INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

The venom from Australian funnel-web spiders (Araneae: Hexathelidae: Atracinae) appears to contain a variety of neurotoxins that target various ionic channels in excitable cells, including calcium (1) and potassium channels (2). The toxins so far identified to be responsible for severe envenomation or lethality in humans, however, were shown to be two peptide neurotoxins that target the voltage-gated sodium channel: -atracotoxin-Ar1¹ (formerly robustoxin) from Atrax robustus and -atracotoxin-Hv1 (formerly versutoxin) from Hadronyche versuta (3, 4). -Atracotoxin-Hv1 and -atracotoxin-Ar1 have been shown to exert their neurotoxicity by slowing or removing tetrodotoxin-sensitive sodium current inactivation in rat dorsal root ganglion neurons (5, 6), an action similar to that of polypeptide scorpion -toxins and sea anemone toxins (7).

Both -atracotoxin-Ar1 and -atracotoxin-Hv1 consist of 42 amino acids and are highly homologous with only 7 substitutions. These toxins show no significant sequence homology with any presently known neurotoxins. Both spider toxins have a high proportion of basic residues and appear to be tightly folded molecules containing four conserved disulfide bonds, including N- and C-terminal cysteines. Recently, the three-dimensional solution structure for -atracotoxin-Hv1 (8) and -atracotoxin-Ar1 (9) has been determined. Both display a small three-stranded anti-parallel -sheet with an "inhibitor cystine knot" motif (10). Interestingly the three-dimensional fold of these toxins is different from the previously determined structures of the scorpion -toxins AaH II (11) and LqhIT (12) despite similar actions on sodium current inactivation (13, 14).

Out of the seven identified neurotoxin receptor sites on the voltage-gated sodium channel, determined by direct radiolabeled toxin studies, at least two receptor sites were shown to bind various peptide toxins from different animal venoms that inhibit sodium current inactivation. Scorpion -toxins and sea anemone toxins such as ATX II bind to the so-called neurotoxin receptor site 3 and -conotoxins which bind to receptor site 6 on the -subunit of the sodium channel (Refs. 15 and 16; for a review see Refs. 7 and 17). Both of these receptor sites have been shown to have complex allosteric interactions with neurotoxin receptor site 2 shown to bind several alkaloid toxins such as batrachotoxin and veratridine (17). The alkaloid toxins induce persistent activation of sodium channels by shifting the voltage dependence of activation to very negative membrane potentials and inhibiting the inactivation process (18, 19). Scorpion -toxins were shown to cooperatively enhance the effect of alkaloid toxins by increasing their binding affinity and activity (20, 21).

Based on apparent similarity in the effect of scorpion -toxins and -atracotoxins, the aim of the present study was to determine the likely receptor binding site of funnel-web spider toxins on the sodium channel and their possible interaction with alkaloid toxin binding and action using both ²²Na⁺ uptake and radiolabeled neurotoxin binding assays. We report that -atracotoxins interact with nanomolar affinities with neurotoxin receptor site 3 in rat brain synaptosomes but exhibit unusual allosteric interactions with the site 2 alkaloid toxin receptor.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Purification of Toxins-- Colonies of male A. robustus and female H. versuta spiders were "milked" by direct aspiration from the chelicerae of live spiders using glass pipettes. Collected crude venom was flushed from pipettes using 0.1% (v/v) trifluoroacetic acid and initial fractionation performed via reverse-phase high performance liquid chromatography (HPLC) using a Vydac semi-preparative column (C₁₈, 10.4 × 250 mm, 15 µm) on a Waters HPLC system. Elution of venom components was performed using a linear gradient of 0-60% acetonitrile, 0.1% trifluoroacetic acid over 36 min with a flow rate of 4 ml/min. The fraction containing -atracotoxin-Ar1 eluted with approximately 43% acetonitrile, whereas -atracotoxin-Hv1 eluted with 40% acetonitrile. These fractions were lyophilized and then resuspended in 25% acetonitrile, 0.1% trifluoroacetic acid in preparation for further purification on a Vydac analytical column (C₁₈, 4.6 × 250 mm, 5 µm). The funnel-web spider toxins were eluted from the analytical column using a linear gradient of 25-50% acetonitrile, 0.1% trifluoroacetic acid over 30 min at a flow rate of 1 ml/min. Purity of eluted toxins was confirmed by SDS-polyacrylamide gel electrophoresis under reducing and alkylating conditions with a Tris/Tricine buffer system. Protein concentration was determined using amino acid composition analysis. Toxins were stored lyophilized at 20 °C until required, at which time they were dissolved in 20 mM HEPES-Tris (pH 6.0, 4 °C).

