©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A New Conotoxin Affecting Sodium Current Inactivation Interacts with the -Conotoxin Receptor Site (*)

(Received for publication, August 15, 1994; and in revised form, November 4, 1994)

Michael Fainzilber (1) (2)(§) Johannes C. Lodder (2) Karel S. Kits (2) Ora Kofman (3) Ilya Vinnitsky (3) Jurphaas Van Rietschoten (4) Eliahu Zlotkin (1) Dalia Gordon (1)(§) (4)

From the  (1)Department of Cell and Animal Biology, Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel, the (2)Graduate School Neurosciences Amsterdam, Research Institute of Neuroscience, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands, the (3)Beer-Sheva Mental Health Center and Department of Behavioral Sciences, Ben-Gurion University, Beer-Sheva, Israel, and the (4)Faculty of Medicine, Department of Biochemistry, University of Aix-Marseille II, Bd. Pierre Dramard, 13916 Marseille Cedex 20, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We describe a new peptide conotoxin affecting sodium current inactivation, that competes on binding with -conotoxin TxVIA (TxVIA). The amino acid sequence of the new toxin, designated conotoxin NgVIA (NgVIA), is SKCFSOGTFCGIKOGLCCSVRCFSLFCISFE (where O is trans-4-hydroxyproline). The primary structure of NgVIA has an identical cysteine framework and similar hydrophobicity as TxVIA but differs in its net charge. NgVIA competes with TxVIA on binding to rat brain synaptosomes and molluscan central nervous system and strongly inhibits sodium current inactivation in snail neurons, as does TxVIA. In contrast to TxVIA, NgVIA is a potent paralytic toxin in vertebrate systems, its binding appears to be voltage-dependent, and it synergically increases veratridine-induced sodium influx to rat brain synaptosomes. TxVIA acts as a partial antagonist to NgVIA in rat brain in vivo. NgVIA appears to act via a receptor site distinct from that of TxVIA but similar to that of Conus striatus toxin. This new toxin provides a lead for structure-function relationship studies in the -conotoxins and will enable analysis of the functional significance of this complex of receptor sites in gating mechanisms of sodium channels.


INTRODUCTION

Voltage-dependent sodium channels are integral plasma membrane proteins responsible for the rapidly rising phase of action potentials in most excitable tissues and as such are specifically targeted by many neurotoxins. These toxins occupy at least six identified (receptor sites 1-5 (Catterall, 1986), and receptor site 6 (Fainzilber et al., 1994)) and two unidentified (CsTx (^1)and GPT (Catterall, 1992)) receptor sites on the rat brain sodium channel and have been used as tools for functional mapping and characterization of the channel (Catterall, 1986, 1992; Fainzilber et al., 1994).

Over the past decade a number of selective toxin ligands have been characterized that compete directly on their binding but by various criteria cannot share precisely the same receptor binding sites (Adams and Olivera, 1994; Gordon et al., 1992). Receptor sites are thought to overlap when they appear to be targeted by a number of different toxins that induce similar pharmacological effects and compete in binding assays. Complexes of such overlapping sites have been termed macrosites (Olivera et al., 1991). We suggest that a receptor site be defined as the combined points of attachment/recognition sites that are directly involved in the binding interactions with a toxin. A macrosite, in this context, may include distinct receptor sites for several toxins, each with its own unique points of attachment. Some of these recognition sites may be shared by different toxins that bind in the same macrosite. For example, receptor site 1 on the sodium channel (Catterall, 1986, 1992) binds the blockers tetrodotoxin, saxitoxin, and µ-conotoxins. We suggest that this is in fact a macrosite, since tetrodotoxin and saxitoxin do not share all of their attachment points (Terlau et al., 1991). Furthermore, µ-conotoxins selectively bind to skeletal muscle sodium channels (Moczydlowski et al., 1986) on which at least part of their attachment points are distinct from those of saxitoxin (Stephan et al., 1994).

