Eur J Biochem 269, 1500–1510 (2002) Ó FEBS 2002 Variations in receptor site-3 on rat brain and insect sodium channels highlighted by binding of a funnel-web spider d-atracotoxin Nicolas Gilles1, Greg Harrison1, Izhar Karbat2, Michael Gurevitz2, Graham M Nicholson3,* and Dalia Gordon2 CEA, De`partement d’Inge`nierie et d’Etudes des Prote`ines, Gif-sur-Yvette, France; 2Department of Plant Sciences, Tel Aviv University, Israel; 3Department of Health Sciences, University of Technology, Sydney, Australia d-Atracotoxins (d-ACTXs) from Australian funnel-web spiders differ structurally from scorpion a-toxins (ScaTx) but similarly slow sodium current inactivation and compete for their binding to sodium channels at receptor site-3 Characterization of the binding of 125I-labelled d-ACTXHv1a to various sodium channels reveals a decrease in affinity for depolarized (0 mV; Kd ¼ 6.5 ±1.4 nM) vs.polarized ()55 mV; Kd ¼ 0.6 ± 0.2 nM) rat brain synaptosomes The increased Kd under depolarized conditions correlates with a 4.3-fold reduction in the association rate and a 1.8increase in the dissociation rate In comparison, ScaTx binding affinity decreased 33-fold under depolarized conditions due to a 48-fold reduction in the association rate The binding of 125I-labelled d-ACTX-Hv1a to rat brain synaptosomes is inhibited competitively by classical ScaTxs and allosterically by brevetoxin-1, similar to ScaTx binding However, in contrast with classical ScaTxs, 125I-labelled dACTX-Hv1a binds with high affinity to cockroach Na+ channels (Kd ¼ 0.42 ± 0.1 nM) and is displaced by the ScaTx, LqhaIT, a well-defined ligand of insect sodium channel receptor site-3 However, d-ACTX-Hv1a exhibits a surprisingly low binding affinity to locust sodium channels Thus, unlike ScaTxs, which are capable of differentiating between mammalian and insect sodium channels, d-ACTXs differentiate between various insect sodium channels but bind with similar high affinity to rat brain and cockroach channels Structural comparison of d-ACTX-Hv1a to ScaTxs suggests a similar putative bioactive surface but a ÔslimmerÕ overall shape of the spider toxin A slimmer shape may ease the interaction with the cockroach and mammalian receptor site-3 and facilitate its association with different conformations of the rat brain receptor, correlated with closed/open and slow-inactivated channel states Australian funnel-web spiders (Araneae: Hexathelidae: Atracinae) are Mygalomorph spiders confined to the south-eastern seaboard of Australia A number of neurotoxins, named atracotoxins (ACTXs) that display various pharmacological properties, have been isolated from the venom of the funnel-web spider subfamily, Atracinae [1–4] Several lethal atracotoxins that modulate sodium channel gating have been assigned to the d-ACTX group because of their ability to induce spontaneous repetitive firing in neuronal cells, accompanied by plateau action potentials [5–8] The d-ACTXs, d-ACTX-Hv1a (formerly versutoxin [9]), the vertebrate-selective toxin d-ACTX-Hv1b [8] from the venom of Hadronyche versuta, and d-ACTX-Ar1 (formerly robustoxin [10]) from the venom of the male Sydney funnel-web spider Atrax robustus, are highly homologous 42-residue polypeptides These toxins contain a high proportion of basic residues and show no significant sequence homology with any presently known neurotoxin They are tightly folded molecules constrained by four conserved intramolecular disulfide bonds, arranged in a unique formation The solution structures of d-ACTXHv1a and -Ar1 have been determined by NMR spectroscopy [11,12] and constitute a small triple-stranded antiparallel b-sheet and a Ôcystine knotÕ motive [13] d-ACTXs slow tetrodotoxin (TTX)-sensitive sodium channel inactivation and produce modest shifts in the voltagedependence of sodium channel activation in insect and mammalian neurons [5–8] in a manner similar to scorpion a-toxins and sea anemone toxins [14,15] Despite the similar effect on sodium current inactivation kinetics, d-ACTXs have a distinct three-dimensional structure, which differs greatly from those of other toxins interacting with recep- Correspondence to D Gordon, Department of Plant Sciences, Tel-Aviv University, Ramat-Aviv, Tel Aviv 69978, Israel Fax: +972 640 6100, E-mail: dgordon@post.tau.ac.il Abbreviations: Aah-II, antimammalian a-toxin II from the venom of the scorpion Androctonus australis hector; d-ACTX-Ar1, d-atracotoxin-Ar1 (formerly robustoxin) from Atrax robustus; d-ACTX-Hv1a, d-atracotoxin-Hv1a (formerly versutoxin) from Hadronyche versuta; ATX-II, toxin II from the sea anemone Anemonia sulcata; [3H]BTX,[3H]batrachotoxinin, A-20a-benzoate; IC50, median inhibitory concentration; Kd, dissociation constant; Ki, inhibitory constant; koff, dissociation rate constant; kon, association rate constant; Lqh-II, Lqh-III, LqhaIT, a classical a-toxin, an a-like toxin, and an a-toxin highly active on insects, respectively, from the venom of the scorpion Leiurus quinquestriatus hebraeus; PbTx-1, brevetoxin-1 from the dinoflagellate Ptychodiscus brevis Enzyme: lactoperoxidase (EC 1.11.1.7) *Present address: Department of Health Sciences, University of Technology, Sydney PO Box 123, Broadway NSW 2007, Australia Fax: +61 9514 2228, E-mail: Graham.Nicholson@uts.edu.au (Received 25 September 2001, revised 15 January 2002, accepted 21 January 2002) Keywords: brevetoxin; sodium channel; spider toxin; synaptosomes; voltage-dependent binding Ó FEBS 2002 Binding of d-atracotoxins on sodium channels (Eur J Biochem 269) 1501 tor site-3 {e.g scorpion a-toxins, Aah-II (toxin II from Androctonus australis hector [16]), LqhaIT (from Leiurus quinquestriatus hebraeus [17]), and the sea anemone toxin, anthopleurin-B [18]} At least seven neurotoxin receptor sites have been identified on the voltage-gated sodium channel by radiolabelled toxin binding studies [19] Scorpion a- and sea anemone toxins such as ATX-II bind to neurotoxin receptor site-3 (for reviews see [19–22]) The binding of classical scorpion a-toxins, such as Aah-II [23] and Lqh-II [24,25], to receptor site-3 on rat brain sodium channels is voltage dependent and allosterically modulated by lipidsoluble sodium channel activators such as brevetoxin, veratridine and batrachotoxin [21,22,27–30] Notably, at nanomolar concentrations d-ACTX-Ar1 and -Hv1a completely inhibit the binding of classical scorpion a-toxins (e.g Aah-II and Lqh-II) to rat brain synaptosomes as well as the binding of LqhaIT to insect sodium channels [29,30] Thus, d-ACTXs constitute a unique group of polypeptides capable of high affinity binding presumably to receptor site-3 on both mammalian and insect voltagegated sodium channels Indeed, they enhance 3H-batrachotoxin binding similarly to scorpion a-toxins; however, they differ from scorpion toxins in that they inhibit, rather than enhance, the activation of sodium channels by batrachotoxin [30] Thus, clarification of d-ACTXs receptor sites on sodium channels requires a detailed analysis of their binding properties Here we provide a detailed characterization of radiolabelled d-ACTX-Hv1a direct binding to rat brain and insect sodium channels We present evidence