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• W2016442704 abstract "Sodium channels posses receptor sites for many neurotoxins, of which several groups were shown to inhibit sodium current inactivation. Receptor sites that bind α- and α-like scorpion toxins are of particular interest since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel. We examined, for the first time, the interaction of different scorpion neurotoxins, all affecting sodium current inactivation and toxic to mammals, with α-scorpion toxin receptor sites on both mammalian and insect sodium channels. As specific probes for rat and insect sodium channels, we used the radiolabeled α-scorpion toxins AaH II and LqhαIT, the most active α-toxins on mammals and insect, respectively. We demonstrate that the different scorpion toxins may be classified to several groups, according to their in vivo and in vitro activity on mammalian and insect sodium channels. Analysis of competitive binding interaction reveal that each group may occupy a distinct receptor site on sodium channels. The α-mammal scorpion toxins and the anti-insect LqhαIT bind to homologous but not identical receptor sites on both rat brain and insect sodium channels. Sea anemone toxin ATX II, previously considered to share receptor site 3 with α-scorpion toxins, is suggested to bind to a partially overlapping receptor site with both AaH II and LqhαIT. Competitive binding interactions with other scorpion toxins suggest the presence of a putative additional receptor site on sodium channels, which may bind a unique group of these scorpion toxins (Bom III and IV), active on both mammals and insects. We suggest the presence of a cluster of receptor sites for scorpion toxins that inhibit sodium current inactivation, which is very similar on insect and rat brain sodium channels, in spite of the structural and pharmacological differences between them. The sea anemone toxin ATX II is also suggested to bind within this cluster. Sodium channels posses receptor sites for many neurotoxins, of which several groups were shown to inhibit sodium current inactivation. Receptor sites that bind α- and α-like scorpion toxins are of particular interest since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel. We examined, for the first time, the interaction of different scorpion neurotoxins, all affecting sodium current inactivation and toxic to mammals, with α-scorpion toxin receptor sites on both mammalian and insect sodium channels. As specific probes for rat and insect sodium channels, we used the radiolabeled α-scorpion toxins AaH II and LqhαIT, the most active α-toxins on mammals and insect, respectively. We demonstrate that the different scorpion toxins may be classified to several groups, according to their in vivo and in vitro activity on mammalian and insect sodium channels. Analysis of competitive binding interaction reveal that each group may occupy a distinct receptor site on sodium channels. The α-mammal scorpion toxins and the anti-insect LqhαIT bind to homologous but not identical receptor sites on both rat brain and insect sodium channels. Sea anemone toxin ATX II, previously considered to share receptor site 3 with α-scorpion toxins, is suggested to bind to a partially overlapping receptor site with both AaH II and LqhαIT. Competitive binding interactions with other scorpion toxins suggest the presence of a putative additional receptor site on sodium channels, which may bind a unique group of these scorpion toxins (Bom III and IV), active on both mammals and insects. We suggest the presence of a cluster of receptor sites for scorpion toxins that inhibit sodium current inactivation, which is very similar on insect and rat brain sodium channels, in spite of the structural and pharmacological differences between them. The sea anemone toxin ATX II is also suggested to bind within this cluster. INTRODUCTIONScorpion venom toxicity to humans has mainly been attributed to the pharmacological properties of toxic polypeptides that interfere with the sodium conductance in mammalian excitable tissues. The principal toxic compounds in scorpion venoms belong to a clearly defined family of homologous proteins, composed of single chain of 63-70 