Other Chemicals-- AaH II and Lqh II were purified according to the methods of Miranda et al. (22) and Sautière et al. (23) respectively. Batrachotoxin was a generous gift from Dr. John Daly (Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda). Lqh II (catalogue number LTX001) was purchased from Latoxan (AP-1724, 05150 Rosans, France). Tetrodotoxin was purchased from Calbiochem (Alexandria, New South Wales, Australia), and veratridine was obtained from Sigma. [³H]BTX and carrier-free ²²Na⁺ were purchased from NEN Life Science Products. All other chemicals were of analytical or HPLC grade.

Electrophysiological Recordings-- Whole cell patch clamp recordings of tetrodotoxin-sensitive sodium currents were made from both dorsal root ganglion and periaqueductal gray neurons. Acutely dissociated dorsal ganglion neurons were prepared from 4- to 12-day-old Wistar rats according to the methods of Nicholson et al. (5, 6). Micropipettes (0.67-2 megohms) were pulled from borosilicate glass capillary tubing and were filled with (in mM) CsF 135, NaCl 10, HEPES 5 with the pH adjusted to 7.0 with 1 M CsOH. The external solution contained (in mM) NaCl 30, MgCl₂ 1, CaCl₂ 1.8, CsCl 5, KCl 5, D-glucose 25, HEPES 5, tetraethylammonium (TEA) chloride 20, tetramethylammonium chloride 70, with the pH adjusted to 7.4 with 1 M TEA hydroxide. Membrane current recordings were made at ambient room temperature (22-25 °C). Large round light dorsal root ganglion cells with diameters of 20-40 µm were selected for experiments.

Neuronal Membrane Preparations-- Synaptosomes for ²²Na⁺ uptake and [³H]BTX binding assays were prepared from brains of male Wistar rats (4-8 weeks, 250-350 g) using a combination of homogenization and differential and density gradient centrifugation according to the method of Gray and Whittaker (26) as described by Tamkun and Catterall (27). Synaptosomes were suspended in a solution consisting of (in mM) choline chloride 130, KCl 5.4, MgSO₄ 0.8, D-glucose 5.5, and HEPES-Tris 50 (pH 7.4, 37 °C) and were stored in liquid nitrogen until required for ²²Na⁺ uptake assays, or used within 3 h of preparation for [³H]BTX binding assays. For ¹²⁵I-Lqh II binding studies, rat brain synaptosomes were prepared according to the method of Kanner (28) using (in mM) D-mannitol 300, EDTA 10, HEPES 10 (pH 7.4, 4 °C), and Ficoll (type 400 Sigma) gradients (14). A combination of proteinase inhibitors consisting of phenylmethylsulfonyl fluoride (50 µg/ml), pepstatin A (1 µM), iodoacetamide (1 mM), and 1,10-phenanthroline (1 mM) was present in all buffers used for this procedure. Synaptosomes were frozen at 80 °C in mannitol buffer until use. Membrane protein concentration was determined using a Bio-Rad protein assay with BSA as a standard.

