. I. M. I. D. Gordon D. J. Neurosci. 1999; PubMed Google Scholar). brain were from as reported Biochemistry. PubMed Scopus Google Scholar). were and as A. N. P. A. C. C. J. Gordon D. Eur. J. Biochem. PubMed Scopus Google Scholar). toxin binding was determined in the of an of of Nerve and frog nerve muscle were (1 h, with 2 in solution and with fluorescein isothiocyanate-conjugated with the were with a on an The were collected in the using a of the are as the were using were to be Purification and Amino Acid of of C. ermineus venom on Sephadex G-50 to the in Fig. muscle applied to isolated frog neuromuscular The was purified to by reverse phase HPLC with C18 Vydac and C18 Merck phase Edman degradation and of the the were with an and three The of the showed the and Therefore, δ-EVIA of 32 amino acid residues and three a similar to that of other from various the of a C. ermineus conotoxin in the of Atlantic with a amino acid sequence similar to that of and M. Gordon D. Hasson A. Spira M.E. Zlotkin E. Eur. J. Biochem. 1991; 202: 589-595Crossref PubMed Scopus (89) Google Scholar, 16Shon K.J. Grilley M.M. Marsh M. Yoshikami D. Hall A.R. Kurz B. Gray W.R. Imperial J.S. Hillyard D.R. Olivera B.M. Biochemistry. 1995; 34: 4913-4918Crossref PubMed Scopus (92) Google previously been reported G. DeLaCruz R. Azimi-Zonooz A. West P. Watkins M. Yoshikami D. Olivera B.M. Biochemistry. 2001; 40: 13201-13208Crossref PubMed Scopus (68) Google and to the of Conus peptides as by is low homology the amino acid sequence of of of action and that of our described δ-EVIA In to other δ-EVIA an acid at 2 and a at a at a at and a of amino acid is also in with i.e. K.S. Lodder J.C. Van Fainzilber M. J. Neurochem. 1996; 67: PubMed Scopus Google and Hasson A. Spira M.E. Gray W.R. Marsh M. Hillyard D.R. Olivera B.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. K.J. Olivera B.M. J. 1996; PubMed Scopus Google of the amino acid sequence and the framework of δ-EVIA with of the The for amino acid residues and are in a new In of Venom and after low of C. ermineus venom and of of of and a of venom of an of the and a of and to and δ-EVIA similar in and of δ-EVIA and in after (1 and in 1 of δ-EVIA on a nerve elicited a muscle action at the of the muscle However, the of δ-EVIA to the of action potentials after a nerve and nerve that the action potentials were by end plate potentials the membrane was to most Na+ a nerve a in the of the However, in the of δ-EVIA, a nerve of at high and in the range of δ-EVIA concentrations were in isolated not Thus, the of δ-EVIA was the of nerve terminal excitability, that a nerve elicited of in of muscle action potentials. We then δ-EVIA directly elicited muscle action potentials using a and nerve muscle in containing 20 to block muscle and the of δ-EVIA did not affect muscle excitability the duration of action potentials at the membrane of muscle was not by the range of δ-EVIA concentrations used for and after toxin four δ-EVIA not act directly on the frog skeletal muscle of δ-EVIA on and in and to the myelinated axons action potentials without the membrane the 1 after toxin the duration of the axonal action at membrane from to The was not of with similar action of δ-EVIA was with spinal neurons not We also characterized the of δ-EVIA on the of tetrodotoxin-sensitive Na+ in myelinated axons and spinal neurons all of the Na+ channels as a of and were at the end of δ-EVIA Na+ channel to of an Na+ The at the end of was of the and the to inactivation of the with δ-EVIA did not the of the and the of δ-EVIA also the of Na+ inactivation as determined using a In the of 0.2 δ-EVIA, a of the Na+ to the described did not and were