amino acid polypeptides cross-linked by four disulfide bridges (Miranda et al., 1970; Kopeyan et al., 1974; Darbon et al., 1982; Gregoire and Rochat, 1983). These sodium channel neurotoxins have been classified into several structural groups on the basis of primary structure (Rochat et al., 1979; Dufton and Rochat, 1984; Posanni, 1985; Watt and Simard, 1984) and immunological (Delori et al., 1981) criteria. The four first groups (I-IV) reveal a good correlation between amino acid sequences and pharmacological properties and contain the α-scorpion toxins active on vertebrates. The other groups contain the β-scorpion toxins and the toxins active on insect sodium channels (excitatory and depressant insect-selective toxins) (reviewed by MartinEauclaire and Couraud(1995)). The specificity of these toxins vary considerably (Zlotkin et al., 1978). Thus toxins specifically active on mammals (Miranda et al., 1970), insects, or crustaceans have been already described (Zlotkin, 1987). All these different toxins affect sodium conductance in various excitable tissues, thus serving as important pharmacological tools for the study of excitability and sodium channel structure.Voltage-dependent sodium channels are integral plasma membrane proteins responsible for the generation and propagation of action potentials in most excitable tissues. Being a critical element in excitability, sodium channels serve as specific targets for many neurotoxins. These toxins occupy different receptor sites on a sodium channel and have been used as tools for functional mapping and characterization of the channel (reviewed by Catterall(1986, 1992)).At least six neurotoxin receptor sites have been identified by direct radiotoxin binding on the mammalian sodium channels and additional, as yet unidentified receptor sites have been noticed (Table 1). Although the identification and characterization of the distinct receptor sites have been predominantly performed using vertebrate excitable preparations (Catterall, 1980, 1986; Strichartz et al., 1987), insect neuronal membranes have been shown to possess similar receptor sites. The presence of receptor sites 1-4 has been indicated by the binding of: [3H]saxitoxin and TTX ( 1The abbreviations used are: TTXtetrodotoxinAaITexcitatory insect-selective toxin from the scorpion Androctonus australis Hector, also called AaH IT1AaH I-IIIα-toxins I, II, and III from the venom of the scorpion A. australis HectorATX IItoxin II from the sea anemone Anemonia sulcataBom III and Bom IVtoxin III and IV from the venom of the scorpion Buthus occitanus mardochei from MexicoBSAbovine serum albuminCss II and Css VIscorpion β-toxins II and VI from the venom of the Mexican scorpion Centruroides suffusus suffususEmmembrane potentialEhholding potentialLqhαIT, α-toxin specific to insectsfrom the venom of the scorpion Leiurus quinquestriatus hebraeusLqhIT2depressant insect-selective toxin from the scorpion L. quinquestriatus hebraeusLqq III-V, α-toxins III, IVand V from the venom of the scorpion L. quinquestriatus quinquestriatus (Lqq V is called also LqTx or ScTx)PbTx-1brevetoxin from the marine dinoflagellate Ptychodiscus brevisTxVIAδ-conotoxin-TxVIA from Conus textile.) (receptor site 1, Gordon et al., 1985); tritiated derivative of batrachotoxin ([3H]batrachotoxin A 20-α-benzoate) and veratridine (receptor site 2, Soderlund et al., 1989; Dong et al., 1993; Church and Knowles, 1993); 125I-α-scorpion (LqhαIT) and 125I-ATX II sea anemone toxins (receptor site 3, Gordon and Zlotkin, 1993; Pauron et al., 1985) and 125I-β-scorpion toxins (Ts VII, Css VI, receptor site 4; Lima et al., 1986, 1989), on locust, cockroach and other insect neuronal membranes. The presence of receptor site 5 has been most recently demonstrated by the electrophysiological activity of brevetoxin on cockroach axons and its allosteric modulation on LqhαIT binding on locust sodium channels (Cestele et al., 1995). The presence of receptor site 6, which binds the δ-conotoxin TxVI, has also been suggested on insect sodium channels. ( 2D. Gordon and M. Fainzilber, unpublished results.) Tabled 1Sodium channels from various excitable tissues and animal phyla contain a major α-subunit of about 240-280 kDa (Catterall, 