Purification of binding protein for Tityus y toxin identified with the gating component of the voltage-sensitive Na ' channel ( Electrophorus electricus electroplax / electrical excitability / ion transport / scorpion toxin / tetrodotoxin )

The gating. component associated with the voltage-sensitive Na+ channel from electroplax membranes of Electrophorus electricus has been purified by using toxin y from the venom of the scorpion Tityus serrulatus serrulatus. The toxinbinding site was efficiently. solubilized with Lubrol PX, resulting in an extract of high initial specific activity. Purification was achieved by adsorption of the toxin-binding component to DEAESephadex A-25 followed by desorption at high ionic strength and chromatography on either wheat germ agglutinin-Ultrogel or Sepharose 6B. Maximal final specific activities were at least 42% of the specific activity expected for a pure toxin-binding component. The purified material exhibited a Stokes radius of 85 A, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated a single polypeptide component of M, 270,000. Furthermore, tetrodotoxin binding activity and Tityus y toxin binding activity copurified, suggesting that the selectivity filter and the gating component of the Na' channel are carried by the same polypeptide chain. Neurotoxic molecules have become essential tools in neurobiological studies (1, 2). A molecule rich in interactions with naturally occurring neurotoxins is the voltage-sensitive Na+ channel which is responsible for the initial rising phase of the action potential in excitable cells. Electrophysiological studies have identified a number of classes of specific neurotoxins that interact with the Na+ channel at discrete receptor sites, and the use of these toxins has afforded a detailed knowledge of the functional characteristics of this molecule. However, the structure and molecular mechanisms responsible for the function of the Na+ channel are less well characterized. Tetrodotoxin (TTX) and saxitoxin (STX), two toxins that are thought to interact at a common site associated with the selectivity filter, have been the most widely used to study the molecular properties of the Na' channel. The TTX/STX binding protein has been solubilized and substantially purified from the electric organ of Electrophorus electroplax (3-5), from rat brain (6), and from the sarcolemma of rat skeletal muscle (7, 8). There is now general agreement that a large glycoprotein, Mr 200,000270,000, is implicated in the Na+ channels from all three tissue sources. Furthermore, for the electric organ Na+ channel, this component has been shown to be associated with TTX binding activity (5). Whether other smaller components with Mr between 32,000 and 64,000 also are involved awaits clarification. Many other neurotoxins interact specifically with the Na+ channel, and four other distinct classes of binding sites, in addition to that of TTX/STX, have now been described on the basis of both biochemical and electrophysiological evidence (913). These include sites for (i) the lipophilic toxins such as veratridine and batrachotoxin, (ii) the pyrethroid toxins, (iii) polypeptide toxins such as Androctonus and Leiurus scorpion toxins and sea anemone toxins, and (iv) polypeptide toxins such as toxin II from Centruroides suffusus suffusus and toxin y from Tityus serrulatus serrulatus (TiTXy). This fourth class of toxins has recently been demonstrated to block Na' channels in frog muscle in a way different from that of TTX (12) and to produce spontaneous Na' channel activity on frog nodes of Ranvier (1416). These actions have been proposed to represent an action on the activation gating mechanism (16). Recent reconstitution studies performed on purified sarcolemmal STX binding component have suggested that at least two of these neurotoxin binding sites-i.e., veratridine and STX-are copurified (8). So far, no information regarding the copurification of the polypeptide toxin binding sites has been presented. In this report we describe the purification of the TiTXy receptor of the Na' channel from E. electricus electroplax and show that this component copurifies with TTX/STX binding activity. Direct biochemical assay of the solubilized receptor, identified with the gating component of the Na' channel, was made possible by use of the high-affinity polypeptide toxin, TiTXy (13). MATERIALS AND METHODS TiTXy was purified by the method of Possani et al (17) as modified by Sampaio et al. (18). Homogeneity of the pure toxin was demonstrated by polyacrylamide gel electrophoresis, isoelectric focusing, high-pressure liquid chromotography, and NH2terminal sequence analysis. TTX was obtained citrate-free from Sankyo. [3H]Ethylenediamine TTX ([3H]en-TTX) was synthesized and characterized according to Chicheportiche et aL (19). The [3H]en-TTX used had a specific radioactivity of 25 Ci/mmol (1 Ci = 3.7 x 1010 Bq) and a radiochemical purity of 90%. Protease inhibitors iodoacetamide (1 mM), pepstatin A (1 p.M), 1, 10-phenanthroline (1 mM), and phenylmethylsulfonyl fluoride (0. 