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Originally published In Press as doi:10.1074/jbc.M309576200 on November 13, 2003

J. Biol. Chem., Vol. 279, Issue 6, 4680-4685, February 6, 2004
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A {delta}-Conotoxin from Conus ermineus Venom Inhibits Inactivation in Vertebrate Neuronal Na+ Channels but Not in Skeletal and Cardiac Muscles*

Julien Barbier,abc Hung Lamthanh,bd Frédéric Le Gall,bde Philippe Favreau,af Evelyne Benoit,a Haijun Chen,g Nicolas Gilles,d Nitza Ilan,h Stefan H. Heinemann,g Dalia Gordon,h André Ménez,d and Jordi Molgóai

From the aLaboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040, CNRS, 91198 Gif-sur-Yvette cedex, France, the dDépartement d'Ingénierie et d'Etudes des Protéines, Centre d'Etudes Nucléaires/Saclay, 91191 Gif sur Yvette cedex, France, the gResearch Unit Molecular and Cellular Biophysics, Medical Faculty of the Friedrich Schiller University Jena, D-07747 Jena, Germany, and the hDepartment of Plant Sciences, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel

Received for publication, August 28, 2003 , and in revised form, November 13, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have isolated {delta}-conotoxin EVIA ({delta}-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 {omega}-, {delta}-, µO-, and {kappa}-conotoxins. However, it displays low sequence homology with the latter conotoxins. {delta}-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 {delta}-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. {delta}-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 {delta}-EVIA was confirmed by showing that a fluorescent derivative of {delta}-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, {delta}-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. {delta}-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Defective nerve conduction is frequently associated with neurological diseases, particularly with those causing axonal demyelinization (1, 2). 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 (3, 4). The {alpha}-subunit of each channel is a large membrane-spanning glycoprotein containing four homologous domains associated with smaller accessory {beta}-subunits (3). Mammals possess at least 10 Na+ channel genes, each encoding a distinct channel subtype (5), 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 (6-10). Thus, the µ-conotoxins GIIIA and GIIIB selectively block tetrodotoxin-sensitive Na+ channels in skeletal muscles (6, 10, 11). 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 (12, 13). The {delta}-conotoxins represent a novel category of Na+ channel probes because their receptor/binding site differs from those of other neurotoxins affecting Na+ channel inactivation (7, 14-18). The goal of our studies was to discover {delta}-conopeptides that exclusively inhibit inactivation of Na+ channel subtypes in the nervous systems of vertebrates.

We report here the isolation and characterization of {delta}-conotoxin EVIA ({delta}-EVIA),1 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, {delta}-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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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. {delta}-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 {alpha}-bungarotoxin ({alpha}-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 {delta}-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 x g, 4 min), and the supernatant fluids from all extractions were pooled, applied to a column (1.8 x 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). {delta}-EVIA was further purified by three steps of reverse phase HPLC with Vydac C18 and Merck C18 columns (see Fig. 1).



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  		FIG. 1.
Reverse phase HPLC of {delta}-EVIA in C. ermineus venom. The excitotoxic fraction obtained by molecular exclusion chromatography (Sephadex G-50) of the venom was applied to and eluted from (flow rate, ~3 ml/min) a C18 Vydac semi-preparative column (10 mm x 25 cm; particle size, 10 µm). The arrow indicates the excitotoxic fraction. Inset, the excitotoxic fraction shown in the main panel was applied to a C18 Vydac analytical column (4.6 mm x 25 cm; particle size, 5 µm), and {delta}-EVIA was eluted (flow rate, ~1 ml/min) with an acetonitrile (Buffer B) gradient, as indicated by the dashed line.