1992; Gordon et al., 1988, 1990, 1993), composed of about 2000 amino acids comprising four homologous repeated domains (I-IV), each containing six putative transmembrane α-helices (for a review, see Gordon(1990) and Catterall(1992)). Insect sodium channels were shown to resemble their vertebrate counterparts by their primary structure (Loughney et al., 1989), topological organization (Gordon et al., 1992; Moskowitz et al., 1994), and basic biochemical (Gordon et al., 1988, 1990, 1992, 1993; Moskowitz et al., 1991, 1994) and pharmacological (Pelhate and Sattelle, 1982; Pelhate and Zlotkin, 1982; Cestele et al., 1995) properties. On the other hand, a possible uniqueness of the insect sodium channels was suggested by the description of two groups of scorpion toxins that modify sodium conductance exclusively in insect neuronal preparations, the excitatory and depressant insect-selective toxins (Pelhate and Zlotkin, 1982; Zlotkin et al., 1985, 1991). These toxins bind selectively to insect sodium channels at two distinct receptor sites (Gordon et al., 1992; Moskowitz et al., 1994) and therefore indicate the existence of unique features in the structure of insect channels, as compared to their mammalian counterparts (Gordon et al., 1984, 1992, 1993). Thus, a comparative study of mammalian and insect neurotoxin receptor sites on the respective sodium channels may elucidate the structural features involved in the binding and activity of the various neurotoxins and may contribute to the clarification of structure-function relationship in sodium channels.Receptor sites for peptide neurotoxins that inhibit sodium current inactivation in neurons (the classical effect induced by α-scorpion and sea anemone toxins; see Table 1) 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). The most studied neurotoxins that induce inhibition of sodium current inactivation are the α-scorpion toxins and sea anemone toxins, which are believed to share receptor site 3 on sodium channels (Couraud et al., 1978; Catterall and Beress, 1978; Catterall, 1980). Several α-scorpion toxins have been identified by their high toxicity to mammals and by a high homology in their amino acid sequence (reviewed by Martin-Eauclaire and Couraud(1995)).In the present study we have used AaH II, the α-scorpion toxin that reveals the highest affinity to rat brain synaptosomes (Jover et al., 1978), and LqhαIT, the α-scorpion toxin that reveals significantly higher activity to insects as compared to vertebrates (Eitan et al., 1990; Gordon and Zlotkin, 1993) as specific probes for receptor site 3 in rat brain and insect sodium channels, respectively. LqhαIT binding characteristics to locust neuronal membranes have been shown to be similar to those described for the α-scorpion toxins Lqq V (Ray et al., 1978) and AaH II (Jover et al., 1978) on rat brain sodium channels, except that its binding is not dependent on membrane potential (Gordon and Zlotkin, 1993). Thus, the receptor site for LqhαIT on insect sodium channels has been considered to be homologous to receptor site 3 in vertebrate sodium channels (Eitan et al., 1990; Gordon and Zlotkin, 1993; Zlotkin et al., 1994).We have compared the toxic activity and binding interactions of various scorpion toxins on mammals and insects. Three different neuronal sodium channel preparations have been chosen: rat brain synaptosomes, which are the most studied; and two different insect central nervous system membranes, locust and cockroach neuronal membranes, which served for neurotoxin binding studies in insects. Cockroach axons have been used as the main preparation for physiological effects of neurotoxins in insects. We have tested binding interactions of several different scorpion toxins, which reveal peculiarity in their toxic and pharmacological behavior, to get some insight into their possible receptor sites on sodium channels.The results of our comparative study suggest that scorpion toxins affecting inactivation of sodium current may be divided into several different groups according to their mammal versus insect activities, each possessing its distinct receptor site on sodium channels. The α-toxin receptor site on sodium channels is suggested to be a macrosite, which includes