that d-ACTX-Hv1a acts similarly to scorpion a-toxins in terms of its interaction with sodium channels at nanomolar affinities, similar voltage dependence and allosteric interaction with brevetoxin-1 on rat brain sodium channels Nevertheless, d-ACTX-Hv1a differs from scorpion a-toxins in its lower voltage dependency and ability to differentiate between receptor site-3 of cockroach and locust sodium channels rather than between rat brain and cockroach pipettes d-ACTX-Hv1a was obtained from adult male or female H versuta spiders while d-ACTX-Ar1 was obtained from adult male A robustus spiders Crude venom was washed from pipettes with 0.1% (v/v) trifluoroacetic acid and d-ACTX-Hv1a and d-ACTX-Ar1 isolated and purified by RP-HPLC Purification was achieved using a Pharmacia HPLC system using a Vydac analytical rpHPLC column ˚ (C18, 250 · 4.6 mm, 300 A, lm particle size) Pooled venom was applied to the column and venom components eluted at a flow rate of mLỈmin)1 using a linear gradient of 5–25% acetonitrile/0.1% trifluoroacetic acid over 22 min, followed by a gradient of 25–50% acetonitrile/0.1% trifluoroacetic acid over 48 Fractions containing d-ACTX-Hv1a or d-ACTX-Ar1 were then purified further using a linear gradient of 23–32% acetonitrile/0.1% trifluoroacetic acid over 20 at a flow rate of 1Ỉml min)1 Toxin quantification was performed using a bicinchoninic acid Protein Assay Kit (Pierce) using BSA as a standard Absorbance was read at 570 nm on a BIO-RAD Model 450 microplate reader The molecular mass was determined by electrospray ionization MS The fractions containing d-ACTX-Hv1a (Mr ¼ 4852) or d-ACTXAr1 (Mr ¼ 4854) were stored lyophilized at )20 °C in 5–10-nmol aliquots When required, spider toxins were dissolved in 10 mM Hepes (N-2-hydroxyethylpiperazine-N2-ethanesulfonic acid)/Tris buffer (pH 6.0) and an aliquot of this stock solution was diluted in the binding solution Any unused d-ACTX stock solution was kept at °C and used within weeks Neuronal membrane preparation The scorpion a-toxin, Lqh-II, from the venom of the scorpion L q hebraeus, was purchased from Latoxan (A.P 1724, 05150 Rosans, France) and, in part, was also a generous gift from Dr P Sautiere (Insitut Pasteur, Lille, ă France) [24] LqhaIT, an a-insect toxin from the scorpion L q hebraeus, was produced in Escherichia coli as described previously [31] Lactoperoxidase (EC 1.11.1.7) was purchased from Sigma Carrier-free Na125I was purchased from Amersham (Buckinghamshire, UK) All other chemicals were of analytical grade Filters for binding assays were GF/ C glass fibre discs (Whatman) preincubated in 0.3% polyethylenimine (Sigma) All buffers used for preparation of neuronal membranes contained a cocktail of proteinase inhibitors composed of: phenylmethylsulphonyl fluoride (50 lgỈmL)1), pepstatin A (1 lM), iodoacetamide (1 mM) and 1,10-phenantroline (1 mM) All membrane preparation steps were performed on ice Rat brain synaptosomes were prepared from adult albino Sprague-Dawley rats ( 300 g, laboratory bred), according to the method described by Kanner [32] The synaptosomes, which were concentrated at the 12 and 16% Ficoll gradient interface, were washed and aliquoted into Eppendorf tubes and stored at )80 °C Before experiments, the synaptosomes were rapidly defrosted for 30 s in a 37 °C water bath, placed on ice and used immediately (for polarized synaptosomes) For experiments carried out under depolarized conditions, synaptosomes were defrosted and incubated at 37 °C for 30 to facilitate ion gradient dissipation, and then kept on ice until used Insect synaptosomes were prepared from entire heads of adult cockroaches Periplaneta americana according to a previously described method [33] Locust synaptosomes were prepared from dissected brains and ventral nerve cords of adult locusts, Locusta migratoria, as described previously [34,35] Frozen synaptosomes were used within months Membrane protein concentration was determined by a BioRad Protein Assay Kit, using BSA as a standard Purification of d-ACTX-Hv1a and d-ACTX-Ar1 Radioiodination of d-ACTX-Hv1, LqhaIT and Lqh-II Crude venom was ÔmilkedÕ by direct aspiration from the chelicerae of live spiders maintained in a colony, using silanized (Coatasil; Ajax Chemicals, Australia) glass Toxins (5 lg) were radioiodinated for using 0.7 IU of lactoperoxidase (EC 1.11.1.7) from bovine milk and 0.5 mCi carrier-free Na125I, in 10 lL H2O2 (diluted : 50 000) and EXPERIMENTAL PROCEDURES Materials Ó FEBS 2002 1502 N Gilles et al (Eur J Biochem 269) 50 lL 20 mM phosphate buffer pH 7.2 The mono-iodotoxins were purified using a Vydac analytical C18 rpHPLC column and an acetonitrile gradient from 45 to 60% B (A ¼ aqueous 0.1% trifluoroacetic acid, B ¼ 0.085% trifluoroacetic acid, 50% acetonitrile, 0.2% B per min) at a flow rate of mlỈmin)1 The peak of the mono-iodo LqhaIT toxin eluted just after the peak of unmodified toxin as described previously [36] The unmodified d-ACTX-Hv1a, which eluted at 26% acetonitrile, was followed by two radiolabelled fractions, eluting at 26.5 and 27.1% acetonitrile As d-ACTX-Hv1a contains only two Tyr residues (positions 22 and 25), we determined the iodinated residue of each mono-iodo 125I-labelled d-ACTX-Hv1a fraction, using approximately 150 000 c.p.m of each fraction in the presence of unlabelled toxin by Edman degradation and sequencing (Applied Biosystem, 477 A protein sequencing), as described in detail for 125I-labelled LqhaIT [36] The N-terminal sequence analysis indicated that the first fraction of 125I-labelled d-ACTX-Hv1a was labelled on tyrosine 22, while the second mono-iodo 125I-labelled d-ACTX-Hv1a was labelled on tyrosine 25 The concentration of the radiolabelled toxins were determined according to the specific activity of 125I corresponding to 3000–2500 d.p.m.Ỉfmol)1 mono-iodotoxin, depending on the age of the radiotoxin and by estimation of its biological activity as described previously ([36], usually 60–70% for 125I-labelled LqhaIT and 35–55% for 125I-labelled d-ACTX-Hv1a) 125I-labelled LqhaIT was used within weeks whereas 125I-labelled d-ACTX-Hv1a was used within days Competition binding studies For competition binding experiments using 125I-labelled d-ACTX-Hv1a on rat brain sodium channels, synaptosomes were thawed at 37 °C (for 30 s) and suspended in 0.2 or 0.6 mL binding buffer containing a low concentration of radiolabelled toxins (see figure legends) Standard binding medium composition was (in mM): choline Cl, 130; CaCl2, 1.8; KCl, 5; MgSO4, 0.8; Hepes, 50; D-glucose 10; and mgỈmL)1 BSA Following incubation for the designated time periods the reaction was terminated by dilution with ml ice-cold wash buffer of the following composition (in mM): choline Cl, 140; CaCl2, 1.8; KCl, 5.4; MgSO4, 0.8; Hepes, 50; pH 7.2 and mg mL)1 BSA Separation of free from bound toxin was achieved by rapid filtration under vacuum using Whatman GF/C filters preincubated with 0.3% polyethylenimine The filter discs were then rapidly washed twice with mL buffer Termination of the reaction and washing lasted 10 s Nonspecific toxin binding was determined in the presence of a high concentration of the unlabelled