 
Amino Acid Sequencing—Aliquots of {delta}-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 {delta}-EVIA—{delta}-EVIA was synthesized by solid phase synthesis methods (Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry). Folding procedures, including oxidation of the three disulfides, will be described elsewhere. The calculated Mr of synthetic {delta}-EVIA agreed with that determined by electrospray ionization mass spectrometry (observed MH+ = 3,288.3; calculated MH+ = 3,288.9; data not shown). N-{alpha}-Rhodaminyl B-labeled {delta}-EVIA ({delta}-REVIA) was synthetized as described above, except that rhodamine B was used as an acetylating reagent. {delta}-REVIA was submitted to amino acid analysis and mass spectrometry (observed MH+ = 3,712.2; calculated MH+ = 3,712.0). Electrospray ionization mass spectra were monitored with a homemade mass spectrometer (C.E.A., Gif-sur-Yvette) comprising an electrospray ion source (Analytica, Branford, CT), a quadrupole mass analyzer (model R10-10; Nermag, France), and data analysis software (Chem-MS Chemstation; Hewlett-Packard).

Bioassays—The bioassays were performed with mosquito fish (Gambusia affinis, 1.0-1.5 g) and Swiss-Webster mice (10-15 g). The fish were injected i.m., and the mice were injected intracerebroventricularly with a stereotaxic system (Harvard/ASI Apparatus, Kent, UK). The ED50 was defined as the dose producing hyperactivity in 50% of the tested mice within 2 h postinjection. Isolated frog (Rana esculenta) nerve muscle preparations were used to detect excitoxin(s) in venom fractions (excitotoxins produce spontaneous muscle contractions) and to characterize the activity of purified {delta}-EVIA.

Electrophysiological Studies—Frogs (R. esculenta, weighing 20-25 g) were killed by double pithing. Cutaneous pectoris nerve muscle preparations were dissected and placed in a standard solution containing 115 mM NaCl, 2.1 mM KCl, 1.8 mM CaCl2, and 5 mM HEPES (pH 7.25). In most experiments, excitation-contraction was uncoupled with 2 M formamide, as previously described (19). Membrane potential and synaptic potentials were recorded at 20-22 °C, with intracellular microelectrodes filled with 3 M KCl (8-12 M{Omega} resistance), using conventional techniques (19). In some experiments, a dual microelectrode current-clamp technique, with the aid of an Axoclamp 2B system (Axon Instruments, Union City, CA), was used to trigger and record action potentials at a membrane potential of –100 mV.

Electrophysiological experiments with nodes of Ranvier were performed at 13-16 °C with single myelinated axons isolated from the frog sciatic nerve. Action potentials and Na+ currents were recorded using current- and voltage-clamp techniques, as previously described (20). Na+ current recordings were performed after suppressing K+ currents with internal 110 mM CsCl and 10 mM external tetraethylammonium chloride. {delta}-EVIA was applied to the nodal compartment of the chamber (for details, see Ref. 20).

Spinal Neuron Cultures—Xenopus cultures were prepared as previously described (21, 22). Briefly, neural tubes and the associated myotomal tissue of Xenopus embryos (20-22 stage) (23) were dissociated for 30-45 min in a Ca2+- and Mg2+-free solution containing 113 mM NaCl, 2 mM KCl, 0.5 mM EDTA, and 5 mM HEPES, (pH 7.8). The cells were plated in a culture medium containing (v/v): 49.5% Leibovitz L-15 medium (Invitrogen), 0.5% fetal calf serum (Invitrogen), and 50% standard solution containing 113 mM NaCl, 2 mM KCl, 0.7 mM CaCl2, and 5 mM HEPES (pH 7.8). The cultures were supplemented with antibiotics and used 24 h after incubation at 19 °C. Spinal neurons were voltage-clamped at 20-22 °C, using the whole cell configuration of the patch-clamp technique with an Axopatch 200B amplifier (Axon Instruments). Patch pipettes made from borosilicate glass and pulled on a P-87 puller (Sutter Instrument Company, Novato, CA) had 1-3 M{Omega} tip resistances. Voltage commands and data acquisition were performed using pCLAMP 8.0 software (Axon Instruments), and Na+ currents were recorded in an extracellular solution containing 140 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM tetraethylammonium chloride, 10 mM HEPES (pH 7.4). The pipette solution contained 130 mM CsCl, 10 mM NaCl, 2 mM MgCl2, 2 mM EGTA, and 10 mM HEPES (pH 7.22).