the LqhαIT/Lqq III receptor site that partially overlaps with both ATX II and AaH II receptor sites. The other groups of α-like scorpion toxins are suggested to bind to distinct receptor sites on both rat brain and insect sodium channels, which interact with receptor site 3. A cluster of receptor sites that preferentially bind scorpion toxins affecting current inactivation is suggested to be present on both rat brain and insect sodium channels. ATX II receptor site is suggested to be included in this cluster.EXPERIMENTAL PROCEDURESToxinsAaH I, AaH II, AaH III, Lqq III, Lqq IV, and Lqq V were purified according to Miranda et al.(1970). Bom III and Bom IV were purified as described (Vargas et al., 1987). LqhαIT, used for radioiodination and saturation curves was a generous gift of Prof. Eliahu Zlotkin (The Hebrew University of Jerusalem, Jerusalem, Israel) and was purified as described (Eitan et al., 1990). LqhαIT used for nonspecific binding determinations and Brevetoxin PbTx-1 were from Latoxan (A.P. 1724, 05150 Rosans, France). ATX II and veratridine were from Sigma. Carrier-free Na125I was from Amersham. All other chemicals were of analytical grade. Filters for binding assays were glass fiber GF/C (Whatman) preincubated in 0.3% polyethyleneimine (Sigma).Neuronal Membrane PreparationsRat brain synaptosomes were prepared from adult albino Wistar rats (about 300 g, laboratory-bred), according to the procedure of Dodd et al.(1981). Insect synaptosomes (P2L preparation) were prepared from the central nervous system of adult locusts (Locusta migratoria) and cockroach (Periplaneta americana) according to established methods (Gordon et al., 1990, 1992; Moskowitz et al., 1994). All buffers contained a mixture of proteinase inhibitors composed of: phenylmethylsulfonyl fluoride (50 μg/ml), pepstatin A (1 mM), iodoacetamide (1 mM), and 1 mM 1,10-phenanthroline. Membrane protein concentration was determined using a Bio-Rad protein assay, with BSA as standard.RadioiodinationAaH II was radioiodinated by lactoperoxidase as described previously (Rochat et al., 1977) using 1 nmol of toxin and 1 mCi of carrier-free Na125I. LqhαIT was iodinated by IODOGEN (Pierce) using 5 μg toxin and 0.5 mCi of carrier-free Na125I, as described previously (Gordon and Zlotkin, 1993). δTxVIA was radiolabeled as described (Fainzilber et al., 1994). The monoiodotoxins were purified according to Lima et al.(1989), using a Merck RP C8 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. The concentration of the radiolabeled toxins was determined according to the specific activity of the 125I corresponding to 2424 dpm/fmol monoiodotoxin.Binding AssayEquilibrium competition and saturation assays were performed using increasing concentrations of the unlabeled toxin in the presence of a constant low concentration of the radioactive toxin. In order to obtain saturation curves (#x0201C;cold” saturation), the specific radioactivity and the amount of bound toxin were calculated and determined for each toxin concentration. In some cases, for comparative purposes, equilibrium saturation curves were generated by increasing concentrations of the labeled toxin and nonspecific binding was determined for each concentration (#x0201C;hot” saturation). Standard binding medium composition was: (in mM): choline chloride 140, CaCl2 1.8, KCl 5.4, MgSO4 0.8, HEPES 25, pH 7.4; glucose 10, BSA 2 mg/ml. Wash buffer composition was (in mM): choline chloride 140, CaCl2 1.8, KCl 5.4, MgSO4 0.8, HEPES 25, pH 7.4, BSA 5 mg/ml.Rat brain synaptosomes (100 μg of protein/ml) or insect synaptosomes (P2L, 50 μg/ml and 3.3 μg/ml, for locust and cockroach, respectively) were suspended in 0.15 or 0.3 ml of binding buffer, containing 125I-AaH II or 125I-LqhαIT, respectively. After incubation for the designated time periods, the reaction mixture was diluted with 2 ml of ice-cold wash buffer and filtered through GF/C filters under vacuum. Filters were rapidly washed with an additional 2 × 2 ml of buffer. Nonspecific toxin binding was determined in the presence of 0.2 μM unlabeled AaH II or 1 μM LqhαIT, respectively, and consist typically of 15-20% of total binding for 125I-AaH II or 125I-LqhαIT, using rat brain or locust membranes, respectively, and