toxin, as specified in figure legends, and comprised typically 5–10% of total binding for 125I-labelled LqhaIT and 30–50% for 125Ilabelled d-ACTX-Hv1a Competition binding experiments using 125I-labelled LqhaIT and 125I-labelled d-ACTX-Hv1a on insect neuronal membranes were performed according to established methods [35,36] under conditions specified in the figure legends Equilibrium and kinetic analysis of binding The median inhibitory concentration (IC50) values for the inhibition of toxin binding were determined by nonlinear regression analysis using the Hill equation using a Hill coefficient (nH) of Mathematical curve fitting was accomplished using KALEIDAGRAPH (Synergy Software, USA) for IC50 determination and the Ki values were calculated [37] Cold saturation assays were performed using increasing concentrations of the unlabelled toxin in the presence of a constant low concentration of the monoiodinated toxin Hot saturation assays were performed using increasing concentrations of the radiolabelled toxin, with the same amount of membranes Data were analysed using the iterative program LIGAND (Elsevier Biosoft) using Ôcold saturationÕ or Ôhot saturationÕ analysis The kinetic data for ligand association and dissociation rates were subjected to analysis by LIGAND, using Ôkinetic analysisÕ Each curve was subjected to multislope analysis to detect the presence of one or two slopes Toxin dissociation curves were initiated by the addition of excess unlabelled toxin and the dissociation rate constant (koff) was determined directly from a first order plot of ligand dissociation vs time The rate of toxin association (kon) was determined from the equation:   ẵRLe kon ẳ kobs ẵLẵRLmax where [L] is the concentration of ligand, [RL]e is the concentration of the complex at equilibrium, [RL]max is the maximum number of receptors present (determined in a parallel saturation experiment) and kobs is the slope of the pseudo-first order plot of ln ([RL]e/{[RL]e–[RL]t}) vs time [38] The concentration of labelled ligand in association kinetic determinations was adjusted to keep the reaction at pseudo-first order conditions and varied according to the Kd values of the toxin under polarized or depolarized conditions [38] Results were compared using a Student’s t-test and all data are expressed as the mean ± SEM from the number of experiments (n) indicated The corresponding affinity (Kd) can be calculate from the kinetics parameters according to the equation Kd ¼ koff/kon RESULTS Both d-ACTX-Hv1a and d-ACTX-Ar1 inhibit completely the binding of 125I-labelled Aah-II [28] and 125I-labelled Lqh-II [29] to rat brain sodium channels In order to characterize the receptor binding site for d-ACTXs on sodium channels, we analysed the binding properties of radioiodinated d-ACTX-Hv1a to rat brain synaptosomes We have first examined the ability of the classical scorpion a-toxin, Lqh-II, to displace 125I-labelled d-ACTX-Hv1a from its binding site and found a Ki value of 0.25 ± 0.03 nM (n ¼ 4; data not shown), supporting the notion that d-ACTXs share receptor site-3 with scorpion a-toxins Evidence that d-ACTX-Hv1a binding to rat brain synaptosomes is voltage dependent Binding of scorpion a-toxins depends on polarization of the synaptosome membrane and therefore is a useful measure in monitoring membrane potential Indeed, a 90% decrease of the initial maximal binding between polarized and depolarized synaptosomes has been shown [39–42] Ó FEBS 2002 Binding of d-atracotoxins on sodium channels (Eur J Biochem 269) 1503 The resting membrane potential of rat brain synaptosomes is approximately )55 mV (at mM [K+]o) due mainly to a high intracellular concentration of K+, which diffuses passively through the membrane [39,41,43] Although depolarization of synaptosomes by elevating [K+]0 is often used for measuring the influence of membrane potential on scorpion a-toxin binding [40,44], we have found that high concentrations of K+ in the binding buffer perturb the binding of 125I-labelled d-ACTX-Hv1a (data not shown) Unlike the situation with the binding of the a-toxin, 125 I-labelled Lqh-II, where nonspecific binding did not change with increasing external K+ concentration (between and 135 mM [42]), the level of nonspecific binding of 125I-labelled d-ACTX-Hv1a varied greatly, posing difficulties for data analysis Therefore, in order to depolarize the membrane without affecting other binding conditions, the synaptosomes were incubated for 30 at 37 °C in normal binding buffer (containing mM K+ see Experimental procedures) prior to addition of the labelled toxin (under such conditions the K+ gradient dissipates, the membrane potential approaches mV and scorpion a-toxin binding is decreased by 90% [42]) The time-course of 125I-labelled d-ACTX-Hv1a binding to polarized rat brain synaptosomes was performed at 22 °C to maintain the resting membrane potential for longer duration (Fig 1A [42]) Maximal binding was achieved after 10–15 and was maintained for an additional 10 before an apparent decrease could be observed A similar decrease in saturable binding was observed with the scorpion a-toxin Lqh-II (Fig 1, inset), suggesting dependency of binding on membrane depolarization for both toxins Therefore, all subsequent experiments on polarized synaptosomes were performed at 22 °C with a 20 incubation time to reach equilibrium binding conditions Despite the similar effect of membrane potential on binding of both toxins, the ratio between 125I-labelled d-ACTX-Hv1a (Fig 1B, left bars) maximal binding to polarized (empty bar) vs depolarized (gray bars) synaptosomes was substantially different from that measured for 125 I-labelled Lqh-II (Fig 1B, right bars) This difference necessitated analysis of the binding affinity d-ACTX-Hv1a to rat brain synaptosomes under polarized and depolarized conditions Affinity of d-ACTX-Hv1a for polarized and depolarized rat brain synaptosomes To study the influence of synaptosome membrane potential on 125I-labelled d-ACTX-Hv1a affinity, 125I-labelled d-ACTX-Hv1a was incubated with polarized or depolarized rat brain synaptosomes in the presence of increasing concentrations of unlabelled toxin (cold saturation) The dissociation constant (Kd) of d-ACTX-Hv1a increased 11-fold between polarized (Kd ¼ 0.57 ± 0.20 nM; n ¼ 3) and depolarized (Kd ¼ 6.5 ± 1.4 nM; n ¼ 5) synaptosomes, whereas the maximum number of receptor sites (Bmax) increased 1.8-fold (P < 0.05; Bmax ẳ 1.24 0.17 pmolặmg protein)1; n ¼ and 2.26 ± 0.05 pmolỈmg protein)1; n ¼ 5, respectively; Fig 2) To assure the significance of the change in Bmax, the experiments under polarized and depolarized conditions were performed in parallel using the same batch of rat brain synaptosomes It is Fig Time-course of 125I-labelled d-ACTX-Hv1a and 125I-labelled Lqh-II binding to rat brain synaptosomes at 22 °C (A) Typical association kinetics of 125I-labelled d-ACTX-Hv1a (75 pM) to polarized synaptosomes (20 lgỈmL)1 membrane protein) Non-specific binding, determined in the presence of lM Lqh-II, was time-invariant, and was subtracted from the experimental data points Maximal binding of 125I-labelled d-ACTXHv1a remained stable