Receptor sites for peptide neurotoxins that inhibit sodium current inactivation are of particular interest for the study of the dynamics of channel gating since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel (Catterall, 1992). These are the macrosite that binds sea anemone and alpha-scorpion toxins (site 3 of Catterall(1986)) and the receptor sites for GPT, CsTx, and TxVIA (Gonoi et al., 1986, 1987; Fainzilber et al., 1994). The latter three receptor sites have been shown to be distinct from each other on the basis of binding and pharmacological studies (Catterall and Beress, 1978; Ray et al., 1979; Gonoi et al., 1986, 1987; Fainzilber et al., 1994). Furthermore, TxVIA has the unique property of distinguishing between phyletic variants of sodium channels on the basis of activity but not binding (Fainzilber et al., 1994). Thus subtle differences in the TxVIA receptor site on different channels may be the cause of significant differences in toxin effects; and conversely sequence variabilities in a family of toxins interacting with a single channel subtype may cause differing agonist/antagonist activities. Therefore, the aim of this study was to find a peptide homologous to TxVIA, which would act as an agonist at its receptor site on the rat brain sodium channel. Screening of piscivorous Conus venoms revealed a new conotoxin that competes with TxVIA on binding to both rat brain and molluscan sodium channels and acts as a full agonist in rat brain. This new toxin, designated NgVIA, will serve as a complementary pharmacological probe for the study of the role(s) and structureof the TxVIA receptor site and related receptor sites in modulation of sodium channel gating.


EXPERIMENTAL PROCEDURES

Materials

108 mg of crude venom was obtained from 32 adult Conus nigropunctatus from the northern Red Sea. TxVIA was purified from Conus textile venom as described previously (Fainzilber et al., 1991). Tetrodotoxin, veratridine, and sea anemone toxin II were from Sigma. All other chemicals were of analytical grade. Filters for binding assays were glass fiber GF/F (Whatman, United Kingdom), and for sodium flux assays 0.45-µm cellulose BA85 (Schleicher & Schuell).

Column Chromatography

50-mg venom aliquots were extracted three times in 0.1 M ammonium acetate, pH 7.5, for 1 h in a rotatory shaker at 4 °C. Supernatants from the three extracts were combined and separated on Sephadex G-50 (Pharmacia Biotech Inc.). HPLC purification of the active fractions was accomplished in two steps of reverse phase chromatography on Vydac C18 and phenyl columns as described in the legend to Fig. 1.


Figure 1: Purification of NgVIA. A, 50 mg of lyophilized venom was extracted as described under ``Experimental Procedures,'' and separated on a Sephadex G-50 column (63 times 1.42 cm), equilibrated, and eluted in 0.1 M ammonium acetate, pH 7.5, at a flow rate of 5 ml/h and a temperature of 4 °C. B, the marked fraction was further fractionated by reverse phase HPLC on a Vydac C18 column (25 times 0.46 cm, 5 µm particle size), eluted at a flow rate of 0.5 ml/min with a gradient of acetonitrile in 0.1% trifluoroacetic acid as shown by the dashed line. C, the active fraction (indicated by the arrow in B) was further purified on a Vydac phenyl column (25 times 0.46 cm, 5 µm particle size) using the same flow rate and solvents. The inset shows complete identity in the superimposed UV spectra sampled by a diode array detector at different time points along the NgVIA peak. D, synthetic NgVIA was prepared as described under ``Experimental Procedures,'' and purified to homogeneity on an Alltech C8 column (25 times 0.46 cm, 5 µm particle size) at a flow rate of 0.5 ml/min, using a linear gradient of 0-60% acetonitrile, 2-propanol (1:1) in 0.1% trifluoroacetic acid. The lower trace (1) is pure synthetic NgVIA, and the upper trace (2) is an equimolar mix of native and synthetic NgVIA.



Amino Acid Analysis

Analysis of amino acid composition after acid hydrolysis and Fmoc derivatization was performed according to Betner and Foldi(1986).

Peptide Sequencing

Purified toxin was reduced and alkylated with 4-vinylpyridine, HPLC-purified, and adsorbed onto polyvinylidene difluoride membranes as described previously (Fainzilber et al., 1991). Amino acid sequence analysis was performed by automated Edman degradation with an Applied Biosystems 475A gas-phase sequencer. The sequence was confirmed in two separate determinations utilizing different batches of peptide.