about 1% using cockroach membranes. The experiments with the rat brain preparation were carried out for 30 min at 37°C and those with insect membranes, for 60 min at 22°C. Equilibrium saturation or competition experiments were analyzed by the iterative computer program LIGAND (Elsevier Biosoft). Each experiment was performed at least three times.Electrophysiological ExperimentRat Neuronal Cells Cultured rat cerebellar granule neurons in 45-mm dishes (Costar) were used at day 7-14 of culture for electrophysiological experiments, which were performed at room temperature (20-22°C) with the single-electrode whole-cell voltage clamp technique, using suction pipettes ranging from 2 to 4 megohms. The Na+ gradient was reversed to eliminate variability in space clamp, allowing recordings of highly reproducible peak currents (Numann et al., 1991; Dargent et al., 1994). The external solution contained 90 mM choline chloride, 15 mM tetraethylammonium chloride, 1 mM MgCl2, 1.5 mM CaCl2, 1 mM KCl, 5 mM glucose, 30 mM HEPES (pH adjusted to 7.3 with TMAOH), 1 mg/ml BSA. The internal solution contained 100 mM NaF, 30 mM NaCl, 20 mM CsF, 0.2 mM CdCl2, and 5 mM HEPES (pH adjusted to 7.3 with CsOH). Currents induced by a 50-ms depolarizing test pulse were recorded using Axon Instrument Axopatch 200A patch-clamp amplifier, low pass-filtered at 2 kHz with an 8-pole Bessel filter, and sampled at 20 kHz using a 12-bit ADC (Labmaster TM 40, Scientific Solution, Foster City, CA). Capacitance and leak currents were subtracted from active currents using a P/4 protocol (Benzanilla and Armstrong, 1977). Data acquisition and analysis were controlled by pCLAMP software (Axon Instrument).Insect AxonAdult male cockroaches (P. americana) were used throughout these experiments. A segment (1.5-2.5 mm) of one giant axon was isolated from a connective linking the 4th and 5th abdominal ganglia and cleaned of adhering fibers. The preparation was transferred to an experimental chamber in which two lateral Ag-AgCl electrodes were in contact with the severed ends of the axon and a central Ag-AgCl electrode was in contact through the external bathing solution with a 100-150-μm segment of the dissected axon. The preparation was immersed in paraffin oil and the #x0201C;artificial node” created by the non-electrolyte (Pichon and Boistel, 1967) was voltage-clamped as described in detail previously (Pelhate and Sattelle, 1982). This axonal preparation contains 2-3 layers of glial cells surrounding the isolated axon, which limit the access of toxin molecules to reach their receptor sites on the axonal membrane (Pichon et al., 1983). As a result, higher concentrations of toxins are required to detect their activity. Normal physiological saline had the following composition (in mM): NaCl 200, KCl 3.1, CaCl2 5.4, MgCl2 5.0, HEPES buffer 1, pH 7.2. Experiments were performed at 19-21°C. When necessary, potassium current was suppressed largely by 0.5 mM 3,4-diaminopyridine.In Vivo Animal BioassaysFifty percent lethal doses (LD50) were established according to Behrens and Karber(1935). The anti-mammal activity was tested by subcutaneous or intracerebroventricular injections into C57 BL/6 mice (20 ± 2 g). Anti-insect activity was evaluated in cockroaches (Blatella germanica, 50 ± 2 mg) using an automatic microsyringe from the Burker Manufacturing Co. (Rickmansworth, United Kingdom).RESULTSCorrelation between Toxicity and Binding of Scorpion ToxinsTable 2 represents the activity in vitro (competition for AaH II binding, the most active α-scorpion toxin on vertebrates,) and in vivo (by intracerebroventricular injections to mice) of several scorpion toxins (Fig. 1). Some of these toxins (belonging to structural groups III and IV) were shown to have much weaker toxic effects on mice, as compared to the AaH II (Table 2). Out of these less active toxins on mice, only Lqq IV and Lqq III have been shown to satisfy the main criterion used for α-scorpion toxin definition (Couraud et al., 1982), namely competition for AaH II binding in rat brain synaptosomes, although at higher concentration (Table 2 and Fig. 2, upper inset).Tabled 1Figure 1:Comparison of scorpion toxin amino acid sequences classified according to their