Na+ Channel Expression and Two-electrode Voltage-Clamp Assay Using Xenopus Oocytes—cRNAs encoding the {alpha}-subunits of rat skeletal muscle (rNaV1.4), human cardiac muscle (hNaV1.5), and rat brain IIa (rNaV1.2a), III (rNaV1.3), and Na6 (rNaV1.6) Na+ channels and the human auxiliary {beta}1-subunit were transcribed in vitro, injected into Xenopus laevis oocytes, and analyzed by a two-electrode voltage-clamp protocol, as described (24). The bath solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES (pH 7.85). A stock solution of {delta}-EVIA (2 mM) was prepared in 50% acetonitrile, 0.05% trifluoroacetic acid, and was diluted 200-fold with bath solution containing bovine serum albumin (1 mg/ml). A BPS-8 perfusion system (ALA Scientific Instruments Inc., Westbury, NY) was used to wash the oocytes with bath solution (0.7 ml/min, flow rate; 4 p.s.i., positive pressure). Approximately 1 ml of toxin-containing solution was perfused over the oocytes mounted in a 200-µl chamber at room temperature. Exposure to the vehicle (0.25% acetonitrile) did not affect the oocytes.

Binding of Radioiodinated Toxins to Synaptosomes—{delta}-Conotoxin TxVIA (1 nmol) was incubated for 2 min at room temperature with 0.5 mCi of carrier-free Na[125I] in potassium phosphate buffer (0.1 M, pH 7.25) containing H2O2 (10 µl of a 1/50,000 solution) and lactoperoxidase (0.7 units, EC 1.11.1.7 [EC] , from bovine milk). The monoiodotoxin was purified with a Vydac C18 column. Lqh-2 was radioiodinated as previously described (25). Rat brain synaptosomes were prepared from adult rats (Sprague-Dawley; 300 g) as reported (26). Equilibrium competition assays were performed and analyzed as before (27). Nonspecific toxin binding was determined in the presence of an excess of unlabeled toxin.

Staining of Nerve Terminals and Confocal Microscopy—Isolated frog cutaneous pectoris nerve muscle preparations were stained (1 h, 22 °C) with 2 µM {delta}-REVIA dissolved in standard solution and double-labeled (15 min, 22 °C) with fluorescein isothiocyanate-conjugated {alpha}-BTX (diluted 1:100). After being washed several times with standard medium, the preparations were examined with a Leica TCS scanning laser confocal system (Leica Microsystems, Mannheim, Germany) mounted on an upright microscope. The images were collected in the sequential acquisition mode, using a water immersion lens (x40, N.A. 0.8).

Data Analysis—All of the data are expressed as the means ± S.E. Statistical comparisons were performed using Student's t test (p values < 0.05 were considered to be statistically significant).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification and Amino Acid Sequencing of {delta}-EVIA—Chromatography of C. ermineus venom on Sephadex G-50 leads to the profile shown in Fig. 1. Only one fraction caused spontaneous muscle contractions when applied to isolated frog neuromuscular preparations (not shown). The fraction was purified to homogeneity by successive reverse phase HPLC with C18 Vydac (Fig. 1, inset) and C18 Merck analytical columns. Gas phase automated Edman degradation and analysis of the pyridylethylated peptide gave the following unambiguous sequence: (H)-DDCIKOYGFCSLPILKNGLCCSGACVGVCADL-(NH2). Electrospray ionization mass spectrometry analyses (observed average MH+ = 3,288.3; calculated average MH+ = 3,288.9) were consistent with an amidated C terminus and three disulfide bridges. The pairing of the disulfide linkages unambiguously showed the arrangement C1-4, C2-5, and C3-6 (not shown). Therefore, {delta}-EVIA consists of 32 amino acid residues and three disulfide linkages forming a four-loop cysteine scaffold similar to that of other conotoxins from various families (Table I). On the basis of cDNA analysis, a 29-amino acid-long C. ermineus conotoxin (collected in the western part of Atlantic Ocean), with a deduced amino acid sequence similar to that of {delta}-PVIA and {delta}-NgVIA (15, 16) has previously been reported (18) and designated according to the current nomenclature of Conus peptides as E6a (encoded by clone E6.4). Interestingly, there is low homology between the amino acid sequence of this E6a peptide of unknown mechanism of action and that of our described {delta}-EVIA (Table I). In contrast to other {delta}-conotoxins, {delta}-EVIA possesses an aspartic acid at positions 2 and 31, a tyrosine at position 7, a glycine at position 8, a small side chain (alanine) at position 24, and a longer loop composed of 9 amino acid residues, which, however, is also observed in conotoxins with unrelated functions; i.e. {omega}-conotoxin PnVIA (28) and µO-conotoxins (29, 30).