Peptide Synthesis

Solid phase synthesis was performed on an Applied Biosystems 430A automated synthesizer, as described previously (Sabatier et al., 1993), using N-butyloxycarbonyl amino acids. Crude peptide was resuspended in 0.2 M Tris-HCl, pH 8.0, 2% beta-mercaptoethanol, and the suspension was vigorously mixed for 1 h at 70 °C. It was then rapidly diluted into 0.2 M Tris, 2 M guanidine hydrochloride for a final peptide concentration of 1 mg/ml and allowed to air oxidize for 48 h at room temperature. The sample was then concentrated on preparative C8 cartridges (Supelco) and purified by two sequential reverse phase HPLC steps using a C8 column, as detailed in Fig. 1D.

Neuronal Membrane Preparations

Mollusc (Helix) central nervous system membrane preparations were prepared as described in Fainzilber et al.(1994). Rat brain synaptosomes were prepared from adult Sabra albino rats (6-8 months old) according to the procedure of Kanner(1978). Membrane protein concentration was determined according to Peterson (1977) with bovine serum albumin used as the standard.

Radioiodination

TxVIA was radioiodinated and purified as described previously (Fainzilber et al., 1994). The monoiodinated toxin fraction was used for binding assays, corresponding to a specific activity of 2.8 times 10^6 dpm/pmol.

Binding Assays

Binding competition assays were performed using increasing concentrations of unlabeled competitor in the presence of a constant low concentration of radioactive TxVIA. Binding and wash media compositions were as described previously (Fainzilber et al., 1994). Helix (30-40 µg of protein/reaction) or rat (20-30 µg of protein/reaction) neuronal membranes were suspended in 0.2 ml of binding medium containing 0.2 nMI-TxVIA.

After incubation for the designated time periods, the reaction mixture was diluted with 2 ml of ice-cold wash buffer and filtered through GF/F (Whatman, U. K.) under vacuum. Filters were rapidly washed an additional two times with 2 ml of buffer. Nonspecific toxin binding was determined in the presence of 1 µM unlabeled TxVIA and typically consisted of 30-35% of total binding, one-third of which was to the filters alone. Binding data were analyzed using the iterative computer program LIGAND (Elsevier Biosoft, U. K.).

Na Flux Assays

Na influx assays in rat brain synaptosomes were carried out as described previously (Tamkun and Catterall, 1981; Fainzilber et al., 1994). Briefly, rat brain synaptosomes (0.3-0.5 mg of membrane protein/reaction vial) were preincubated for 10 min with toxins at twice their final concentrations in 100 µl of Na-free preincubation buffer at 37 °C. Flux was initiated by adding 100 µl of influx buffer containing 2.66 mM NaCl and 1.5 µCi/ml NaCl. After 30 s at 37 °C, flux was terminated by the addition of 2 ml of ice-cold wash buffer and rapid filtration through BA85 nitrocellulose membrane filters under vacuum. Background Na influx was determined in the presence of 2 µM tetrodotoxin and subtracted from all data points (background was always less than 20% of total flux after subtraction of blank filters).

Electrophysiology

Whole cell voltage clamp recordings were taken from dissociated caudodorsal neurons from laboratory bred adult Lymnaea stagnalis. The cells were isolated by mechanical dissociation after 30 min of incubation in 0.2% trypsin (type III, Sigma) in HEPES-buffered saline at 37 °C, as described previously (Dreijer and Kits, 1994). Cells were used within 7 h of isolation. For recording of sodium currents, HEPES-buffered saline was washed out and replaced under constant perfusion with a solution containing 40 mM NaCl, 4 mM CaCl(2), 1.5 mM CdCl(2), 10 mM HEPES, 2 mM 4-aminopyridine, pH 7.8, adjusted with NaOH. The internal pipette solution was composed of 29 mM CsCl, 2.3 mM CaCl(2), 10 mM HEPES, 11 mM EGTA, 2 mM MgATP, 0.1 mM Tris-GTP, pH 7.4, adjusted with CsOH; the calculated free calcium concentration was 10M. Conotoxins were applied by means of a picospritzer system that allows rapid application of drugs. Toxin applications commenced 2 s before the test pulse and ended immediately after it. Whole cell voltage clamp experiments were performed and data analyzed as described previously (Dreijer and Kits, 1994) except that an Axopatch 2A amplifier (Axon Instruments, Inc.) was used. Current recordings were filtered at 2 kHz and sampled at >5 kHz.