for 10 before decreasing due to spontaneous depolarization of synaptosomes The time-course of 60 pM 125I-labelled Lqh-II binding to polarized synaptosomes (20 lgỈmL)1) is presented in the inset as per cent of maximal specific binding (B) Comparison of the maximal binding of 125I-labelled d-ACTX-Hv1a (left bars) and 125I-labelled Lqh-II (right bars) to polarized (empty bars) and to depolarized (gray bars) synaptosomes 125I-labelled d-ACTX-Hv1a binding was performed as described for panel (A) Maximal binding under polarized (5.5 ± 0.3 pM and 5.1 ± 0.2 pM bound 125I-labelled d-ACTX-Hv1a or 125I-labelled Lqh-II, respectively) and depolarized synaptosomes pretreated at 37 °C for 30 (3.8 ± 0.6 pM and 0.75 ± 0.15 pM, for 125I-labelled d-ACTX-Hv1a or 125I-labelled Lqh-II, respectively), corresponds to the level of 125I-labelled toxin binding after 20 and 60 of incubation for polarized and depolarized conditions, respectively 1504 N Gilles et al (Eur J Biochem 269) Ĩ FEBS 2002 1.84 ± 0.2 · 106ỈM)1Ỉs)1 (n ẳ 3) and 1.1 0.1 Ã 10)3ặs)1 (n ¼ 3), respectively The corresponding Kd (0.6 ± 0.1 nM) calculated from the kinetic values, was comparable with the values obtained at equilibrium (Fig 2) Equilibrium of d-ACTX-Hv1a binding to depolarized synaptosomes was achieved after longer incubation (Fig 3A, open symbols), and dissociation was induced after 60 of association by adding lM unlabelled d-ACTX-Hv1a (Fig 3B, open symbols) The calculated kon and koff under depolarized membrane conditions were 0.43 0.13 Ã 106ặM)1ặs)1 (n ẳ 3) and 2.0 ± 0.3 · 10)3 s)1 (n ¼ 3), respectively The corresponding calculated Kd was 4.7 ± 2.1 nM, which fitted the value obtained at equilibrium These results indicate that d-ACTX-Hv1a binding is dependent on the membrane potential of synaptosomes Interestingly, synaptosome depolarization had a minute effect on koff but decreased fourfold the kon These results support our recent studies using the classical scorpion a-toxin, Lqh-II [42], and seem to provide a different interpretation to that suggested previously for scorpion a-toxins, which attributed the change in binding affinity under depolarized conditions mainly to an increase in the dissociation rate [15,26,39,40,44–47] Allosteric modulation of d-ACTX-Hv1a binding site on rat brain synaptosomes Fig Scatchard plots of 125I-labelled d-ACTX-Hv1a binding to rat brain synaptosomes (A) 92 pM 125I-labelled d-ACTX-Hv1a incubated at 22 °C for 20 with polarized synaptosomes (28.8 lgỈmL)1) and (B) 168 pM 125I-labelled d-ACTX-Hv1a incubated at 22 °C for 60 with depolarized synaptosomes (36.5 lgỈmL)1), in the presence of increasing concentrations of unlabelled toxin (cold saturation) (see Experimental procedures) Analysis of a typical experiment is presented Nonspecific binding, determined in the presence of 0.2 lM (A) or lM (B) d-ACTX-Hv1a, was subtracted Equilibrium binding parameters were calculated using the program LIGAND (see Experimental procedures) The dissociation constants (Kd) were 0.57 ± 0.2 nM and 6.5 ± 1.4 nM and the maximum number of binding sites (Bmax) were 1.24 ± 0.17 pmolỈmg protein)1 and 2.26 ± 0.05 pmolỈmg protein)1, under polarized (n ¼ 3) and depolarized (n ¼ 5) conditions, respectively noteworthy, that in order to maintain a pseudo-first order reaction conditions, toxin and receptor (membrane protein) concentrations were adjusted as a function of the change in 125 I-labelled d-ACTX-Hv1a affinity (Fig [38]) Kinetic constants of d-ACTX-Hv1a binding to rat brain synaptosomes 125 I-Labelled d-ACTX-Hv1a was incubated with synaptosomes and the association binding kinetics were monitored until equilibrium had been reached (Fig 3A, closed symbols for polarized, and open symbols for depolarized synaptosomes, respectively) After 10 incubation with polarized synaptosomes, toxin dissociation was initiated by adding lM unlabelled d-ACTX-Hv1a (Fig 3B, closed symbols) The calculated association and dissociation rate constants, kon and koff, under polarized membrane conditions were Brevetoxin-1 (PbTx-1) from a marine dinoflagellate, inhibits allosterically the binding of the scorpion a-toxin, Aah-II, to rat brain synaptosomes [27,28] To examine the similarity in binding to receptor site-3 between scorpion a-toxins and d-ACTXs, we analysed the effect of PbTx-1 on 125I-labelled d-ACTX-Hv1a binding to rat brain synaptosomes Similarly to the effect of PbTx-1 on Aah-II binding [27,28], this brevetoxin substantially inhibited the binding of 125 I-labelled d-ACTX-Hv1a with an IC50 of 50 nM (data not shown) We also analysed the effect of deltamethrin, a pyrethroid insecticide known to modulate sodium channels, and like scorpion a-toxins [48] found that it had no allosteric effect on d-ACTX-Hv1a binding d-ACTX-Hv1a differentiates between cockroach and locust sodium channels d-Atracotoxins are unique in their potency to displace scorpion a-toxins from their binding sites on both rat brain and cockroach sodium channels [29,30] Therefore, the interaction of 125I-labelled d-ACTX-Hv1a with scorpion a-toxins on binding to cockroach neuronal membranes was examined Competition binding experiments using increasing concentrations of the scorpion toxins, Lqh-II, LqhaIT, and Lqh-III as well as the related spider toxin, d-ACTXAr1, revealed complete inhibition of 125I-labelled d-ACTXHv1a binding by all toxins tested (Fig 4, main panel) Scatchard transformation of the competition binding curve of d-ACTX-Hv1a to cockroach neuronal membranes (cold saturation, Fig 4, inset) provided a Kd value of 0.42 ± 0.1 nM (n ¼ 3), which was highly similar to the affinity of d-ACTX-Hv1a binding to polarized rat brain synaptosomes (Table 1) The receptor site capacity (Bmax ¼ 2.1 ± 0.5 pmolặmg protein)1; n ẳ 3) was similar to that obtained previously for LqhaIT binding to cockroach neuronal membranes [35,36] Ó FEBS 2002 Binding of d-atracotoxins on sodium channels (Eur J Biochem 269) 1505 Fig Association (A) and dissociation (B) kinetics of 125I-labelled d-ACTX-Hv1a binding to polarized and depolarized rat brain synaptosomes Fifty and 200 pM 125I-labelled d-ACTX-Hv1a were incubated at 22 °C (in 200 lL) in the presence of 37 or 73 lgỈmL)1 polarized or depolarized synaptosomes, respectively, for various periods of time Nonspecific binding, determined in the presence of 200 nM or lM Lqh-II (for polarized and depolarized conditions, respectively) was time-invariant and was subtracted from the experimental points 125I-labelled d-ACTX-Hv1a dissociation was initiated by addition of 200 nM or lM unlabelled toxin after 10 or 60 association under polarized and depolarized conditions, respectively (B) A typical experiment is presented Kinetic constants, representing the mean of three experiments, were: kon ¼ 1.84 ± 0.2 · 106 s1ặM)1 and ko ẳ 1.1 0.1 Ã 103 s)1 under polarized conditions; kon ¼ 0.43 ± 0.13 · 106 s)1ặM)1 and ko ẳ 2.0 0.3 Ã 103 s)1 under depolarized conditions Unexpectedly, however, no specific binding of I-labelled d-ACTX-Hv1a to locust neuronal membranes could be detected As we have recently demonstrated that 125 Fig Binding interaction of 125I-labelled d-ACTX-Hv1a with cockroach sodium channels Competition for 125I-labelled