In the toxin the to inactivation by In δ-EVIA did not affect the of Na+ channel i.e. we did not a in δ-EVIA but and Muscle of δ-EVIA for neuronal Na+ channels was confirmed by the of on rat neuronal Na+ channels (subtypes rNaV1.3, and rNaV1.6) that are of the nervous Na+ i.e. the of the at the end of steps to the was The of 10 δ-EVIA inhibited Na+ inactivation in the three neuronal channel subtypes without affecting the of the In the of δ-EVIA, the for rNaV1.3, and were and in Fig. δ-EVIA did not affect the rat skeletal muscle (rNaV1.4) and the human cardiac muscle (hNaV1.5) Na+ channel subtypes. δ-EVIA was also on rNaV1.4 channels in as previously described H. Gordon D. 2000; PubMed Scopus Google Scholar), in the of inactivation. Thus, we that in the range of concentrations used, δ-EVIA a on neuronal Na+ channels in vertebrates. with that inhibit Na+ channel inactivation to receptor site 3 6 of Na+ channels S. Catterall W.A. Biochimie (Paris). 2000; 82: 883-892Crossref PubMed Scopus (595) Google Scholar). Therefore, were with a that to receptor site 3 N. C. I. M. I. D. Gordon D. J. Neurosci. 1999; PubMed Google Scholar), and with a conotoxin that to receptor site 6 (7Fainzilber M. Lodder J.C. Kits K.S. Kofman O. Vinnitsky I. Van Rietschoten J. Zlotkin E. Gordon D. J. Biol. Chem. 1995; 270: 1123-1129Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 17Shichor I. Fainzilber M. Pelhate M. Malecot C.O. Zlotkin E. Gordon D. J. Neurochem. 1996; 67: 2451-2460Crossref PubMed Scopus (20) Google Scholar). δ-EVIA did not at high concentrations not it inhibited binding of δ-EVIA to receptor site in a The from were to for δ-EVIA and for of Nerve in with The neuronal specificity of δ-EVIA to be used to Na+ channels in nerve and skeletal muscle. of motor nerve of neuromuscular was after to 2 but was not in muscle fibers. to fluorescein isothiocyanate-conjugated muscle that the of was distinct from that of confirmed the specific of the toxin with and of the neuromuscular We have that C. ermineus venom contains the toxin δ-EVIA, of 32 amino acid residues and the six-cysteine/four-loop framework as previously characterized δ-, μO-, ω-, and δ-EVIA an specificity for neuronal Na+ channels. was first using amphibian δ-EVIA inhibits neuronal Na+ channel increases the duration of action potentials in myelinated axons and spinal neurons, and it considerably nerve terminal excitability and synaptic efficacy at the neuromuscular However, it not directly muscle excitability in directly elicited muscle action potentials in the range of concentrations The property of δ-EVIA in amphibian was confirmed by our that derivative of frog motor nerve but not skeletal muscle fibers. the of our studies with rat and human Na+ channel subtypes in and that the toxin also differentiates neuronal and muscle Na+ channels in Thus, δ-EVIA inhibited inactivation in the three rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but it did not affect rat skeletal muscle and human cardiac muscle subtypes and Therefore, we that δ-EVIA a action on neuronal Na+ channels in vertebrates. The of δ-EVIA in Na+ channels from amphibian spinal neurons, of and motor nerve Na+ channels in may be in in Na+ channels present in Other δ-conotoxins also inhibit Na+ channel inactivation. The first and only affect Na+ channels in neuronal they also high binding to Na+ channels in and brain and muscle (7Fainzilber M. Lodder J.C. Kits K.S. Kofman O. Vinnitsky I. Van Rietschoten J. Zlotkin E. Gordon D. J. Biol. Chem. 1995; 270: 1123-1129Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 15Fainzilber M. Gordon D. Hasson A. Spira M.E. Zlotkin E. Eur. J. Biochem. 