In Vivo Animal Bioassays

Animal bioassays were performed as described by Fainzilber and Zlotkin(1992). Effects of toxins on rat brain in vivo were studied using male Sprague-Dawley rats implanted intracerebroventricularly in the lateral ventricle with guide cannulae, according to Kofman et al.(1993). Toxins were dissolved in artificial cerebrospinal fluid and introduced via the guide cannulae. Correct placement of cannulae was verified by injections of Giemsa stain immediately following the experiments, after decapitation of the animals.


RESULTS

Our primary aim was to identify a small peptide ligand that might act as an agonist at the -conotoxin receptor site in rat brain. Preliminary assays on piscivorous Conus venoms revealed that the venom of C. nigropunctatus contained a <4-kDa fraction that displaced TxVIA from its binding sites on both rat brain synaptosomes and molluscan central nervous system. Therefore this venom was chosen for further fractionation. Fractions were assayed in parallel for toxicity to fish and inhibition of TxVIA binding in both rat and mollusc neuronal membrane preparations.

Purification, Characterization, and Toxicity of Conotoxin NgVIA

The major peptide peak obtained in the initial fractionation of C. nigropunctatus venom on Sephadex G-50 (Fig. 1A) was refractionated by reverse phase HPLC on a Vydac C18 column as shown in Fig. 1B. The active fraction indicated by the arrow was purified on a Vydac phenyl column (Fig. 1C). Homogeneity of the purified toxin was indicated by complete identity of the UV spectra along the elution profile of the final active peak (Fig. 1C, inset). The homogeneity of this peptide, designated conotoxin NgVIA, was confirmed by Edman sequencing and amino acid analysis (see below). Two attempts to obtain a clear mass measurement of the peptide by electrospray-mass spectrometry failed, perhaps due to low ionization efficiencies on the extremely hydrophobic sample.

The amino acid sequence of the toxin was determined by gas-phase automated Edman sequencing after reduction and pyridylethylation. A single unambiguous sequence of 31 amino acid residues was obtained in two separate runs (Table 1) and was in good correlation with the amino acid composition analysis of the toxin (Table 2). Interestingly, the amino acid sequence of the new toxin included a cysteine framework identical to that of TxVIA, with identical numbers of residues in the intercysteine loops (Fig. 2). Two other residues are identical, and there are a number of similarities in the positioning of hydrophobic residues, although the overall net charges of the peptides are contrasting (Fig. 2).






Figure 2: Sequence comparison of conotoxins that inhibit sodium channel inactivation. Identical residues in all four toxins are shown in bold type and are boxed. The standard one-letter code for amino acid residues (except O = trans-4-hydroxyproline) is used. Spacers (bullet) are inserted to show maximal homology. , net hydrophobicity calculated according to Fauchere et al.(1988). References for sequences: NgVIA, this paper; GmVIA, Shon et al.(1994); TxVIA/B, Fainzilber et al.(1991).



As we were unable to verify the amino acid sequence data by mass spectrometry, the peptide was synthesized with free C-terminal and folded as described under ``Experimental Procedures.'' As expected from such a hydrophobic sequence the final yield of active (i.e. correctly folded) peptide was low, averaging 3 nmol of active toxin/10 mg of crude synthetic peptide (folding efficiencies ranged from 0.1 to 0.5%). The final purified product co-eluted with native NgVIA in reverse phase HPLC (Fig. 1D) and had the same activity as native toxin in electrophysiological tests and in vivo assays in rat brain (see below).

The paralytic activity of NgVIA was examined in bioassays on fish (Gambusia), snails (Patella), and fly larvae (Sarcophaga). Although the toxin has potent paralytic activity on fish (ED = 2.8 pmol/100 mg of body weight) and snails (ED = 14.5 pmol/100 mg), there were no observable effects on fly larvae at doses of up to 250 pmol/100 mg.