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  		TABLE I
Comparison of the amino acid sequence and the framework of {delta}-EVIA with members of the O superfamily The standard one-letter codes for amino acid residues (except O = trans-4-hydroxyproline and {gamma} = {gamma}-carboxyglutamate) are used.

 
In Vivo Activity of Venom and {delta}-EVIA—2-5 min after intracerebroventricular injection, low doses of C. ermineus venom induced rapid running and jumping of mice (ED50 = 0.25 µg/g of body mass), whereas higher doses (>0.4 µg/g of body mass) caused convulsions and death within a few minutes. Intramuscular injection of venom (0.5 µg/g of body mass) into mosquito fish produced an immediate extension of the dorsal and caudal fins, a loss of balance control, and violent convulsions just prior to death. Native and synthetic {delta}-EVIA exhibited similar excitatory activity in mice (ED50 = 1.8 and 1.9 pmol/g of body mass, respectively). {delta}-EVIA (40 pmol) produced hyperactivity, rapid running, and seizures in mice 1-3 min after intracerebroventricular injection. Higher doses (1 nmol) induced convulsions and death in about 1 min.

Effects of {delta}-EVIA on Frog Neuromuscular Preparations— Under control conditions, a single nerve stimulus elicited a single muscle action potential recorded at the junctional region of the muscle fiber (Fig. 2A, top trace). However, the addition of {delta}-EVIA (0.2-1 µM) to the medium induced trains of repetitive action potentials after a single nerve stimulus (Fig. 2A, middle trace), and continued nerve stimulation (0.5 Hz) revealed that the repetitive action potentials were triggered by repetitive end plate potentials (EPPs) (Fig. 2A, bottom trace). When the membrane was preset to –45 mV (to inactivate most voltage-dependent Na+ channels), a single nerve stimulus evoked a single EPP (Fig. 2B, upper trace) in the absence of the toxin. However, in the presence of {delta}-EVIA, a single nerve stimulus evoked trains of EPPs (Fig. 2B, lower trace) at high frequency. Similar effects and in the same range of {delta}-EVIA concentrations were obtained in isolated mouse phrenic-hemidiaphragm preparations (data not shown). Thus, the main effect of {delta}-EVIA was the enhancement of nerve terminal excitability, so that a single nerve stimulus elicited trains of repetitive EPPs, which in turn triggered trains of repetitive muscle action potentials.



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  		FIG. 2.
Effect of {delta}-EVIA on muscle and nerve action potentials, and on synaptic potentials. A, action potentials evoked by a single nerve stimulus and recorded in the same neuromuscular junction before (top trace) and after (middle and bottom traces) adding 1 µM {delta}-EVIA to the medium. The resting membrane potential was –90 mV. B, EPPs recorded in another junction, at a preset membrane potential of –45 mV, in response to a single nerve stimulus before (upper trace) and during (lower trace) the action of 1 µM {delta}-EVIA. C, muscle action potentials evoked by direct stimulation under control conditions (left trace) and in the presence of 1 µM {delta}-EVIA (right trace). The membrane potential was preset at –100 mV. D, action potentials (elicited by 0.5 ms of depolarizing stimuli at 1 Hz) recorded in a myelinated axon from a resting potential of –70 mV, before (trace a) and 5 s (trace b), 10 s (trace c), 15 s (trace d), 5 min (trace e), 10 min (trace f), and 15 min (trace g) after the addition of 0.2 µM {delta}-EVIA to the external standard medium.