d-ACTX-Hv1a (120 pM) binding to neuronal membranes (7 lgỈmL)1) by various neurotoxins Nonspecific binding, determined in the presence of 200 nM LqhaIT, was subtracted Bound 125I-labelled d-ACTX-Hv1a is expressed as the percentage of maximal specific binding in the absence of competitor toxins The competition curves were fitted by the nonlinear Hill equation (with a Hill coefficient of 1) to determine IC50 values (see Experimental procedures) Typical curves are presented The Ki values (in nM) and the number of experiments (n) are: LqhaIT, 0.12–0.16 (n ¼ 2); Lqh-III, 0.12–0.14 (n ¼ 2); d-ACTX-Hv1a, 2.6–3.0 (n ¼ 2); d-ACTX-Ar1, 1.5–2.5 (n ¼ 2); Lqh-II, 9.5–14.1 nM (n ¼ 2) Inset: Scatchard transformation of 55 pM 125I-labelled d-ACTX-Hv1a binding to cockroach neuronal membranes (8.7 lgỈmL)1) in a volume of 600 lL using increasing concentrations of unlabelled d-ACTX-Hv1a (Ôcold saturationÕ) The equilibrium binding parameters were calculated using the program LIGAND Data represents the mean of two cold- and two hot-saturation experiments (see Experimental procedures),which showed no significant differences Kd ¼ 0.42 ± 0.1 nM; Bmax ẳ 2.1 0.5 pmolặmg protein)1 iodination of one Tyr residue in the a-like toxin, Lqh-III, impairs binding to locust but not cockroach sodium channels [36], we identified which Tyr residue was iodinated on 125I-labelled d-ACTX-Hv1a Amino acid sequence analysis of the two radiolabelled peaks obtained during toxin radioiodination (see Experimental procedures) identified an iodinated Tyr22 in the first peak and an iodinated Tyr25 in the second peak Both iodinated derivatives did not differ in their binding properties to cockroach neuronal membranes (data not shown) In order to eliminate the possibility that the lack of 125I-labelled d-ACTX-Hv1a binding to locust neuronal membranes was consequent on its iodination per se, we examined the binding of d-ACTX-Hv1a to locust sodium channels indirectly, by its ability to compete for 125I-labelled LqhaIT binding (Fig 5) Interestingly d-ACTX-Hv1a competed for LqhaIT binding only at high concentrations (Ki ¼ 67 ± 17 nM; n ¼ 3) with a Ki value 160-fold higher than the Kd for cockroach sodium channels (Fig 4) Thus, in contrast with the scorpion a-like toxin Lqh-III [36] and despite its high binding affinity for cockroach sodium channels, d-ACTX-Hv1a is a weak ligand on locust sodium channels (Fig 5) DISCUSSION Effect of membrane depolarization on kinetics of toxin binding to receptor site-3 The binding properties of the spider toxin, d-ACTX-Hv1a, to rat brain synaptosomes resemble those of scorpion a-toxins, thereby suggesting a common receptor binding site on the sodium channel This resemblance is substantiated by a similar, yet nonidentical, decrease in binding affinity at polarized ()55 mV) and depolarized (0 mV) membrane potentials (Kd increase of 11.4-fold for d-ACTX-Hv1a and 33-fold for the classical a-toxin, Lqh-II, Table [42,49]) The increase in Kd of d-ACTX-Hv1a binding correlates with a 4.3-fold lower association rate and a 1.8-fold increase in the dissociation rate (Table 1) The more profound increase Ó FEBS 2002 1506 N Gilles et al (Eur J Biochem 269) Table Comparison between equilibrium and kinetic binding parameters of 125I-labelled d-ACTX-Hv1a and the scorpion a-toxin, Lqh-II Binding to polarized (membrane potential of )55 mV) and depolarized (0 mV) synaptosomes is performed as described in Figs and (see text for details) Data are means ± SEM values n ¼ number of independent experiments Data for Lqh-II binding parameters are from Gilles et al [42] Toxin Synaptosomes d-ACTX-Hv1a Polarized Depolarized Lqh-II Polarized Depolarized Kd (nM) kon (106 0.57 ± 0.20 (n ¼ 3) 6.5 ± 1.4 (n ¼ 4) 0.18 ± 0.04 (n ¼ 3) 5.85 ± 0.5 (n ¼ 4) 1.84 ± 0.2 (n ¼ 3) 0.43 ± 0.13 (n ¼ 3) 12.0 ± 4.0 (n ¼ 6) 0.25 ± 0.03 (n ẳ 4) )1 )1 M ặs ) ko (103ặs)1) 1.1 ± 0.1 (n ¼ 3) 2.0 ± 0.3 (n ¼ 3) 0.82 ± 0.06 (n ¼ 3) 1.12 ± 0.08 (n ¼ 3) conditions hinder Lqh-II association to a greater extent than d-ACTX-Hv1a, and may be related to their different structures A slow inactivated channel state prevails in depolarized synaptosomes Fig Competition of d-ACTX-Hv1a for 125I-labelled LqhaIT binding to locust sodium channels 125I-labelled LqhaIT (0.2 nM) was incubated for 60 at 22 °C with increasing concentrations of LqhaIT or d-ACTX-Hv1a, and the binding to locust neuronal membranes (60 lgỈmL)1) was determined Nonspecific binding, determined in the presence of 200 nM LqhaIT, was subtracted Bound 125I-labelled LqhaIT is expressed as the percentage of the maximal specific binding in the absence of competitor A typical experiment is presented The competition curves were fitted by a nonlinear Hill equation (with a Hill coefficient of 1) to determine IC50 values (see Experimental procedures) The calculated Ki values [37] were: LqhaIT, 1.2–2.2 nM (n ¼ 2); d-ACTX-Hv1a, 67 ± 17 nM (n ¼ 3) in Kd of Lqh-II binding under similar steady-state conditions may be related to the 48-fold decrease in its association rate constant (Table 1) Thus, the conformational alteration induced by depolarization at receptor site-3 appears to affect toxin binding by two mechanisms The first involves steric (architectural) and/or electrostatic (long-range) changes, which are unfavourable for d-ACTX-Hv1a access and even more so for Lqh-II, thus reducing substantially the kon The difference in kon suggests that the two toxins bind in a nonidentical manner to overlapping receptor sites The second mechanism involves a change in the surface of receptor site-3, which destabilizes its close fit with the bound toxin, thus increasing the off-rate Surprisingly, this change is much smaller as the off-rate of both toxins increased less then twofold between polarized and depolarized steadystate conditions but affects d-ACTX-Hv1a binding affinity more than that of Lqh-II (Table 1) Hence, depolarization d-ACTX-Hv1a binding to rat brain synaptosomes reveals an increase in Kd (11.4-fold) and in Bmax (1.8-fold) between polarized and depolarized conditions (Fig 2) The increase in Bmax may be attributed to a change in the ratio between sodium channels at high and low affinity states for toxin binding We assume that at resting membrane potentials, only  60% of site-3 receptors are in a high affinity conformation enabling toxin binding (presumably on sodium channels in closed states [47]), whereas the remaining channels are in a low affinity conformation associated with the slow-inactivated state This suggestion is supported by our study with Lqh-II, using both binding and electrophysiological analyses [42] and the study of Smith & Goldin [50] which implied that, at )55 mV, most rat brain subtype I (rBI) channels, which comprise  20% of sodium channels in synaptosomes [51], were available for activation presumably by being in closed, resting states [50] Nevertheless they showed that at identical membrane potential more than 50% of brain subtype IIA (rBIIA) channels that constitute the majority ( 80%) in synaptosomes [51], were in an inactivated state Our electrophysiological analysis of rBII channels expressed in mammalian cells supports this conclusion [42] Thus, a