Conotoxin NgVIA Effects on Sodium Current in Molluscan Neurons

We examined the effects of NgVIA in a molluscan neuronal system similar to that used to define the electrophysiological effects of TxVIA (Hasson et al., 1993). For this purpose caudodorsal neurons of the snail L. stagnalis were used in the whole cell voltage clamp mode. Characteristics of the sodium currents in these cells have been described by Brussaard et al.(1991). We first verified the effects of TxVIA in this system. As can be seen in Fig. 3A, application of TxVIA (1.2 µM) immediately inhibited sodium current inactivation, resulting in a marked slowing of the current decay. A slight increase in the peak sodium current was also observed. The effect was rapidly reversible, with the cell recovering its former sodium current kinetics within 1 min of the wash. Application of NgVIA (1 µM) in this system also caused a strong inhibition of sodium current inactivation, which was accompanied by an increase in the peak sodium current (Fig. 3B). Despite the more dramatic effects of NgVIA, the neuron recovered rapidly upon wash, as seen with TxVIA. Application of synthetic NgVIA (0.8 µM) produced effects similar to those observed with venom-derived toxin (Fig. 3C).


Figure 3: Inhibition of voltage-dependent sodium current inactivation by TxVIA and NgVIA. Sodium currents at 10 mV were recorded from caudodorsal neurons of the snail L. stagnalis in the whole cell voltage clamp mode. The effects of 1.2 µM TxVIA (A), 1 µM venom-derived NgVIA (B), and 0.8 µM synthetic NgVIA (C) are shown. Left panels are control currents, middle panels show currents recorded in the presence of the toxin, and right panels show currents recorded after 1 min of wash out of the toxin. The data shown are representative results from a number of different cells. Capacitive transients were clipped in the illustrations. Calibration bars are 50 ms and 0.5 nA.



Effects of Conotoxin NgVIA on TxVIA Binding and on Sodium Flux

In order to quantify the effects of NgVIA on binding of TxVIA, we performed competitive binding experiments on rat brain synaptosomes and molluscan central nervous system membranes. Fig. 4A shows that NgVIA is able to inhibit TxVIA binding in a dose-dependent manner in both rat and mollusc preparations. As binding of TxVIA is not dependent on membrane potential (Fainzilber et al., 1994), in contrast to the other toxins that inhibit sodium current inactivation, it was of interest to determine whether NgVIA competition with TxVIA binding was affected by membrane potential. The IC of NgVIA in lysed rat brain synaptosomes is 4-fold higher than the IC in intact synaptosomes (Fig. 4A), strongly suggesting that NgVIA binding is dependent on membrane polarization.


Figure 4: Pharmacology of NgVIA in rat brain and snail central nervous system membrane preparations. A, inhibition of I-TxVIA binding by NgVIA. Neuronal membranes were incubated with 0.2 nMI-TxVIA and increasing concentrations of NgVIA. The amount of I-TxVIA bound at each data point is expressed as a percentage of the maximal specific binding in the system. Circles,Helix central nervous system (IC = 59.4 ± 11.4 nM), full triangles, rat brain synaptosomes (IC = 4.9 ± 1.2 nM, empty triangles, lysed rat brain synaptosomes (IC = 18.7 ± 4.4 nM). B, NgVIA and TxVIA effects on Na influx in rat brain synaptosomes. Synaptosomes were incubated with toxins as described under ``Experimental Procedures,'' and net influx of Na after 30 s was determined. Enhancement of veratridine (Ver)-induced flux examined at 2 µM veratridine and 1 µM TxVIA or NgVIA. Results are shown as a percentage of the control flux induced by 2 µM veratridine (1.2 0.3 nmol of sodium/min/mg of protein). The maximal flux obtainable is shown by the rightmost bar (effect of 200 µM veratridine).



The effects of NgVIA on rat brain sodium channels in vitro were examined by Na influx assays in rat brain synaptosomes. NgVIA alone was not able to initiate sodium influx (Fig. 4B), in common with other inactivation-inhibiting toxins (Catterall and Beress, 1978; Fainzilber et al., 1994). However, NgVIA synergically increased the veratridine-stimulated uptake of Na to approximately 3-fold above control levels (Fig. 4B). This is similar to the effect previously obtained with CsTx in this system (Fainzilber et al., 1994).