 
We then examined whether {delta}-EVIA affects directly elicited muscle action potentials using a dual microelectrode current-clamp technique and nerve muscle preparations equilibrated in standard medium containing 20 µM tubocurarine to block muscle nicotinic acetylcholine receptors and neutralize the presynaptic effects of {delta}-EVIA. Under these conditions, {delta}-EVIA (0.2-2 µM) did not affect muscle excitability or modify the amplitude, overshoot, or duration of action potentials (n = 6) recorded at –100 mV (Fig. 2C). Furthermore, the membrane potential of muscle fibers was not significantly affected by the range of {delta}-EVIA concentrations used (–88.8 ± 3.6 mV for controls and –90.7 ± 4.7 mV after toxin treatment; n = 20, four different muscles). Clearly, {delta}-EVIA does not act directly on the frog skeletal muscle membrane.

Effects of {delta}-EVIA on Action Potentials and Na+ Currents in Frog Myelinated Axons and Xenopus Spinal Neurons—{delta}-EVIA (0.2 µM) added to the medium bathing current-clamped single myelinated axons consistently prolonged action potentials without modifying the resting membrane potential or the peak amplitude (Fig. 2D). Within 1 min after toxin exposure, the duration of the axonal action potentials, measured at 50% membrane repolarization, increased from 1.2 ± 0.1 to 11.4 ± 1.3 ms (n = 3). The effect was not reversible within 30 min of superfusion with standard toxin-free solution. A similar action of {delta}-EVIA was observed with current-clamped spinal neurons (n = 6; data not shown).

We also characterized the effects of {delta}-EVIA (0.2 µM) on the time dependence of tetrodotoxin-sensitive Na+ currents in myelinated axons (Fig. 3A) and spinal neurons (Fig. 3B). Under control conditions, all of the Na+ channels inactivated as a function of time and thus were closed at the end of depolarizing steps. {delta}-EVIA significantly suppressed time-dependent Na+ channel inactivation, which led to maintenance of an inward Na+ current. The sustained current, measured at the end of depolarizing steps, was 20.7 ± 1.7% (n = 6) of the peak current, and the time corresponding to 50% inactivation of the inactivatable peak current increased 3.6 ± 1.6-fold (n = 6) when compared with controls. {delta}-EVIA exposure did not significantly alter the amplitude of the peak current and the kinetics of current activation.



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  		FIG. 3.
Effect of {delta}-EVIA on the time and voltage dependence of Na+ current activation and inactivation in myelinated axons and spinal neurons. A and B, Na+ currents recorded in a myelinated axon (A) and in a spinal neuron (B) during a depolarization to –20 mV, preceded by a 50-ms hyperpolarization to –140 mV applied from a holding potential of –60 mV, before and after adding 0.2 µM {delta}-EVIA to the external medium. Na+ currents were characterized by their sensitivity to tetrodotoxin (1 µM). C, voltage dependence of Na+ current activation (circles) and inactivation (triangles). The peak current was measured in a spinal neuron during depolarizations of various amplitudes (circles) or to –20 mV (triangles), preceded by 50 ms hyperpolarizations to –140 mV (circles) or of various amplitudes (triangles) applied from a holding potential of –60 mV, before (closed symbols) and after (open symbols) treatment with 0.2 µM {delta}-EVIA. To determine steady-state inactivation-voltage relationships, the peak current was normalized to its maximum value at large prehyperpolarizations. To determine activation-voltage relationships, the conductance (g) was calculated from the peak current (I), according to: g = I/(V – ENa), where V is the test pulse potential, and ENa is the current reversal potential, and was normalized to its maximum value at large depolarizations.

 
{delta}-EVIA also altered the voltage dependence of steady-state Na+ current inactivation (Fig. 3C), as determined using a two-pulse protocol. In the presence of 0.2 µM {delta}-EVIA, a fraction of the Na+ current (corresponding to the sustained current described above) did not inactivate and remained constant when prehyperpolarizations were more positive than –45 mV. In addition, the toxin shifted the prepulse potential corresponding to 50% steady-state inactivation by –14.9 ± 1.9 mV (n = 4). In contrast, {delta}-EVIA (0.2 µM) did not affect the voltage dependence of Na+ channel activation, i.e. we did not observe a significant change in peak conductance-voltage relationships (Fig. 3C).