substantial fraction of sodium channels would occupy the slow-inactivated states at polarized synaptosomes and thus display a low affinity conformation for toxin binding [42] The observed increase in Aah-II binding to rat brain sodium channels in the presence of TTX [28] may be attributed to shifting receptor site-3 from low to high affinity conformation by binding of TTX to the external vestibule of the slow-inactivated channel pore In so far as the 125I-labelled d-ACTX-Hv1a concentrations used in the binding studies were low compared to the Kd (Figs and 3), the low affinity binding sites in polarized synaptosomes were undetectable, because only a small fraction of site-3 receptors were occupied Conversely, in depolarized synaptosomes, most sodium channels are in the slow-inactivated states, thus available for toxin binding only at low affinity conformations of receptor site-3 In this situation and under proper ligand concentra- Ó FEBS 2002 Binding of d-atracotoxins on sodium channels (Eur J Biochem 269) 1507 tions, maximum binding capacity is observed (Fig 2, and see [42]) The coexistence of (at least) two distinct conformational states of receptor site-3 among sodium channels in polarized synaptosomes gains further support from the 1.9-fold increase in receptor site capacity for scorpion a-toxins binding in the presence of batrachotoxin, an alkaloid toxin binding to receptor site-2 [41] This result suggests that batrachotoxin allosterically affects receptor site-3 by shifting it from the low to the high affinity state, increasing both scorpion a-toxin affinity and receptor site capacity [20,21,28] Together these results indicate that the low affinity conformation of receptor site-3 involves changes in external channel regions, which are affected by alterations in membrane potential or binding of toxins to topologically distinct receptor sites on the channel protein [28] The mechanisms involved in this affinity change are different, however, as depolarization affects mainly the association rate whereas allosteric modulation by other toxins affects mainly the dissociation rate constant [21,27,41] Resemblance of the putative bioactive surfaces of LqhaIT, Aah-II, and d-ACTX-Hv1a Despite the difference in three-dimensional structure, sequence, and size, the similarity in binding properties of scorpion a-toxins and d-ACTX-Hv1a suggests some structural resemblance at the bioactive surface However, the variations in kon and unusual binding selectivity of the toxins to receptor site-3 may result from either variations at the bioactive surface, or other, yet unidentified, structural differences In search for possible resemblance of molecular exteriors, we compared LqhaIT [17], Aah-II (which is almost identical to Lqh-II [16,24]) and d-ACTX-Hv1a [1] focusing on residues shown in LqhaIT to constitute the bioactive surface (Fig 6, left [52,53]) A number of bioactive residues of LqhaIT appear also on the surface of Aah-II in a similar position, and interestingly, also appear on the surface of d-ACTX-Hv1a (Fig 6) The positively charged Lys3, Lys4, Arg5, and Lys10 of d-ACTX-Hv1a are oriented similarly to Lys8, Arg58, Lys62, and Arg18 of LqhaIT The aromatic Trp7 and Tyr25 in d-ACTX-Hv1a resemble to some extent Trp38 and Phe17 in LqhaIT or Trp38 and Phe15 in Aah-II The nonpolar Asn6 in d-ACTX-Hv1a occupies a similar position to Asn44 in LqhaIT or Aah-II Finally, the negatively charged Glu12 in d-ACTX-Hv1a resembles Glu24 in both LqhaIT and Aah-II Despite this similarity, the shape of d-ACTX-Hv1a at the angle presented in Fig 6, is slimmer than that of the scorpion toxins, which may explain its accessibility, with high binding affinity, to both rat brain and cockroach receptor site-3 This possibility may also explain the smaller decrease in association rate to receptor site-3 between polarized and depolarized rat brain synaptosomes (Fig 2) compared to Lqh-II Conformational changes in the sodium channel states that are associated with a shift from the high to the low affinity state of receptor site-3 by depolarization may involve steric hindrance for toxin access, which is less pronounced for the slimmer d-ACTX This hypothesis is in concert with the smaller depolarization effect on the association rate, kon (Fig 3) of d-ACTX compared to that of Lqh-II (Table 1) In light of the structural resemblance at the putative bioactive surface between the spider and scorpion toxins, the subtle variations in action and binding properties [7,30], suggest that d-ACTXs interact with the sodium channel at a nonidentical, yet overlapping site to that of scorpion a-toxins Differences between cockroach and locust receptor site-3 All of the site-3 toxins that compete for classical scorpion a-toxin binding to rat brain sodium channels compete for LqhaIT binding to cockroach sodium channels but with different potencies [29,30,35] The spider d-ACTXs are unique in that they bind with equally high affinity to receptor site-3 of both rat brain and cockroach sodium channels (Figs and [29,30]) Such broad potency for sodium channels of distant phyla could be related to the slimmer shape of the spider toxin compared with the bulkier appearance of scorpion a-toxins (Fig 6) d-ACTXs are similar to LqhaIT in toxicity symptoms of injected insects [30,35,54], and the inactivation of the sodium current in cockroach neuronal preparations [5,54] Therefore, the substantial difference in d-ACTX-Hv1a binding affinity for cockroach vs locust sodium channels is surprising Structural differences between the two insect Fig Structural comparison of scorpion a-toxins and d-atracotoxin-Hv1a The structures for Aah-II ([16], PDB accession code 1PTX), d-ACTXHv1a ([11]; 1VTX); and LqhaIT ([17]; 1LQH) are presented with a similar orientation of their putative bioactive surfaces Residues reported to participate in the bioactive surface of LqhaIT [52,53] together with topologically related residues in Aah-II and d-ACTX-Hv1a are highlighted and colour coded: blue, positively charged; green, aromatic; magenta, Asn; yellow, negatively charged (see text for details) Toxin models were prepared USING SWISS-PDB VIEWER [57] Ó FEBS 2002 1508 N Gilles et al (Eur J Biochem 269) sodium channels have been previously inferred from allosteric modulations of LqhaIT binding Brevetoxin (site-5 toxin) and veratridine (site-2 toxin) increase the binding of LqhaIT to locust but not to cockroach sodium channels [27,35,55] In addition, we have shown that LqhIII, which binds to locust and cockroach receptor site-3 equally well, lost its binding capacity to locust sodium channels in its iodinated form [36] Regardless of iodination, however, the spider toxin binds with high affinity to cockroach and rat brain channels and with low affinity to locust receptor site-3 Of note is that LqhaIT and Lqh-III bind very poorly to rat brain sodium channels [49,56] in contrast with the high affinity binding of d-ACTX-Hv1a (Table 1) Hence, various toxin probes may expose subtle differences at receptor site-3 Still, the structural basis for these selective interactions requires determination of contact surfaces between the various ligands and