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Binding of Sea Anemone Toxin to Receptor Sites Associated with Gating System of Sodium Channel in Synaptic Nerve Endings in Vitro (Scorpion Neurotoxin/Synaptosomes) J

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Binding of Sea Anemone Toxin to Receptor Sites Associated with Gating System of Sodium Channel in Synaptic Nerve Endings in Vitro (Scorpion Neurotoxin/Synaptosomes) J Proc. Nati. Acad. Sci. USA Vol. 77, No. 3, pp. 1646-1650, March 1980 Neurobiology Binding of sea anemone toxin to receptor sites associated with gating system of sodium channel in synaptic nerve endings in vitro (scorpion neurotoxin/synaptosomes) J. P. VINCENT, M. BALERNA, J. BARHANIN, M. FOSSET, AND M. LAZDUNSKI Centre de Biochimie, Universit6 de Nice, Parc Valrose, 06034 Nice cedex, France Communicated by Heinz Fraenkel-Conrat, October 9, 1979 ABSTRACT Iodination of toxin II from the sea anemone nel. These polypeptide toxins (17-19) selectively alter the Anemonia sulcata gives a labeled monoiododerivative that re- functioning of the gating system of the channel without inter- tains 80% of the original neurotoxicity. This derivative binds fering with the binding of tetrodotoxin or saxitoxin at or near specifically to rat brain synaptosomes at 20'C and pH 7.4 with a second-order rate constant of association ka = 4.6 X 104 M-' the selectivity filter (4, 8). They have been shown to interact sect and a first-order rate constant of dissociation kd = 1.1 X with a large variety of excitable membranes including my- 10-2 sect. The binding occurs on the Na+ channel at a binding elinated and nonmyelinated axons (4, 5), neuronal cells in site distinct from that of other gating system toxins like batra- culture (8), cardiac and skeletal muscle cells in culture (7), and chotoxin, veratridine, grayanotoxin, aconitine, and pyrethroids. nerve terminals (20). The maximal binding capacity Bmax is 3.2 pmol/mg of protein We describe in this paper the preparation and purification (i.e., about two sea anemone toxin binding sites per tetrodotoxin of a labeled monoiododerivative (['251]ATX11) of toxin II from binding site) and the Kd is 240 nM for the monoiododerivative This derivative and 150 nM for the native toxin. Corresponding binding pa- the sea anemone Anemonia sulcata (ATX11). rameters for the association of a 1251-labeled derivative of toxin has been used to study properties of interaction between ATXII II from the scorpion Androctonus australis Hector are Bmax = and rat brain synaptosomes. Results are compared with those 0.3 pmol/mg of protein and Kd = 1 nM, whereas the Kd of the obtained when using a labeled iododerivative ([1251]AaH11) of unmodified scorpion toxin is 0.6 nM. Competition experiments toxini from the scorpion Androctonus australis Hector involving scorpion toxins, sea anemone toxins, and synapto- somes demonstrate that, although the sea anemone toxin is able (AaH1I). to displace the scorpion toxin bound to synaptosomes, the MATERIALS AND METHODS scorpion toxin does not displace the sea anemone toxin. The sea anemone toxin but not the scorpion toxin binds to depolarized Toxins I, II, and III from the sea anemone Anemonia sulcata synaptosomes. Differences between binding properties of the and a purified fraction from the venom of the scorpion Leiurus two polypeptide toxins are analyzed in the discussion. quinquestriatus (venom obtained from Sigma) were prepared as described (16, 21). Toxin II from the scorpion Androctonus A number of toxic molecules are now available for the analysis australis Hector was kindly given to us by S. Lissitzky's group of the structural organization and function of the voltage- (Marseille, France). dependent Na+ channel in excitable membranes. These toxins The guanidine side chain of the only arginine residue of the include (i) tetrodotoxin and saxitoxin, which bind at or near the ATX11 sequence, Arg-14, has been modified by 1,2-cyclohex- selectivity filter of the Na+ channels (1, 2); (ii) veratridine, anedione, using the method of Toi et al. (22) as described for batrachotoxin, aconitine, and grayanotoxin, which depolarize the modification of the bee venom neurotoxin apamin (23). excitable membranes by causing a persistent activation of the Amino acid analysis of the ATX11 derivative obtained after Na+ channels (1); and (iii) sea anemone and scorpion neuro- cyclohexanedione treatment indicates a selective and complete toxins, which slow down the closing (inactivation) of Na+ modification of Arg-14; none of the other amino acid residues channels in axons (3-5) and in various excitable cells in culture of thie toxin was modified. Total modification of Arg-14 results (6-10). in complete loss of toxicity. The biochemical characterization of receptors that selectively lodination of Toxins. ATXII was iodinated by the chlora- recognize each class of toxins is an important step towards the mine-T method (24). ATX11 (5 mg) was incubated with 5 mCi understanding of the