Effects of Conotoxin NgVIA in Rat Brain

We had previously characterized the effects of CsTx and TxVIA in rat brain in vivo by intracerebroventricular injections. In contrast to CsTx, TxVIA induces no toxic effects in rat brain (Fainzilber et al., 1994). It was therefore of interest to study the effects of NgVIA in this system and to examine its interactions with the silently binding TxVIA. Rats implanted with guide cannulae were injected intracranially with toxins dissolved in artificial cerebrospinal fluid. Behavior and activity of the toxin-injected rats were observed for up to 1 h postinjection. The results summarized in Fig. 5show that NgVIA caused toxic symptoms peaking with paralysis and seizures. Severity of the effects is dose-dependent, leading to death within 10 min at doses of 100 pmol/rat and above (Fig. 5A). As shown previously, TxVIA alone had no effect on the rats even when injected at a dose of 30 nmol/rat (Fainzilber et al., 1994). Upon simultaneous injection of the two toxins, 20 nmol of TxVIA were not able to completely mitigate the toxic effects of 40 pmol of NgVIA (Fig. 5B) in contrast to previous results with CsTx (Fainzilber et al., 1994). However, when TxVIA was injected 20 min before administering 40 pmol of NgVIA the toxic symptoms were clearly delayed and reduced to less than those shown after administration of 20 pmol of NgVIA (Fig. 5B). This result indicates that TxVIA acts as a partial antagonist to NgVIA in rat brain.


Figure 5: Effects of NgVIA in rat brain in vivo. A, rats were injected intracranially with NgVIA, and their reactions were followed for up to 20 min postinjection. Circles, 100 pmol of NgVIA (n = 2); triangles, 40 pmol of NgVIA (n = 3); squares, 20 pmol of NgVIA (n = 3). B, upon simultaneous injection of 40 pmol of NgVIA with 20 nmol of TxVIA (full triangles, n = 3), the full repertoire of toxic symptoms was seen without very marked change relative to the control of 40 pmol of NgVIA alone (empty triangles). However, when 20 nmol of TxVIA were injected 20 min before administration of 40 pmol of NgVIA (full diamonds, n = 4), the toxic effects were clearly delayed and reduced.



Application of lethal doses of synthetic NgVIA to rats revealed a similar progression of symptoms to that seen with the venom-derived toxin. The rats underwent rapid paralysis and commenced seizures, with death occurring within 10 min. A sublethal dose caused transient hyperactivity and pronounced shaking movements, as seen previously with native NgVIA.


DISCUSSION

In the present study we have identified and characterized a novel peptide conotoxin that affects sodium channel inactivation. As will be detailed below this toxin provides an important complement of the pharmacological tools required for understanding the functional role of different receptor sites in gating mechanisms of sodium channels.

Binding and Pharmacology of Conotoxin NgVIA

The available evidence strongly suggests that conotoxin NgVIA binds at a receptor site distinct from that of TxVIA. Although the interaction of NgVIA with the TxVIA receptor site is obvious on the basis of its competition with TxVIA in two neuronal membrane preparations (Fig. 4A) and the partial antagonism of its toxic effects by TxVIA in rat brain (Fig. 5), the following lines of evidence suggest that the receptor site of NgVIA must be distinct from that of TxVIA. 1) Its inhibition of TxVIA binding is partially dependent on membrane potential in rat brain synaptosomes (Fig. 4A), whereas binding of TxVIA is not voltage-dependent (Fainzilber et al., 1994). 2) It exhibits positive synergism with veratridine in sodium flux assays (Fig. 4B) in contrast to TxVIA (Fig. 4B and Fainzilber et al.(1994)). 3) Its antagonism by TxVIA in rat brain in vivo is relatively weak and requires a significant time advantage for the antagonist (Fig. 5).

There are also qualitative differences in the effects of the two toxins on the sodium current, for example the increase of peak sodium current by NgVIA (Fig. 3). The partial antagonistic effects of TxVIA versus NgVIA might be explained by postulating a higher efficacy of NgVIA activity and/or differences in their binding kinetics. Another possibility is that NgVIA identifies an additional minor population of sodium channels in rat brain, which is not recognized by TxVIA. The lower capacity of TxVIA receptors in rat brain synaptosomes (Fainzilber et al., 1994) as compared with saxitoxin receptors (Ray et al., 1978) is consistent with this possibility. These aspects should be examined in the future when labeled analogs of NgVIA become available.

Binding of NgVIA appears to be at least partially voltage-dependent (Fig. 4A) as is that of CsTx (Gonoi et al., 1987) and the other toxins that inhibit sodium current inactivation. Depolarization of the membrane by lysis of synaptosomes causes a 4-fold increase in the IC for inhibition of I-TxVIA binding by NgVIA. This is comparable with the 5-fold increase in K(0.5) for sea anemone toxin II action in depolarized neuroblastoma cells (Catterall and Beress, 1978).