Measurement of ²²Na⁺ Uptake-- The effect of -atracotoxins on ²²Na⁺ uptake in rat brain synaptosomes was measured using the method described by Tamkun and Catterall (27). Briefly, rat brain synaptosomes (500 µg of membrane protein) were preincubated for 10 min with toxins in 100 µl of sodium-free preincubation medium at 37 °C. The preincubation ²²Na⁺ uptake medium contained (in mM) choline chloride 130, KCl 5.4, MgSO₄ 0.8, glucose 5.5, HEPES-Tris (pH 7.4) 50, and 1 mg/ml BSA. Uptake was initiated by adding 150 µl of assay medium containing (in mM) choline chloride 128, KCl 5.4, MgSO₄ 0.8, glucose 5.5, HEPES-Tris (pH 7.4) 50, NaCl 2.66, ouabain 5, 1 mg/ml BSA, and 0.9 µCi/ml carrier-free ²²NaCl. After 5 s at 37 °C, uptake was terminated by the addition of 4 ml of ice-cold wash solution and rapid filtration through 0.45-µm nitrocellulose membrane filters (Millipore, Sydney, Australia). The wash solution consisted of (mM) choline chloride 163, CaCl₂ 1.8, MgSO₄ 0.8, HEPES-Tris (pH 7.4) 5, and 1 mg/ml BSA. Maximal ²²Na⁺ uptake was determined in the presence of 1 µM batrachotoxin, whereas uptake not mediated by the voltage-gated sodium channel was determined in the presence of 1 µM TTX. Nonspecific ²²Na⁺ uptake was typically less than 35% of total uptake.

Binding Assays-- Equilibrium competition and saturation assays were performed using increasing concentrations of the unlabeled toxin in the presence of a constant low concentration of the radiolabeled toxin. In order to obtain saturation curves ("cold" saturation), the specific radioactivity and the amount of bound toxin were calculated and determined for each toxin concentration. Equilibrium saturation or competition experiments were analyzed by the iterative computer program LIGAND, using "Cold saturation" and "Drug" analysis, respectively (Elsevier Biosoft, UK). The composition of the binding medium for ¹²⁵I-Lqh II binding was (in mM) choline Cl 140, CaCl₂ 1.8, KCl 5.4, MgSO₄ 0.8, HEPES 25 (pH 7.4); D-glucose 10, BSA 2 mg/ml. Wash buffer composition was (in mM) choline Cl 140, CaCl₂ 1.8, KCl 5.4, MgSO₄ 0.8, HEPES 25 (pH 7.4), BSA 5 mg/ml. [³H]BTX binding experiments used the preincubation medium and wash solution as for ²²Na⁺ uptake experiments (see above). After incubation, reactions were terminated by dilution with 4 ml of ice-cold wash solution for [³H]BTX binding experiments or 2 ml of ice-cold wash solution for ¹²⁵I-Lqh II binding experiments. Separation of free from bound toxin was achieved by rapid filtration under vacuum using Whatman GF/C filters. The filter discs were washed with a further 2 × 4 ml of wash solution for [³H]BTX binding or 2 × 2 ml washes for ¹²⁵I-Lqh II binding.

[³H]BTX Binding Experiments-- Experiments were performed according to a modification of the method described by Catterall and colleagues (29). Rat brain synaptosomes (350 µg of protein/ml) were suspended in 0.2 ml of buffer containing 15 nM [³H]BTX (0.8 µCi). After incubation for 50 min at 37 °C, the reaction mixture was diluted with 4 ml of ice-cold wash buffer and filtered. Nonspecific binding was determined in the presence of 300 µM veratridine and was typically 5-15% of total binding. Equilibrium saturation experiments were analyzed using EBDA and LIGAND computer programs (Elsevier Biosoft, UK).