their receptor binding sites Concluding remarks These results suggest that toxin selectivity for receptor site-3 may be conferred not only by structural variables at the direct interacting surface, but also by external channel elements that may affect toxin access to the binding site We have shown that toxins with different shapes or electrostatic surface potentials are capable of reaching the same or overlapping receptor sites (this study and see also [35,42,56]) Thus, structural differences in channel regions that flank the putative binding site could also be involved in toxin binding This assumption is supported by the difference between the association rates of Lqh-II and d-ACTX to polarized synaptosomes (which mainly account for the difference in their affinity) and the changes detected in kon to different channel states (see Table 1) Also, the differences in association rates of Lqh-II, Lqh-III and LqhaIT to rat skeletal muscle channels, expressed in mammalian cells [46], support this notion The rational design of subtype-specific compounds will take into account such architectural considerations, which may facilitate the design of subtype specific drugs and insecticides 10 11 12 13 14 ACKNOWLEDGEMENTS This work was supported in part by an Australian Research Council research grant and an UTS internal research grant (to G M N.), by a research grant from the Israeli Science Foundation (508/00, to D G.) and by grants from BARD, The United States-Israel Binational Agricultural Research & Development (IS-2901–97C, to M G and IS-3259–01 to D G.) 15 REFERENCES 17 Fletcher, J.I., Smith, R., O’Donoghue, S.I., Nilges, M., Connor, M., Howden, M.E.H., Christie, M.J & King, G.F (1997) The structure of a novel insecticidal neurotoxin, omega-atracotoxinHV1, from the venom of an Australian funnel web spider Nat Struct Biol 4, 559–566 Wang, X.-H., Smith, R., Fletcher, J.I., Wilson, H., Wood, C.J., Howden, M.E.H & King, G.F (1999) Structure–function studies of omega-atracotoxin, a potent antagonist of insect voltage-gated calcium channels Eur J Biochem 264, 488–494 Szeto, T.H., Wang, X.-H., Smith, R., Connor, M., Christie, M.J., Nicholson, G.M & King, G.F (2000) Isolation of a funnel web 16 18 19 spider polypeptide with homology to mamba intestinal toxin and the embryonic head inducer Dickkopf1 Toxicon 38, 429–442 Wang, X.-H., Connor, M., Smith, R., Maciejewski, M.W., Howden, M.E.H., Nicholson, G.M., Christie, M.J & King, G.F (2000) Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge Nat Struct Biol 7, 505–513 Grolleau, F., Stankiewicz, M., Birinyi-Strachan, L.C., Wang, X.-H., Nicholson, G.M., Pelhate, M & Lapied, B (2001) Electrophysiological analysis of the neurotoxic action of a funnel-web spider toxin, d-atracotoxin-Hv1a, on insect voltage-gated sodium channels J Exp Biol 204, 711–721 Nicholson, G.M., Willow, M., Howden, M.E.H & Narahashi, T (1994) Modification of sodium channel gating and kinetics by versutoxin from the Australian funnel-web spider Hadronyche versuta Pflugers Arch 428, 400–409 Nicholson, G.M., Walsh, R., Little, M.J & Tyler, M.I (1998) Characterisation of the effects of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, on sodium channel activation and inactivation Pflugers Arch 436, 117–126 Szeto, T.H., Birinyi-Strachan, L.C., Wang, X.-H., Smith, R., Connor, M., Christie, M.J., King, G.F & Nicholson, G.M (2000) Isolation and pharmacological characterisation of d-atracotoxinHv1b, a vertebrate-selective sodium channel toxin FEBS Lett 470, 293–299 Brown, M.R., Sheumack, D.D., Tyler, M.I & Howden, M.E.H (1988) Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus Biochem J 250, 401–405 Sheumack, D.D., Claassens, R., Whiteley, N.M & Howden, M.E.H (1985) Complete amino acid sequence of a new type of lethal neurotoxin from the venom of the funnel-web spider Atrax robustus FEBS Lett 181, 154–156 Fletcher, J.I., Chapman, B.E., Mackay, J.P., Howden, M.E.H & King, G.F (1997) The structure of versutoxin (d-atracotoxinHv1): implications for binding of site-3 toxins to the voltage-gated sodium channel Structure 5, 1525–1535 Pallaghy, P.K., Alewood, D., Alewood, P.F & Norton, R.S (1997) Solution structure of robustoxin, the lethal neurotoxin from the funnelweb spider Atrax robustus FEBS Lett 419, 191–196 Pallaghy, P.K., Neilsen, K.J., Craik, D.J & Norton, R.S (1993) A common structural motif incorporating a cystine knot and a triplestranded beta-sheet in toxic and inhibitory polypeptides Protein Sci 3, 1833–1839 Hanck, D.A & Sheets, M.F (1995) Modification of inactivation in cardiac sodium channels: ionic current studies with anthopleurin-A toxin J Gen Physiol 106, 601–616 Strichartz, G.R & Wang, G.K (1986) Rapid voltage-dependent dissociation of scorpion a-toxins coupled to Na channel inactivation in amphibian myelinated nerves J Gen Physiol 88, 413–435 Fontecilla-Camps, J.-L., Habersetzer-Rochat, C & Rochat, H (1988) Orthorhombic crystals and three dimensional structure of the potent toxin II from the scorpion Androctonus Australis Hector Proc Natl Acad Sci USA 85, 7443–7447 Tugarinov, V., Kustanovich, I., Zilberberg, N., Gurevitz, M & Anglister, J (1997) Solution structures of a highly insecticidal recombinant scorpion alpha-toxin and a mutant with increased activity Biochemistry 36, 2414–2424 Monks, S.A., Pallaghy, P.K., Scanlon, M.J & Norton, R.S (1995) Solution structure of the cardiostimulant polypeptide anthopleurin-B and comparison with anthopleurin-A Structure 3, 791–803 Gordon, D (1997) A new approach to insect-pest control – combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity Invertebr Neurosci 3, 103–116 Ó FEBS 2002 Binding of d-atracotoxins on sodium channels (Eur J Biochem 269) 1509 20 Catterall, W.A (1986) Molecular properties of voltage-sensitive sodium channels Annu Rev Biochem 55, 953–985 21 Catterall, W.A (1992) Cellular and molecular biology of voltagegated sodium channels Physiol Rev 72 (Suppl.), S15–S48 22 Gordon, D (1997) Sodium channels as targets for neurotoxins: mode of action and interaction of neurotoxins with receptor sites on sodium channels In Toxins and Signal Transduction (Lazarowici, P & Gutman, Y., eds), pp 119–149 Harwood Press, Amsterdam, the Netherlands 23 Martin-Eauclaire, M.-F & Couraud, F (1995) Scorpion neurotoxins: Effects and mechanisms In Handk Neurotoxicology (Chang, L.W & Dyer, R.S., eds), pp 683–716 Marcel Dekker, New York 24 Sautiere, P., Cestele, S., Kopeyan, C., Martinage, A., Drobecq, H., ă ă Doljansky, Y & Gordon, D (1998) New toxins acting on sodium channels from the scorpion Leiurus quinquestriatus hebraeus suggest a clue to mammalian vs insect selectivity Toxicon 36, 1141–1154 25 Gordon, D., Savarin, P., Gurevitz, M & Zinn-Justin, S (1998) Functional anatomy of scorpion toxins affecting sodium channels J Toxicol Toxin Rev 17, 131–159 26 Catterall, W.A (1977) Activation of the action potential Na+ ionophore by neurotoxins J Biol Chem 252, 8669–8676 27 Cestele, S., Ben Khalifa, R., Pelhate, M., Rochat, H & Gordon, ă D (1995) a-Scorpion toxins binding on rat brain and insect sodium channels reveal divergent allosteric modulations by brevetoxin and veratridine J Biol Chem 270, 15153–15161 28 Cestele, S & Gordon, D (1998) Depolarization dierentially ă aects allosteric modulation by neurotoxins of scorpion alphatoxin binding on voltage-gated sodium channels J Neurochem 70, 1217–1226 29 Little, M.J., Wilson, H., Zappia, C., Cestele, S., Tyler, M.I., ă Martin-Eauclaire, M.