{delta}-EVIA Affects Xenopus Oocyte-Expressed Mammalian Neuronal Na+ Channels but Not Skeletal and Cardiac Muscle Na+ Channels—The selectivity of {delta}-EVIA for vertebrate neuronal Na+ channels was confirmed by the results of its effects on several heterologously expressed rat neuronal Na+ channels (subtypes rNaV1.2a, rNaV1.3, and rNaV1.6) that are typical of the central nervous system. Under control conditions, Na+ currents fully inactivated, i.e. the ratio of the sustained current at the end of depolarizing steps (Is) to the peak current (Ip) was <1%. The addition of 10 µM {delta}-EVIA inhibited Na+ current inactivation in the three neuronal channel subtypes (Fig. 4) without affecting the amplitude of the peak current. In the presence of {delta}-EVIA, the Is/Ip ratios for rNaV1.2a, rNaV1.3, and rNaV1.6 were 11.1 ± 2.1% (n = 3), 3.7 ± 0.23% (n = 3), and 5.6 ± 0.14% (n = 3), respectively. As shown in Fig. 4, {delta}-EVIA did not affect the rat skeletal muscle (rNaV1.4) and the human cardiac muscle (hNaV1.5) Na+ channel subtypes. {delta}-EVIA (5 µM) was also tested on rNaV1.4 channels expressed in HEK 293 cells as previously described (31), yielding no effect in the time course of inactivation. Thus, we conclude that in the range of concentrations used, {delta}-EVIA has a predominant effect on neuronal Na+ channels in vertebrates.



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  		FIG. 4.
Effects of {delta}-EVIA on mammalian Na+ channel subtypes expressed in Xenopus oocytes. The {alpha}-subunits of neuronal Na+ channels (rNaV1.2a, rNaV1.6, and rNaV1.3) and the muscle Na+ channels (rNaV1.4 and hNaV1.5) were co-expressed with the human auxiliary {beta}1-subunit. The currents were elicited by depolarizing pulses from –80 to –20 mV, in the absence (control) and in the presence of 10 µM

{delta}-EVIA.

 
Binding Studies with Rat Brain Synaptosomes—Toxins that inhibit Na+ channel inactivation bind to either receptor site 3 or 6 of Na+ channels (4). Therefore, competition experiments were performed with [125I]Lqh-2, a scorpion {alpha}-toxin that binds to receptor site 3 (25), and with [125I]{delta}-TxVIA, a conotoxin that specifically binds to receptor site 6 (7, 17). {delta}-EVIA did not compete [125I]Lqh-2, even at high concentrations (1-10 µM, data not shown), whereas, like unlabeled {delta}-TxVIA, it fully inhibited binding of [125I]{delta}-TxVIA (Fig. 5). Clearly, {delta}-EVIA bound to receptor site 6, in a dose-dependent manner. The Ki values deduced from these data were equal to 475 ± 75 nM for {delta}-EVIA (n = 3) and 1.9 ± 0.4 nM for {delta}-TxVIA (n = 3).



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  		FIG. 5.
Competition curves for [125I]{delta}-TxVIA binding inhibition by {delta}-EVIA. Synaptosomes (65.5 µg/ml) were incubated (60 min, 22 °C) with 180 pM [125I]{delta}-TxVIA and increasing concentrations of {delta}-EVIA. Nonspecific binding determined in the presence of 1 µM {delta}-TxVIA (8,000 cpm) was subtracted from the total binding (17,000 cpm). The amount of bound [125I]{delta}-TxVIA is expressed as the percentage of maximal specific binding without additional toxin. The slopes of the curves were respectively 0.95 for {delta}-TxVIA and 1.1 for {delta}-EVIA, indicating that the toxins bind to a single class of sites in rat brain synaptosomes. Competition curves were fitted by the nonlinear Hill equation (coefficient of 1) to determine the IC50 values.