molecular mechanisms of action of these (1 Ci = 3.7 X 0'10 becquerels) of Na'251 (Commissariat a toxins and the mode of functioning of the Na+ channel in ex- l'Energie Atomique or Amersham) and 0.1 ,Imol of unlabeled citable tissues. Receptors that have been best characterized NaI in 0.25 ml of 20 mM Tris-HCl (pH 8.6) at room tempera- biochemically are those of tetrodotoxin and saxitoxin (2, 11, 12). ture. Three aliquots (10 ul) of a 10 mM solution of chloramine-T Radiolabeled scorpion toxins have also been prepared and used (Merck) were added at 2-min intervals. The mixture was in- to identify Na+ channels (13-15). The binding of these poly- cubated for 30 more min after addition of the last chloramine-T peptide toxins seems to be strongly voltage dependent (16) and aliquot. Bovine serum albumin (200 Atl of a 1% solution) was for that reason will probably be difficult to use in biochemical then added and the mixture was immediately loaded on a studies of the Nab channel that necessitate working with SP-Sephadex C-25 column (2 X 21 cm) equilibrated and eluted membranes or detergent-solubilized fractions. with a 50 mM NaH2PO4 buffer (pH 5.5) containing 160 mM Sea anemone toxins are among the most interesting tools for analysis of the properties of the voltage-dependent Na+ chan- Abbreviations: ATX11, toxin II from the sea anemone Anemonia sul- cata; [l25I]ATXII, radiolabeled monoiododerivative of ATXI with 1 The publication costs of this article were defrayed in part by page + 0.1 (SEM) iodine atom per mol of ATX~J; AaH11, toxin II from the charge payment. This article must therefore be hereby marked "ad- scorpion Androctonus australis Hector; [l2I]AaHIl, radiolabeled io- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate doderivative of AaH1j with 0.69 + 0.1 (SEM) iodine atom per mol of this fact. AaH11; LD50, mean lethal dose. 1646 Downloaded by guest on September 23, 2021 Neurobiology: Vincent et al. Proc. Natl. Acad. Sci. USA 77 (1980) 1647 NaCI. The chromatography was followed by automatic re- RESULTS cording of the absorbance at 230 and 280 nm with a Seive Characterization of 125I-Labeled ATXI and AaHj1. ATXI1 elugraph and by measurement of the radioactivity in each is a single-chain protein of 47 amino acids crosslinked by three fraction with a Packard Tri-Carb liquid scintillation spectrometer disulfide bridges (18). Although there is no tyrosine residue in (model 2450 or 3390). AaHII was iodinated by the lactoperox- the sequence, there are two histidine residues, His-32 and idase method and purified by gel filtration as described (25). His-37, which can be iodinated. ATX11 was iodinated by the Different specific radioactivities (from 12.5 to 2000 Ci/mmol) chloramine-T method with a mixture of labeled (125I) and un- were obtained for both iodotoxins by varying the ratio of Na'25I labeled (1271) NaI. Fig. 1 shows that a chromatography of the to Na'271 used in the iodination procedures. iodination mixture on sp-Sephadex C-25 at pH 5.5 not only LD5o Measurements. LD50 measurements on mice or crabs separates the toxin from excess reagent (iodine and chlora- were carried out as described (25, 26). mine-T) but also divides the toxin into two different fractions. Synaptosome Preparation. Synaptosomes were prepared In five different series of experiments, the number of iodine from brains of male Sprague-Dawley rats (200-250 g body atoms per mol of toxin was found to be 1i 0.1 in the peak that weight) by the method of Gray and Whittaker (27) as modified is centered around fraction 24. The peak around fraction 43 by Abita et al. (20). Protein concentration was determined by consists of unmodified ATX1I. The mean lethal dose (LD5o) of the method of Hartree (28) with bovine serum albumin as a the monoiodo ATXI1 derivative is 0.21 mg/kg on mice and 2.2 standard. ,ag/kg on crabs compared to 0.17 mg/kg and 2.0 ,ag/kg, re- Binding Experiments. Cellulose acetate filters (Sartorius SM spectively, for the native toxin. Accordingly, we have prepared 11107, 0.2 1Am pore size) were used in binding experiments and purified a monoiododerivative of ATX11 that possesses more involving synaptosomes and either ['25I]ATXII or [125I]AaHII. than 80% of the original neurotoxicity. They were kept in 1% bovine serum albumin for 2 hr and then Scorpion toxin II from Androctonus australis Hector was washed once with5 ml of the incubation buffer before use. The iodinated by the lactoperoxidase method and purified by fil- standard incubation buffer consisted of 140 mM choline chlo- tration on Sephadex as described (25). The number of iodine ride, 5.4 mM KCI, 2.8 mM CaCI2, 1.3 mM MgSO4, 0.1% bovine atoms per mol of toxin is 0.69 : 0.1. We have checked that the serum albumin, and 20 mM Tris-HCI, pH 7.4 (choline medi- specific toxicity of the scorpion neurotoxin was not modified um). As indicated in the text, identical concentrations of NaCI by iodination under the conditions used, as reported (25).
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