The similarity in binding characteristics and interactions of NgVIA with TxVIA in vitro and in vivo to those reported for CsTx (Fainzilber et al., 1994), both qualitatively and quantitatively, suggests that they may bind to closely related sites (see below and Fig. 6). Thus the IC values of both toxins on both rat and mollusc neuronal preparations are similar, as is their synergic effect with veratridine on sodium flux. It is tempting to suggest that NgVIA may have some attachment points in common with those of CsTx; however, the differences between them as regards antagonism in vivo by TxVIA ( Fig. 5and Fainzilber et al.(1994)) suggest that their receptor sites are probably not identical. These differences might also be attributable to different kinetics of binding or efficacy in action of these two toxins. It will be of interest to study these possibilities in the future by direct binding experiments with NgVIA.


Figure 6: A model visualizing locations of peptide neurotoxins affecting sodium current inactivation, bound to their putative receptor sites. The sodium channel extracellular surface is shown from above, with the four homologous repeat domains represented by shaded outlines (I-IV). A, summary of the binding inhibition among the different peptide neurotoxins. The arrows are approximately proportional to the inhibition caused by each toxin on the binding of radiolabeled alpha-scorpion toxin (alphaScTx) or TxVIA in rat brain synaptosomes. B, illustration of the peptide toxins bound at their putative receptor sites. The receptor site for the sodium channel blocker tetrodotoxin (TTX) has been localized to the extracellular vestibule of the ion-conducting pore (Terlau et al., 1991) and is indicated in the center of the model to facilitate orientation. See text for explanations. ATXII, sea anemone toxin II.



Structures of Conotoxins Affecting Sodium Channel Inactivation

To date four different conotoxins affecting sodium channel inactivation have been described: NgVIA (this work), conotoxin GmVIA from Conus gloriamaris (Shon et al., 1994), and the isotoxins TxVIA/B (Fainzilber et al., 1991; Hillyard et al., 1989). The primary structures of these toxins are compared in Fig. 2, and it is immediately apparent that the cysteine framework and overall hydrophobicity of these peptides are conserved. Apart from this the sequences are extremely variable, and the only absolutely conserved residue besides the cysteines is the glycine in the center of the first loop (Fig. 2). A glycine residue in the same position was found to be crucial for maintaining the solution structure of -conotoxin GVIA (Skalicky et al., 1993; Pallaghy et al., 1993). As Shon et al.(1994) have recently shown that the disulfide bond arrangement of the - and -conotoxins is identical, it is possible that the conserved glycine is necessary to stabilize conformations also in the -conotoxin group.

NgVIA is unique in the group of toxins shown in Fig. 2in that it is the only one so far found with significant activity in vertebrates. Indeed it is more potent than CsTx in rat brain by 1 order of magnitude (compare Fig. 5with Fainzilber et al.(1994)). The potent paralytic activity of NgVIA on rats and molluscs and its effects on sodium currents in rat brain synaptosomes and Lymnaea neurons indicate that NgVIA is an agonist of both mammalian and molluscan sodium channels. However, NgVIA cannot be considered a wide range cross-phyletic agonist since it has no discernible paralytic activity on insects. It seems that changes in the composition of the intercysteine loops on a conserved -conotoxin framework may cause significant differences in activity on one variant of sodium channels (i.e. in rat brain), while relatively little change is observed for another variant (i.e. in mollusc central nervous system). NgVIA should be a valuable reference for structure-function analyses of this group of toxins, since it enables a rational analysis of the structural elements necessary for agonist activity of these toxins on the rat brain sodium channel. However, the primary structure variability in this group (Fig. 2) suggests that three-dimensional structural data will be necessary to tackle this question.