 
Staining of Frog Motor Nerve Terminals in Situ with {delta}-REVIA— The observed neuronal specificity of {delta}-EVIA prompted us to determine whether {delta}-REVIA could be used to distinguish between Na+ channels in nerve terminals and skeletal muscle. Indeed, staining of motor nerve terminals of neuromuscular junctions was observed after exposure to 2 µM {delta}-REVIA (Fig. 6A), but staining was not detected in muscle fibers. Subsequent exposure to fluorescein isothiocyanate-conjugated {alpha}-BTX (Fig. 6B) (which labels muscle nicotinic ACh receptors) revealed that the region of {delta}-REVIA staining was distinct from that of {alpha}-BTX staining (Fig. 6C), which confirmed the different, specific interactions of the toxin with presynaptic and postsynaptic elements of the neuromuscular junction.



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  		FIG. 6.
{delta}-REVIA selectively stains motor nerve terminals of the frog neuromuscular junction. A, {delta}-REVIA staining of motor nerve terminals in a neuromuscular junction. B, staining of muscle nicotinic ACh receptors with fluorescein isothiocyanate-conjugated {alpha}-BTX. C, overlay of nerve terminal labeling with {delta}-REVIA and nicotinic ACh receptor staining with {alpha}-BTX. Note that the distal part of the nerve terminals (arrow) is not stained by {delta}-REVIA. Calibration, 20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that C. ermineus venom contains the toxin {delta}-EVIA, which consists of 32 amino acid residues and has the same six-cysteine/four-loop framework as previously characterized {delta}-, µO-, {omega}-, and {kappa}-conotoxins (Table I). {delta}-EVIA possesses an unusual specificity for neuronal Na+ channels. This selectivity was first observed using amphibian preparations. {delta}-EVIA inhibits neuronal Na+ channel inactivation, which increases the duration of action potentials in myelinated axons and spinal neurons, and it considerably enhances nerve terminal excitability and synaptic efficacy at the neuromuscular junction. However, it does not directly enhance muscle excitability or induce detectable changes in directly elicited muscle action potentials in the range of concentrations used. The neuronal-selective property of {delta}-EVIA detected in amphibian tissues was confirmed by our observation that {delta}-REVIA (a fully active, rhodaminated derivative of {delta}-EVIA) labels frog motor nerve terminals but not skeletal muscle fibers. Furthermore, the results of our subsequent studies with rat and human Na+ channel subtypes (heterologously expressed in Xenopus oocytes and HEK 293 cells) indicated that the toxin also differentiates between neuronal and muscle Na+ channels in mammals. Thus, {delta}-EVIA inhibited inactivation in the three rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) tested, but it did not discernibly affect rat skeletal muscle and human cardiac muscle subtypes (rNaV1.4 and hNaV1.5, respectively). Therefore, we conclude that {delta}-EVIA has a predominant action on neuronal Na+ channels in vertebrates. The different potency of {delta}-EVIA in native Na+ channels from amphibian spinal neurons, nodes of Ranvier, and motor nerve terminals versus heterologously expressed mammalian Na+ channels in oocytes may be due to: (i) differences in lipid environment and/or (ii) differences in Na+ channels regulatory proteins present in native versus expressed cells.

Other {delta}-conotoxins also inhibit Na+ channel inactivation. The first discovered, {delta}-TxVIA and {delta}-GmVIA (Table I), only affect Na+ channels in mollusc neuronal membranes, even though they also exhibit high affinity binding to Na+ channels in invertebrate and vertebrate brain and muscle tissues (7, 15-17, 32-34). Also, although three {delta}-conotoxins ({delta}-PVIA, {delta}-NgVIA, and {delta}-SVIE) have been reported to be active on Na+ channels in excitable cells of vertebrates (7, 17, 18), not one has been reported to possess the unique selectivity of {delta}-EVIA. Furthermore, the sea anemone Anemonia sulcata toxin II (35), the {alpha}-like scorpion toxin Lqh-3 (20), and the spider {delta}-atracotoxins (36, 37), known to inhibit neuronal Na+ channel inactivation, also act on skeletal muscle Na+ channels (27, 38) in contrast to {delta}-EVIA. Therefore, to the best of our knowledge, {delta}-EVIA is the first conotoxin found to inhibit neuronal Na+ channel inactivation without affecting Na+ channels in skeletal and cardiac muscles.