structural homology. A, the structural group is marked on the left (I-IV). The sequences were aligned for maximum similarity by eye inspection. B, a table presenting the percentage of identical and conserved (in brackets) residues calculated for maximum homology between each pair of protein sequences.View Large Image Figure ViewerDownload (PPT)Figure 2:Correlation between the toxicity to mice (intracerebroventricular) of different scorpion toxins and the concentration required to inhibit the binding of 125I-AaH II to rat brain synaptosomes (IC50), relative to the toxicity and IC50 of AaH II. The data are from Table 2. Abcissa, LD50 values of each toxin divided by the LD50 of AaH II; ordinate, IC50 values of each toxin divided by IC50 of AaH II. Upper inset, competitive inhibition curves of several toxins for 125I-AaH II binding to rat brain synaptosomes. The results are presented as percent of AaH II maximal specific binding with no competitor. Nonspecific binding, measured in the presence of 200 nM AaH II, was subtracted from all data points. Lower inset, enlargement of the correlation curve (main panel, lower left corner), presenting the correlation between toxicity and binding inhibition of some classical α-scorpion toxins.View Large Image Figure ViewerDownload (PPT)Examination of the correlation between toxicity to mice and the toxins' potency in competing for binding of AaH II on rat brain sodium channels reveals a certain peculiarity (Table 2 and Fig. 2). The graphic presentation of this correlation (Fig. 2) suggests that toxins related to α-scorpion toxins comprise at least four groups: 1) the #x0201C;classical”α-toxins, such as AaH I-III and Lqq V (belonging to structural groups I and II), which reveal a perfect correlation between their toxicity and binding inhibition properties in rat brain (Fig. 2, lower inset); 2) Lqq IV, which exhibits a lower toxicity (54-fold less toxic than AaH II) and inhibits AaH II at significantly higher concentrations than other α-toxins (Table 2) (this toxin holds an intermediate position on the correlation curve; Fig. 2); 3) Lqq III, which is 2200-fold less toxic to mice than AaH II, and inhibits the binding of AaH II at very high concentration (Fig. 2 and Table 2) (Lqq III is highly homologous to the anti-insect α-toxin LqhαIT (see Fig. 1) and holds a unique place in this correlation curve); 4) toxins belonging to structural group III, represented by Bom III and Bom IV, which are toxic to mice but do not compete for AaH II binding and consequently do not reveal any correlation between these parameters (Table 2). The peculiarity of these toxins prompt us to re-examine their toxicity and pharmacology by a comparative approach, using sodium channels from rat and insect central nervous system.Electrophysiological Activity of α-Like Scorpion ToxinsThe toxins presented in Table 2 intoxicate mice (by intracerebroventricular injection) in a similar manner, leading to paralysis and death at different doses (see Table 2). To examine whether the two peculiar scorpion toxins, Bom III and Bom IV, belong to the same category of neurotoxins as the α-scorpion toxin group, namely are able to induce inhibition of sodium current inactivation, we tested their physiological effects on cultured neuronal cells from rat brain (Fig. 3) and on an isolated axon from cockroach central nervous system (Fig. 4).Figure 3:Action of AaH II, Bom III, and Bom IV on isolated rat cerebellar granule cells in culture, under voltage clamp conditions. Outward Na+ currents from cerebellar granule cells were detected before and 3 min after addition of 0.5 nM AaH II (A), 2.5 or 5 nM Bom III (B and C), and 10 or 25 nM Bom IV (D and E). The cells were held at −90 mV, and depolarization was induced by a 50-ms test pulse to −20 mV. Superimposed traces before and after addition (arrow) of toxins are shown. Note the evident toxin effect on slowing the current inactivation and the slight decrease on Na+ peak current (A, C, and E). F and G, I-V activation curves obtained by 8 mV voltage steps from −60 mV to +60 mV, before (black circles) and after (open circles) addition of 0.5 nM AaH II (F) or 25 nM Bom IV (G). No difference in the activation threshold was observed, but the slope