¹²⁵I-Lqh II Binding Assays-- Lqh II was iodinated by IODO-GEN (Pierce) using 5 µg of toxin and 0.5 mCi of carrier-free Na¹²⁵I according to Gordon and Zlotkin (30). Competition binding experiments using ¹²⁵I-Lqh II were performed according to the methods of Gordon et al. (31). The monoiodotoxin was purified via reverse-phase HPLC using a Vydac C₁₈ column and a gradient of 5-90% B (A = 0.1% trifluoroacetic acid, B = acetonitrile, 0.1% trifluoroacetic acid) at a flow rate of 1 ml/min, as described by Cestèle et al. (32). The concentration of the radiolabeled toxins was determined according to the specific activity of the ¹²⁵I corresponding to 4200-3450 dpm/fmol monoiodotoxin, depending on the time of use (the age of the iodine). Rat brain synaptosomes (40-50 µg protein/ml) were suspended in 0.2 ml of binding buffer, containing ¹²⁵I-Lqh II. After incubation for 15 min at 22 °C, the reaction mixture was diluted with 2 ml of ice-cold wash buffer and filtered. Nonspecific toxin binding was determined in the presence of 100 nM unlabeled Lqh II and consisted typically of 5-10% of total binding.

Data Analysis-- The IC₅₀ values obtained for inhibition of ¹²⁵I-Lqh II binding were converted to K_i values according to the relationship described by Cheng and Prusoff (33). Equilibrium saturation of [³H]BTX binding data was fitted according to the following single-site hyperbolic equation mathematically equivalent to the Langmuir isotherm.

(Eq. 1)

where B_max is the maximal number of moles of [³H]BTX bound, K_d is the dissociation constant for [³H]BTX, and x is the total concentration of batrachotoxin.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Effect of -Atracotoxins on Sodium Currents-- Electrophysiological studies using whole cell patch clamp of dorsal root ganglion neurones have shown that -atracotoxins slow sodium current inactivation and shift the voltage dependence of activation in a manner similar to scorpion -toxins (5, 6). As shown in Fig. 1, A and B, 30 nM -atracotoxin-Hv1 slows TTX-sensitive sodium current inactivation in both dorsal root ganglion neurons and periaqueductal gray neurons from rat brain. In addition, 30 nM -atracotoxin-Hv1 shifts the threshold of sodium channel activation by approximately 10 mV to more negative membrane potentials (Fig. 1, C and D). These actions are similar to those reported for scorpion -toxins (13, 34). However, -atracotoxin-Hv1 caused differential actions on peak sodium current amplitude in these two populations of neurons. In dorsal root ganglion neurons -atracotoxin-Hv1 caused a marked reduction in peak sodium current (Fig. 1A), whereas in periaqueductal gray neurons there was no significant change in peak sodium current (Fig. 1B). This is in contrast to scorpion -toxins that typically increase peak sodium current (5, 6, 13, 35, 36). Given these similar actions on sodium currents, we further compared the actions of -atracotoxins with those of scorpion -toxins to produce positive cooperativity with [³H]BTX binding, enhancement of alkaloid toxin-stimulated ²²Na⁺ uptake, and competition in radioiodinated scorpion toxin binding assays.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Actions of -atracotoxin-Hv1 on whole cell tetrodotoxin-sensitive sodium currents in rat dorsal root ganglion and periaqueductal gray neurones. Upper panels show the slowing of sodium current inactivation induced by -atracotoxin-Hv1 in dorsal root ganglion neurons (A) and periaqueductal gray neurons from rat brain (B); a represents the control trace, and b shows the current recorded in the presence of 30 nM -atracotoxin-Hv1. A, currents were activated by depolarizing test pulses from 80 mV to 10 mV for 50 ms every 10 s. B, currents were activated by depolarizing test pulses from 90 mV to 0 mV for 6 ms every 10 s. C and D show the I/V relationships recorded before () and after () 30 nM -atracotoxin-Hv1 from the same neurons as in A and B, respectively. Note the negative shift in the voltage dependence of activation in the presence of -atracotoxin-Hv1.