A Model for Localization of Receptor Sites of Peptide Neurotoxins That Inhibit Sodium Current Inactivation

On the background of the available pharmacological information we propose a model that may illustrate the relative locations of the receptor sites for different peptide toxins affecting sodium current inactivation on the extracellular domains of a sodium channel. A graphic representation of the model is shown in Fig. 6, where the arrows (Fig. 6A) represent binding inhibitions between the different toxins (Ray et al., 1978; Catterall and Beress, 1978; Gonoi et al., 1986, 1987; Fainzilber et al., 1994; and this paper). As the receptor site for alpha-scorpion toxin has been partially localized using site-directed antibodies (Thomsen and Catterall, 1989) and found to include attachment points on the extracellular loops of domains I and IV of the channel, it serves as an ``anchor'' for the model. alpha-Scorpion toxin and sea anemone toxin II share the same macrosite and may occupy a similar space (Loret et al., 1994); thus they are shown in the same region on the model.

For clarity of presentation, we have placed the toxins that do not bind to macrosite 3 (CsTx, GPT, TxVIA) in different domains of the channel. GPT competes with both alpha-scorpion toxin (Gonoi et al., 1986) and TxVIA (Fainzilber et al., 1994) but at concentrations 25-fold higher than its K(0.5) on mammalian neurons, suggesting that it binds to a distinct site. Therefore GPT is positioned in proximity to both to allow steric interference that may cause the competition between them. CsTx, in contrast, competes at very low concentrations with TxVIA and is completely antagonized by TxVIA in rat brain (Fainzilber et al., 1994). Therefore the receptor sites of CsTx and TxVIA are proposed to partially overlap (Fig. 6B) but are not identical since these two toxins reveal opposite allosteric interactions with alkaloid toxins bound at site 2, such as batrachotoxin and veratridine (Gonoi et al., 1987; Fainzilber et al., 1994).

The partial protection observed by administration of TxVIA prior to NgVIA in rat brain in vivo (Fig. 5) is in contrast to the complete protection or antagonism observed by simultaneous injection of CsTx and TxVIA (Fainzilber et al., 1994). This suggests that the NgVIA and TxVIA receptor sites are partially overlapping, similar to CsTx and TxVIA, but may share different points of attachment (Fig. 6B). It should be noted, however, that steric interference (with no overlap) cannot be excluded (Gordon et al., 1992; Moskowitz et al., 1994). The interaction of NgVIA with the TxVIA receptor site is very similar to that of CsTx (see above). Moreover, the toxic effects of NgVIA are at least partially antagonized by the presence of TxVIA (Fig. 5). On this basis we suggest that the receptor site of NgVIA is partially overlapping with both the CsTx and TxVIA receptor sites (Fig. 6B). The similarity in the binding properties (dependence on membrane polarization, competition with TxVIA, and interaction with veratridine) and activity in vivo between NgVIA and CsTx suggest substantial overlap in their receptor sites.

The model presented in Fig. 6gives a graphic visualization of the different peptide toxins bound to their putative receptor sites on the outer surface of sodium channels while emphasizing the lack of structural information on the molecular level on these receptor sites. All -conotoxins inhibit sodium channel inactivation and as exemplified by NgVIA and TxVIA may do so via binding at distinct receptor sites. Localization of the attachment points comprising these receptor sites may shed light on the mechanism of action of toxins that modify sodium channel gating. The use of TxVIA and NgVIA as pharmacological sensors for minor differences in sodium channel variants provides a rational approach to this complex problem and may contribute to the elucidation of structure-function relationships in sodium channels.


FOOTNOTES

*
This work was supported by a research grant from the Smith Family Laboratory for Psychobiology (to O. K. and M. F.) and by Grant 93-00294 from the U.S.A.-Israel Binational Science Foundation (to E. Z. and D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Molecular Neurobiology, Vrije University, De Boelelaan 1087, 1081 HV Amersterdam, The Netherlands. Fax: 31-20-4447123; mike{at}bio.vu.nl.

(^1)
The abbreviations used are: CsTx, Conus striatus toxin; NgVIA, conotoxin NgVIA from C. nigropunctatus; TxVIA, -conotoxin TxVIA from C. textile; GPT, Goniopora coral toxin; HPLC, high performance liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl.


ACKNOWLEDGEMENTS

Our sincere thanks to Dan Corcos and Solly Singer for help in Conus collection, to Dr. Ariel Gaathon (Bletterman Laboratory for Macromolecular Research, Hebrew University) for peptide sequencing, and to Fini Silfen (Interdepartment Equipment Unit, Hebrew University) for amino acid composition analyses. We also thank Prof. Baldomero Olivera (University of Utah) for sharing data with us prior to publication.


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