1991; 202: 589-595Crossref PubMed Scopus (89) Google Scholar, 16Shon K.J. Grilley M.M. Marsh M. Yoshikami D. Hall A.R. Kurz B. Gray W.R. Imperial J.S. Hillyard D.R. Olivera B.M. Biochemistry. 1995; 34: 4913-4918Crossref PubMed Scopus (92) Google Scholar, 17Shichor I. Fainzilber M. Pelhate M. Malecot C.O. Zlotkin E. Gordon D. J. Neurochem. 1996; 67: 2451-2460Crossref PubMed Scopus (20) Google Scholar, M. Kofman O. Zlotkin E. Gordon D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. Fainzilber M. Gordon D. Zlotkin E. Spira M. Eur. J. Neurosci. PubMed Scopus Google Scholar, K.J. Hasson A. Spira M.E. Cruz L.J. Gray W.R. Olivera B.M. Biochemistry. PubMed Scopus Google Scholar). three δ-conotoxins and have been reported to be on Na+ channels in of (7Fainzilber M. Lodder J.C. Kits K.S. Kofman O. Vinnitsky I. Van Rietschoten J. Zlotkin E. Gordon D. J. Biol. Chem. 1995; 270: 1123-1129Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 17Shichor I. Fainzilber M. Pelhate M. Malecot C.O. Zlotkin E. Gordon D. J. Neurochem. 1996; 67: 2451-2460Crossref PubMed Scopus (20) Google Scholar, 18Bulaj G. DeLaCruz R. Azimi-Zonooz A. West P. Watkins M. Yoshikami D. Olivera B.M. Biochemistry. 2001; 40: 13201-13208Crossref PubMed Scopus (68) Google Scholar), not been reported to possess the unique of the toxin C. E. PubMed Scopus (129) Google Scholar), the toxin E. Gordon D. 2001; PubMed Scopus Google Scholar), and the C. N. M. Gordon D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. G.F. 2000; PubMed Scopus Google Scholar), to inhibit neuronal Na+ channel also act on skeletal muscle Na+ channels A. N. P. A. C. C. J. Gordon D. Eur. J. Biochem. PubMed Scopus Google Scholar, M. E. J. Biol. 1996; PubMed Scopus Google in to Therefore, to the of our δ-EVIA is the first conotoxin found to inhibit neuronal Na+ channel inactivation without affecting Na+ channels in skeletal and cardiac muscles. least have been on the of voltage-sensitive Na+ channels. of 3 and are to be binding for toxins that inhibit Na+ channel inactivation S. Catterall W.A. Biochimie (Paris). 2000; 82: 883-892Crossref PubMed Scopus (595) Google Scholar). binding studies that δ-EVIA only to receptor site that are in receptor site 6 of neuronal and muscle Na+ channels. is the role of δ-EVIA in the venom of C. a cone δ-conotoxins are to be for the of B.M. Mol. Biol. Cell. 1997; 8: 2101-2109Crossref PubMed Scopus (323) Google Scholar, 18Bulaj G. DeLaCruz R. Azimi-Zonooz A. West P. Watkins M. Yoshikami D. Olivera B.M. Biochemistry. 2001; 40: 13201-13208Crossref PubMed Scopus (68) Google Scholar, H. K.J. Grilley M. M. Olivera B.M. 1996; PubMed Scopus Google Scholar, Gall F. P. G. Molgó J. 1999; PubMed Scopus Google Scholar). Thus, our that δ-EVIA selectively on neuronal Na+ channels that it to the and in by C. ermineus venom. also the that δ-EVIA may be for not of the of the venom muscle in isolated frog neuromuscular Gall F. P. G. Molgó J. 1999; PubMed Scopus Google Scholar). studies with δ-EVIA may be of in various it is a to channel the with the fluorescent derivative of the toxin the that Na+ channels are in the of frog motor nerve is with showing conduction only in the and of the A. PubMed Scopus Google Scholar). the determined solution of δ-EVIA L. H. J. N. Molgó J. A. J. Biol. Chem. 2003; may a for of action and to the of neuronal voltage-sensitive Na+ channels. because of the specificity of the toxin for neuronal Na+ channels in it may be in designing drugs to treat diseases characterized by defective nerve conduction. We are to M. and A. S. of for We are to R. G. of for the of rNaV1.4 and and to A.L. of for the of rNaV1.3, and We and the from the for in the of the C. ermineus used in