Enhancement of [³H]BTX Binding by -Atracotoxin-Hv1-- Scorpion -toxins have been shown to act in a positive allosteric fashion to increase the binding of site 2 alkaloid toxins such as batrachotoxin (27, 29). We therefore assessed the ability of -atracotoxins to enhance [³H]BTX binding to rat brain synaptosomes. -Atracotoxin-Hv1 was found to significantly enhance the binding of [³H]BTX approximately 12-fold with a concentration required for half-maximal enhancement (EC₅₀) of 25.2 ± 4.5 nM (n = 4, Fig. 2A). In comparison, AaH II-enhanced [³H]BTX binding under the same conditions gave a 5.6-fold increase in the maximal response (E_max) with an EC₅₀ of 14.4 ± 2.3 nM (Fig. 2A, inset). In order to show that the enhancement of [³H]BTX binding represents an increase in binding to neurotoxin receptor site 2, -atracotoxin-Hv1-enhanced [³H]BTX binding was measured in the presence of increasing concentrations of unlabeled batrachotoxin or veratridine. Both alkaloid toxins were able to completely inhibit [³H]BTX binding enhanced by 3 µM -atracotoxin-Hv1 in a concentration-dependent manner indicating the enhancement is specifically mediated through receptor site 2 (Fig. 2B).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Enhancement of [3H]BTX binding by -atracotoxin-Hv1 in rat brain synaptosomes. A, enhancement of [3H]BTX binding by increasing concentrations of -atracotoxin-Hv1. Rat brain synaptosomes (350 µg of protein) were preincubated with 15 nM [3H]BTX for 50 min at 37 °C in the presence of increasing concentrations of -atracotoxin-Hv1 (main panel) or AaH II (inset). The data points represent the mean ± S.E. of four experiments (see "Experimental Procedures" for details). B, inhibition of -atracotoxin-Hv1 enhanced [3H]BTX binding by veratridine and batrachotoxin. Rat brain synaptosomes were incubated with 15 nM [3H]BTX and 3 µM -atracotoxin-Hv1 in the presence of a range of concentrations of batrachotoxin () and veratridine () under the same conditions as in A. Data are presented as a percentage of maximal [3H]BTX binding in the presence of 3 µM -atracotoxin-Hv1. Values represent the mean ± S.E. of three experiments for both veratridine and batrachotoxin. B, inset, lack of enhancement of -atracotoxin-Hv1-enhanced [3H]BTX binding by AaH II. Increasing concentrations of AaH II were incubated with 15 nM [3H]BTX in the absence (gray columns) and presence (black columns) of a saturating concentration of -atracotoxin-Hv1 (10 µM) under the same conditions as A. Values represent the mean ± S.E. of three experiments.

View this table: [in this window] [in a new window]   Table I Effect of AaH II and -atracotoxin-Hv1 on [3H]BTX binding Rat brain synaptosomes were incubated with 15 nM [3H]BTX and increasing concentrations of unlabeled batrachotoxin in the presence of the indicated concentrations of AaH II or -atracotoxin-Hv1 as described under "Experimental Procedures." Data have been presented as the means ± S.E. (n = 3) as determined from nonlinear regression (see Equation 1 under "Experimental Procedures"). Statistical analysis using a one-way analysis of variance revealed no significant differences between any of the data.