-F., Gordon, D & Nicholson, G.M (1998) d-Atracotoxins from Australian funnel-web spiders compete with scorpion a-toxin binding on both rat brain and insect sodium channels FEBS Letters 439, 246–252 30 Little, M.J., Zappia, C., Gilles, N., Connor, M., Tyler, M.I., Martin-Eauclaire, M.-F., Gordon, D & Nicholson, G.M (1998) d-Atracotoxins from Australian funnel-web spiders compete with scorpion a-toxin binding but differentially modulate alkaloid toxin activation of voltage-gated sodium channels J Biol Chem 273, 27076–27083 31 Zilberberg, N., Gordon, D., Pelhate, M., Adams, M.E., Norris, F., Zlotkin, E & Gurevitz, M (1996) Functional expression and genetic modification of an alpha scorpion neurotoxin Biochemistry 35, 10215–10222 32 Kanner, B.I (1978) Active transport of gamma-aminobutyric acid by membrane vesicles isolated from rat brain Biochemistry 17, 1207–1211 33 Krimm, I., Gilles, N., Sautiere, P., Stankiewicz, M., Pelhate, M., Gordon, D & Lancelin, J.M (1999) NMR structures and activity of a novel alpha-like toxin from the scorpion Leiurus Quinquestriatus Hebraeus J Mol Biol 285, 1749–1763 34 Gordon, D., Moskowitz, H & Zlotkin, E (1990) Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies Biochim Biophy Acta 1026, 80– 86 35 Gordon, D., Martin-Eauclaire, M.-F., Cestele, S., Kopeyan, C., ă Carlier, E., Ben-Khalifa, R., Pelhate, M & Rochat, H (1996) Scorpion toxins affecting sodium channel current inactivation bind to distinct homologous receptor sites on rat brain and insect sodium channels J Biol Chem 271, 8034–8045 36 Gilles, N., Krimm, I., Bouet, F., Froy, O., Gurevitz, M., Lancelin, J.M & Gordon, D (2000) Structural implications on the interaction of scorpion alpha-like toxins with the sodium channel receptor site inferred from toxin iodination and pH-dependent binding J Neurochem 75, 1735–1745 37 Cheng, Y & Prusoff, W (1973) Relationship between inhibition constant (KI) and the concentration of an inhibitor which causes 50% inhibition (IC50) of an enzymatic reaction Biochem Pharmacol 22, 3099–3108 38 Weiland, G.A & Molinoff, P.B (1981) Quantitative analysis of drug–receptor interactions I Determination of kinetic and equilibrium properties Life Sciences 29, 313–330 39 Catterall, W.A (1976) Purification of a toxic protein from scorpion venom which activates the action potential Na+ ionophore J Biol Chem 251, 5528–5536 40 Jover, E., Courand, F & Rochat, H (1980) Two types of scorpion neurotoxins characterized by their binding to two separate receptor sites on rat brain synaptosomes Biochem Biophys Res Comm 95, 1607–1614 41 Ray, R & Catterall, W.A (1978) Membrane potential dependent binding of scorpion toxin to the action potential sodium ionophore Studies with a 3-(4-hydroxy 3[125I] iodophenyl) propionyl derivative J Neurochem 31, 397–407 42 Gilles, N., Leipold, E., Chen, H., Heinemann, S.H & Gordon, D (2001) Effect of depolarization on binding kinetics of scorpion a-toxin highlights conformational changes of rat brain sodium channels Biochemistry 40, 14576–14584 43 Blaustein, M.P & Goldring, J.M (1975) Membrane potentials in pinched-off presynaptic nerve terminals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials J Physiol 247, 589–615 44 Ray, R., Morrow, C.S & Catterall, W.A (1978) Binding of scorpion toxin to receptor sites associated with voltage-sensitive sodium channels in synaptic nerve ending particles J Biol Chem 253, 7307–7313 45 Catterall, W.A., Morrow, C.S & Hartshorne, R.P (1979) Neurotoxin binding to receptor sites associated with voltage-sensitive sodium channels in intact, lysed, and detergent-solubilized brain membranes J Biol Chem 254, 11379–11387 46 Chen, H., Gordon, D & Heinemann, S.H (2000) Modulation of cloned skeletal muscle sodium channels by the scorpion toxins Lqh-II, Lqh-III, and LqhaIT Pflugers Arch 439, 423–432 47 Rogers, J.C., Qu, Y., Tanada, T.N., Scheuer, T & Catterall, W.A (1996) Molecular determinants of high affinity binding of a-scorpion toxin and sea anemone toxin in the S3–S4 extracellular loop in domain IV of the Na+ channel a subunit J Biol Chem 271, 15950–15962 48 Trainer, V.L., McPhee, J.C., Boutelet-Bochan, H., Baker, C., Scheuer, T., Babin, D., Demoute, J.P., Guedin, D & Catterall, W.A (1997) High affinity binding of pytethrods to the a subunit of brain sodium channels Mol Pharmacol 51, 651–657 49 Gilles, N., Chen, H., Wilson, H., Legall, F., Montoya, G., Molgo, J., Schnherr, R., Nicholson, G., Heinemann, S.H & Gordon, D ˆ (2000) Scorpion a- and a-like toxins differentially interact with sodium channels in mammalian CNS and periphery Eur J Neurosci 12, 2823–2832 50 Smith, R.D & Goldin, A.L (1998) Functional analysis of the rat I sodium channel in xenopus oocytes J Neurosci 18, 811–820 51 Gordon, D., Merrick, D., Auld, V., Dunn, R., Goldin, A.L., Davidson, N & Catterall, W.A (1987) Tissue-specific expression of the RI and RII sodium channel subtypes Proc Natl Acad Sci USA 84, 8682–8686 52 Zilberberg, N., Froy, O., Cestele, S., Loret, E., Arad, D., Gordon, D & Gurevitz, M (1997) Elucidation of the putative toxic-surface of a highly insecticidal scorpion a-neurotoxin affecting voltage-sensitive sodium channels J Biol Chem 272, 14810–14816 53 Gurevitz, M., Gordon, D., Ben-Natan, S., Turkov, M & Froy, O (2000) Diversification of neurotoxins by C-tail ÔwigglingÕ – a scorpion recipe for survival FASEB J 15, 1201–1205 1510 N Gilles et al (Eur J Biochem 269) 54 Eitan, M., Fowler, E., Herrmann, R., Duval, A., Pelhate, M & Zlotkin, E (1990) A scorpion venom neurotoxin paralytic to insects that affects sodium current inactivation: purification, primary structure, and mode of action Biochemistry 29, 5941– 5947 55 Gordon, D & Zlotkin, E (1993) Binding of an a-scorpion toxin to insect sodium channels is not dependent on membrane potential FEBS Lett 315, 125–128 Ó FEBS 2002 56 Gilles, N., Blanchet, B., Shichor, I., Zaninetti, M., Lotan, I., Bertrand, D & Gordon, D (1999) A scorpion a-like toxin active on insects and mammals reveals an unexpected specificity and distribution of sodium channel subtypes in rat brain neurons J Neurosci 19, 8730–8739 57 Guex, N & Peitsch, M.C (1997) SWISS-MODEL and the SwissPdbViewer: An environment for comparative protein modeling Electrophoresis 18, 2714–2723  ... and batrachotoxin [21,22,27–30] Notably, at nanomolar concentrations d-ACTX-Ar1 and -Hv 1a completely inhibit the binding of classical scorpion a- toxins (e.g Aah-II and Lqh-II) to rat brain synaptosomes... using increasing concentrations of the unlabelled toxin in the presence of a constant low concentration of the monoiodinated toxin Hot saturation assays were performed using increasing concentrations... analysis of the binding affinity d-ACTX-Hv 1a to rat brain synaptosomes under polarized and depolarized conditions Affinity of d-ACTX-Hv 1a for polarized and depolarized rat brain synaptosomes To study