of the curve was decreased after toxin action (G). Steady-state inactivation curves were determined using a 200-ms prepulse from −110 mV to +20 mV in 10-mV steps, followed by a test pulse to +40 mV, before (black squares) and after (open squares) addition of 0.5 nM AaH II (F) or 25 nM Bom IV (G). Note the left shift of the curves.View Large Image Figure ViewerDownload (PPT)Figure 4:The effects of Bom III on an isolated cockroach axon under current and voltage clamp. A, superimposed records of action potentials evoked by a short current pulse (0.5 ms, 10 nA) during a Bom III (5 μg/ml, 0.625 μM) superfusion. The short control action potential is progressively transformed into a #x0201C;plateau” potential seen also in B. B, after 12 min of Bom III application. C, control Na+ current associated to a 5 ms in duration voltage pulse to Em = −20 mV from a holding potential Eh = −60 mV after blockage of Ik by 10 mM 3-4 diaminopyridine. Note the complete inactivation of INa after less than 2 ms. D, superimposed recordings every 15 s during the application of 0.5 μg/ml (62.5 nM) Bom III; note the progressive slowing of the current tracks accompanied here by a slight increase in the peak current. At the end of the voltage pulse, the maintained Na+ current turns off rapidly. E, the peak as well as the maintained Na+ current are blocked by a 60-s application of TTX (1 μM). Near each trace the time of TTX application is marked in seconds. F, potassium current associated to a voltage pulse to Em = +20 mV (Eh = −60 mV), after blockage of INa by 1 μM TTX: after a 10-min application of Bom III (62.5 nM), no significant change is detected in the magnitude as well as in the kinetics of Ik.View Large Image Figure ViewerDownload (PPT)In cerebellar granule cells under voltage-clamp conditions, extracellular addition of 0.5 nM AaH II induced a classical α-scorpion toxin effect, namely a slight, progressive decrease of the Na+ peak current accompanied by an evident slowing of inactivation time course (Fig. 3A). In the same experimental conditions, the main effect induced by Bom III and Bom IV was slowing down the decline of Na+ currents (Fig. 3, B-E), similarly to the one observed with AaH II (Fig. 3A), but Bom IV affects the sodium conductance at higher concentration (Fig. 3, D and E). The higher concentration of Bom III and IV needed for maximal effects are in concert with the lower activity of these toxins on mice (see Table 2). Steady-state inactivation curves obtained before and after addition of 0.5 nM AaH II or 25 nM Bom IV showed a notable shift to the left, to more hyperpolarized potentials for both AaH II and Bom IV (Fig. 3, F and G). However, examination of the current changes induced by AaH II compared to Bom toxins reveals that the latter affect the Na+ conductance in an additional manner, namely slowing the activation kinetics. Although we did not quantitatively analyzed the activation kinetics of the sodium currents, they appear to be slowed by both Bom toxins (Fig. 3, C and E) but not by AaH II (Fig. 3A), as indicated by the rising phase and time-to-peak current. Unlike AaH II, Bom IV reduced the slope of the activation curve (Fig. 3G). These discrepancies between the two groups of toxins could indicate that Bon IV may modify additional properties of the channel. Since plural mechanisms may account for slowing the decline of sodium current, including reopening of channels that are closed along the inactivation pathway as well as those with slowed or modified activation, further experimentation would be necessary to determine the exact nature of the mechanism involved. Thus, both Bom III and IV induce an apparent phenomenologically similar effect to that of the α-scorpion toxin AaH II on the slowed decline of sodium currents in mammalian neurons, but reveal diffe" @default.
• W2016442704 created "2016-06-24" @default.
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• W2016442704 date "1996-04-01" @default.
• W2016442704 modified "2023-10-07" @default.
• W2016442704 title "Scorpion Toxins Affecting Sodium Current Inactivation Bind to Distinct Homologous Receptor Sites on Rat Brain and Insect Sodium Channels" @default.
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