Activation of ²²Na⁺ Uptake by -Atracotoxins-- To determine whether -atracotoxins can directly activate voltage-gated sodium channels, their ability to increase ²²Na⁺ uptake by rat brain synaptosomes was examined. Both -atracotoxins were able to activate ²²Na⁺ uptake in a concentration-dependent manner to 30 and 28% of maximum, with concentrations for half-maximal activation (EC₅₀) of 25 ± 8 and 44 ± 18 nM for -atracotoxin-Hv1 and -atracotoxin-Ar1, respectively (Fig. 3). Maximal ²²Na⁺ uptake was determined in the presence of 1 µM BTX, and the direct stimulation of ²²Na⁺ uptake by -atracotoxins was expressed as a percentage of maximal flux. Moreover the -atracotoxin-activated ²²Na⁺ uptake was TTX-sensitive (Fig. 3B, inset) confirming that it was mediated via the voltage-gated sodium channel. Similarly, the scorpion -toxin AaH II was also able to directly activate ²²Na⁺ uptake to 14 ± 3% at a concentration of 30 nM (Fig. 3A, inset).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Activation of 22Na+ uptake by funnel-web spider toxins in rat brain synaptosomes. Synaptosomes (500 µg) were incubated with a range of concentrations of -atracotoxin-Hv1 (A) and -atracotoxin-Ar1 (B and C) for 30 min at 37 °C, and 22Na+ uptake was measured after a 5-s period. Nonspecific 22Na+ uptake in the presence of 1 µM TTX was subtracted, and data were presented as a percentage of maximal uptake as determined in the presence of 1 µM batrachotoxin. A, inset, activation of 22Na+ uptake by AaH II. Synaptosomes were incubated with a range of concentrations of AaH II and 22Na+ uptake measured under the same conditions as above (n = 3). B, inset, inhibition of -atracotoxin-activated 22Na+ uptake by TTX. Black columns show 22Na+ uptake recorded in the presence of 10 µM -atracotoxin-Hv1, and the gray columns show data recorded in the presence of 1 µM -atracotoxin-Ar1. It was found that 1 µM TTX reduced 22Na+ uptake activated by -atracotoxin-Hv1 and -atracotoxin-Ar1 to 3 ± 2% (n = 3) and 1 ± 1% (n = 3), respectively. C, effect of AaH II on 22Na+ uptake activated by -atracotoxin-Ar1. Synaptosomes were incubated with a range of concentrations of -atracotoxin-Ar1 in the absence () and presence () of 30 nM AaH II. All values represent the mean ± S.E. (n = 3-13 for A, n = 3-6 for B, and n = 3 for C).

Low Concentrations of -Atracotoxins Cooperatively Enhance Batrachotoxin- and Veratridine-activated ²²Na⁺ Uptake-- Since -atracotoxins enhance [³H]BTX binding, we assessed the effect of 6 nM -atracotoxin on batrachotoxin-activated ²²Na⁺ uptake (Fig. 4). This concentration of -atracotoxin-Hv1 gives minimal direct activation of ²²Na⁺ uptake (Fig. 3); nevertheless, it is able to significantly shift the EC₅₀ of batrachotoxin to activate ²²Na⁺ uptake from 155 ± 36 to 15 ± 10 nM (Fig. 4A). Unexpectedly, this low concentration of -atracotoxin-Hv1 also produced a 30 ± 7% inhibition of the maximum ²²Na⁺ uptake, activated by 1 µM BTX (Fig. 4A).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Enhancement of batrachotoxin- and veratridine-stimulated 22Na+ uptake by -atracotoxins. A, increasing concentrations of batrachotoxin were incubated with rat brain synaptosomes in the absence () and presence of 6 nM () -atracotoxin-Hv1 (n = 3). The EC50 of batrachotoxin was reduced from 154 ± 21 to 9.0 ± 4.4 nM in the presence of -atracotoxin-Hv1. Note the 32% inhibition in batrachotoxin-activated 22Na+ uptake. B, enhancement of veratridine-activated 22Na+ uptake by -atracotoxin-Hv1. A range of concentrations of veratridine was incubated with rat brain synaptosomes in the absence () and presence of 6 nM -atracotoxin-Hv1 () (n = 3). The EC50 of veratridine was reduced from 4.0 ± 0.3 to 0.6 ± 0.4 µM in the presence of -atracotoxin-Hv1, and the maximal 22Na+ uptake was increased. For conditions of incubation see "Experimental Procedures." All data are presented as a percentage of maximal uptake as determined in the presence of 1 µM batrachotoxin.