At least seven ligand-binding sites have been identified on the {alpha}-subunits of voltage-sensitive Na+ channels. Two of them, sites 3 and 6, are known to be binding sites for toxins that inhibit Na+ channel inactivation (4). Our binding competition studies revealed that {delta}-EVIA binds only to receptor site 6, which suggests that there are structural differences in receptor site 6 of neuronal and muscle Na+ channels.

What is the role of {delta}-EVIA in the venom of C. ermineus, a fish-hunting cone snail? {delta}-conotoxins are thought to be essential for the rapid immobilization of quickly moving prey (8, 18, 39, 40). Thus, our observation that {delta}-EVIA selectively acts on neuronal voltage-dependent Na+ channels suggests that it contributes to the excitotoxic shock and tetanic paralysis produced in fish by C. ermineus venom. Our findings also support the idea that {delta}-EVIA may be responsible for part, if not all, of the excitatory effects of the venom (i.e. spontaneous muscle contractions) observed in isolated frog neuromuscular preparations (40).

Future studies with {delta}-EVIA may be of interest in various domains. First, it is a useful pharmacological probe to study channel distribution. Indeed, the results obtained with the fluorescent derivative of the toxin ({delta}-REVIA) support the current notion that Na+ channels are absent in the distal part of frog motor nerve endings, which is consistent with data showing active conduction only in the proximal and median parts of the terminals (41). Second, the recently determined three-dimensional solution structure of {delta}-EVIA (44) may provide a basis for understanding its molecular mechanism of action and its capacity to modulate the activity of neuronal voltage-sensitive Na+ channels. Finally, because of the specificity of the toxin for neuronal Na+ channels in mammals, it may be useful in designing drugs to treat diseases characterized by defective nerve conduction.


   FOOTNOTES
 
* This work was supported in part by Direction des Systèmes de Forces et de la Prospective Grant 02 60 65 093 (to J. M.), by European Community Grant QLK3-CT-2000-00204 (to A. M. and D. G.), by United States-Israel Binational Agricultural Research and Development Grant IS-3259-01 (to D. G.), and by Deutsche Forschungsgemeinschaft Grant HE 2993/5 (to S. H. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

b These authors contributed equally to this work. Back

c Supported by a fellowship from DSP-CNRS. Back

e Supported by a fellowship from CEN/Saclay. Present address: L'Oréal Recherche, 92583 Clichy cedex, France. Back

f Present address: Atheris Laboratories, case postale 314, Ch-1233 Bernex, Switzerland. Back

i To whom correspondence should be addressed. Fax: 33-1-69-82-94-66; E-mail: Jordi.Molgo{at}nbcm.cnrs-gif.fr.

1 The abbreviations used are: {delta}-EVIA, {delta}-conotoxin EVIA; {alpha}-BTX, {alpha}-bungarotoxin; HPLC, high pressure liquid chromatography; {delta}-REVIA, {alpha}-rhodaminyl B-labeled {delta}-EVIA; EPP, end plate potential. Back


   ACKNOWLEDGMENTS
 
We are indebted to M. Benveniste (Tel Aviv University, Tel Aviv, Israel) and A. S. Kreger (University of Maryland) for useful discussions. We are grateful to R. G. Kallen (University of Pennsylvania, Philadelphia, PA) for the kind gift of rNaV1.4 and hNaV1.5 clones and to A.L. Goldin (University of California, Irvine, CA) for kindly providing the clones of rNaV1.2a, rNaV1.3, and rNaV1.6. We thank Haidar El Alil and the divers from the Oceanium (Dakar, Senegal) for help in the collection of the C. ermineus specimens used in this study.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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