-Atracotoxins Differentially Modify Batrachotoxin- and Veratridine-activated ²²Na⁺ Uptake-- To analyze further the interactions of -atracotoxins with alkaloid toxin-stimulated ²²Na⁺ uptake, we examined the effect of the funnel-web spider toxins on maximal ²²Na⁺ uptake stimulated either by batrachotoxin or veratridine. As previously noted in Fig. 4A, increasing concentrations of -atracotoxins strongly inhibited the maximal ²²Na⁺ uptake stimulated by 1 µM batrachotoxin (Fig. 5A) with IC₅₀ values of 10 ± 2 and 14 ± 2 nM for -atracotoxin-Hv1 and -atracotoxin-Ar1, respectively. Notable was the inhibition of batrachotoxin-activated ²²Na⁺ uptake to a level comparable with that which the -atracotoxins activate ²²Na⁺ uptake in the absence of batrachotoxin (see Fig. 3, A and B). As with flux activated directly by -atracotoxins, the remaining ²²Na⁺ uptake in the presence of both 1 µM batrachotoxin and saturating concentrations of -atracotoxins could be inhibited by 1 µM TTX (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Differential effect of -atracotoxins on batrachotoxin- and veratridine-stimulated 22Na+ uptake. A, inhibition of batrachotoxin-activated 22Na+ uptake by -atracotoxins. Rat brain synaptosomes were incubated for 10 min at 37 °C with 1 µM batrachotoxin and a range of concentrations of either -atracotoxin-Hv1 (, n = 3-8) or -atracotoxin-Ar1 (, n = 3-5). B, enhancement of veratridine-activated 22Na+ uptake by increasing concentrations of -atracotoxin-Hv1. Synaptosomes were incubated with the indicated concentrations of -atracotoxin-Hv1 in the absence (black columns) and presence (gray columns) of 300 µM veratridine. The left-hand (striped) column shows the activation of 22Na+ uptake by 300 µM veratridine alone. All data are presented as a percentage of maximal 22Na+ uptake as determined in the presence of 1 µM batrachotoxin (n = 3).

Inhibition of Scorpion -Toxin Binding by -Atracotoxins-- AaH II and Lqh II, which differ by only 2 amino acid residues, were shown to have similar LD₅₀ values in mice and reveal identical IC₅₀ values in competition binding studies with ¹²⁵I-AaH II binding to rat brain synaptosomes (22). Similarly, AaH II and Lqh II compete with identical K_i values for the binding of ¹²⁵I-Lqh II (Fig. 6). Likewise -atracotoxin-Hv1 and -atracotoxin-Ar1 are able to completely inhibit the binding of ¹²⁵I-Lqh II to rat brain sodium channels with K_i values of 0.85 ± 0.03 and 1.07 ± 0.2 nM, respectively (Fig. 6). These data indicate that -atracotoxins may share a common binding site with scorpion -toxins.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Competition for binding of 125I-Lqh II by -atracotoxins. Rat brain synaptosomes (43 µg/ml) were incubated for 20 min at 22 °C in the presence of 0.1 nM 125I-Lqh II with increasing concentrations of Lqh II (), AaH II (), -atracotoxin-Hv1 () and -atracotoxin-Ar1 (). Nonspecific binding, measured in the presence of 100 nM Lqh II, was subtracted from all data points. The curves were fitted using the Logistic equation for inhibition using a Hill number (nH) of 1 with regression coefficients (R2) higher than 0.976. The calculated Ki values for inhibition of 125I-Lqh II were as follows: Lqh II, 0.17 ± 0.04 nM; AaH II, 0.2 ± 0.03 nM; -atracotoxin, 0.85 ± 0.03 nM; -atracotoxin, 1.07 ± 0.2 nM.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

-Atracotoxins Affect Sodium Channels in a Unique Manner-- The present study shows that -atracotoxins slow sodium current inactivation and shift the voltage dependence of activation in peripheral and central neurons in a similar manner to scorpion -toxins. Unlike scorpion -toxins, which typically cause a slight increase or do not alter peak sodium currents (13), -atracotoxins were also shown to decrease peak sodium currents in rat dorsal root ganglion but not periaqueductal gray neurons (5, 6). This suggests that the actions of -atracotoxins may be, at least in part, subtype-specific as the sodium channel subtypes in sensory and rat brain neurons have been shown to be distinct (37). This assumption deserves further study, but clearly the most prominent effect to slow sodium current inactivation is similar in the two preparations (Fig. 1). One of the most intriguing results of the present study, however, was the observation that -atracotoxins markedly increase [³H]BTX binding but decrease batrachotoxin-activated ²²Na⁺ uptake (Figs. 2A and 5A). The latter is in marked contrast to the effect of scorpion -toxins on batrachotoxin-activated ²²Na⁺ uptake in rat brain synaptosomes (27, 34).

-Atracotoxins May Induce Sub-conductance States-- Alkaloid toxins, which bind to receptor site 2, are considered to be persistent activators of sodium channels (7). Their mode of action, however, is very complex; they induce a strong shift in the voltage dependence of activation to very negative membrane potentials, inhibit or remove inactivation, alter ion selectivity, and reduce single channel conductance (18, 19, 36, 38). Thus despite the persistent activation, sodium conductance through the permanently open sodium channels is reduced to around 50% or 15-25% of normal in the presence of batrachotoxin or veratridine, respectively, indicating that these two alkaloids stabilize two discrete sub-conductance states (19, 39, 40). It is conceivable that the decrease in sodium conductance observed with -atracotoxins in dorsal root ganglia neurons may be due to stabilization or induction of a distinct sub-conductance state, in addition to actions on sodium current inactivation (5, 6). The presence of sub-conductance states is suggested to occur also in central neurons in the presence of batrachotoxin, as indicated by the inhibition of batrachotoxin-activated ²²Na⁺ uptake induced by -atracotoxins (Fig. 5A). -Atracotoxins can induce 30% of maximal ²²Na⁺ uptake and inhibit the maximal uptake stimulated by batrachotoxin to 20-30% (Figs. 3 and 5A). This may indicate that the sub-conductance state induced by -atracotoxins in the presence of batrachotoxin is about 30% that induced by batrachotoxin alone. Moreover, this sub-conductance state should dominate the open state of the channel even in the simultaneous presence of both batrachotoxin and -atracotoxins. Our present data, however, cannot provide evidence for this assumption. Clarification of the mechanistic basis for the inhibition of sodium current awaits single channel analysis of -atracotoxin-modified currents.

-Atracotoxins Differentially Interact with Batrachotoxin and Veratridine Receptor Sites-- An interesting finding of the present study was that [³H]BTX binding was increased despite a decrease in batrachotoxin-activated ²²Na⁺ uptake. This suggests an uncoupling between the binding of batrachotoxin and its action to activate sodium channels. In support of this hypothesis is the recent study of Wang and Wang (41) that used site-directed mutagenesis in the S6 transmembrane segment of domain I of sodium channels to study the receptor-binding site of batrachotoxin. They revealed that certain mutants could separate the inhibition of channel inactivation from the induction of a sub-conductance state by batrachotoxin. Their results indicate a possible uncoupling between the binding and some of the effects of batrachotoxin on the sodium channel as noted in the present study.

Implications for the -Atracotoxin Receptor Site-- Competition binding experiments revealed that both -atracotoxins were able to completely displace the ¹²⁵I-scorpion -toxin Lqh II in a concentration-dependent manner. This suggests that -atracotoxins bind to at least a partially overlapping receptor site with that of scorpion -toxins. The above considerations suggest, however, that despite competitive interactions in binding studies (Fig. 6), the actual binding interactions may differ, at least partially, between -atracotoxins and scorpion -toxins. This is supported by the differential modulation by the -atracotoxin receptor site, shown to be on the extracellular side of the channel (5, 6), of the receptor site for batrachotoxin and that for veratridine. Hence, -atracotoxins and scorpion -toxins may bind to partially overlapping sites on the sodium channel localized within the area of receptor site 3. In support, another group of scorpion toxins, the "-like" toxins, which also inhibit sodium current inactivation, were suggested to bind to a partially overlapping receptor site with scorpion -toxins and sea anemone toxins (31). Despite the initial insight into the receptor site 3 area (42, 43), the regions of the sodium channel involved in binding of these peptide toxins still await to be discovered.