1 mM) were included in all buffers that came into contact with TiTXy receptor. Additionally, p-bromophenacyl bromide (0.1 mM), an inhibitor of phospholipase A2, was included in all buffers used in purification procedures. Lubrol PX (10%) was deionized on a mixed-bed Amberlite MB3 resin before use. Wheat germ agglutinin (WGA) and L-phosphatidylcholine were obtained from Sigma; glutaraldehyde-activated Ultrogel was from Industrie Biologique Frangaise (IBF). WGA-Ultrogel (10 mg/ml) was prepared according to the IRF literature. Abbreviations: TTX, tetrodotoxin; [3H]en-TTX, [3H]ethylenediamine tetrodotoxin; STX, saxitoxin; TiTXy, Tityus serrulatus serrulatus y toxin; WGA, wheat germ agglutinin. 4164 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 80 (1983) 4165 Radioiodination of TiTXy. Pure TiTXy was radioiodinated by the lactoperoxidase/H202 method as described (13). After iodination, the 1251-labeled toxin ('25I-TiTXy) was purified by a modified procedure using a column (4 ml) of SP-Sephadex C25 preequilibrated in 150 mM NaCl/3 mM CaC12/0.2% bovine serum albumin/20 mM Tris'HCl, pH 8.7. The column was washed with 3-4 vol of the same buffer and the adsorbed 1251TiTXy was eluted with buffer adjusted to 350 mM in NaCl. Assay of Toxin Receptors. Assays of solubilized TiTXy receptors (final volume, 160 /.l) were performed in the presence of saturating concentrations (3-4 nM) of 125I-TiTXy in buffer containing 100 mM choline chloride, 0.02% phosphatidylcholine, 0.1% Lubrol PX, 2 mM CaC12, 0.1% bovine serum albumin, and 20 mM Hepes Tris at pH 7.4 (assay medium). After incubation for 30 min at 40C, 60-jul aliquots of the reaction mixture were loaded onto small columns (120 p.1) of DEAE-Sephadex A-25 preequilibrated in assay medium. Unbound 125I-TiTXy was removed by washing columns with 360 Ml of assay medium containing 175 mM choline chloride. The bound 1'5I-TiTXyreceptor complex was eluted with 480 Al of 600 mM choline chloride in assay medium, and the eluate was assayed in a -y counter. Nonspecific binding of 125I-TiTXy was estimated in parallel incubations containing an excess (30 nM) of unlabeled TiTXy. The specific receptor for TTX was assayed by using [3H]enTTX as described (20). Membrane Preparation and Solubilization. Electroplax membrane fragments were prepared in 20 mM Hepes Tris at pH 7.4 as described by Agnew et al. (3), and the resultant pellet was resuspended (2.5 ml/g) in the same buffer and stored at -70°C until used. The membrane preparation was solubilized by addition of 0.1 vol of 10% Lubrol PX and homogenization by five strokes in a Potter-Elvehjem homogenizer. After incubation for 30 min at 4°C the mixture was centrifuged at 150,000 x g for 30 min at 2°C to yield the detergent extract. Purification of TiTXy Receptor. Lubrol PX extract (30 ml) was adjusted to contain 150 mM KCI, 5 mM CaCl2, 0.3 MM TTX, and 0.1 MM 125I-TiTXy. After incubation for 10 min at 4°C to allow formation of 15I-TiTXy-receptor complex, the mixture was loaded onto a column (1 X 10 cm) of DEAE-Sephadex A-25 preequilibrated with medium A (20 mM Hepes Tris, pH 7.4/150 mM KCI/5 mM CaCl2/0.1% Lubrol PX/0.02% phosphatidylcholine/0.3 MM TTX) containing protease inhibitors. The column was washed with 30 ml of medium A and eluted with 60 ml of medium A containing 600 mM KC1. Fractions containing the peak of eluted radioactivity were pooled and diluted 1:2 in medium A containing no KCL. A column (8 ml) of WGA-Ultrogel was loaded with the diluted eluate, washed with 30 ml of medium A, and eluted with 48 ml of a linear gradient of 0-300 mM N-acetylglucosamine in medium A. Copurification of TiTXy and TTX Receptors. Detergent extract (30 ml) was adjusted to contain 150 mM KC1 and loaded onto a column (10 ml) of DEAE-Sephadex A-25. The column was washed with 30 ml of medium A and eluted with 40 ml of a linear gradient of 150-500 mM KCI in medium A. Fractions containing both TiTXy and TTX binding activity were pooled and incubated for 15 min at 4°C with 10 nM [3H]en-TTX. The incubation medium was loaded on a column (8 ml) of WGA-Ultrogel. The column was washed with 30 ml of 500 mM KC1 in medium A followed by 10 ml of medium A and eluted with 48 ml of a linear gradient of 0-300 mM N-acetylglucosamine in medium A. t3H]en-TTX was present at 10 nM throughout the wash and elution procedures. Nonspecific binding of [3H]enTTX was assessed throughout the purification by incubation of aliquots in an excess of unlabeled TTX (1 uM) for 15 min at 4°C and gel filtering under normal assay conditions (20). Protein Assay. Protein concentrations were determined by the method of Peterson (21) with bovine serum albumin as standard. NaDodSO4 Gel Electrophoresis. Electrophoresis was performed under reducing conditions in slab (14 x 13 X 0.15 cm) polyacrylamide gels with a 4-12% linear acrylamide gradient according to Laemmli (22). Gels were stained for protein by the silver staining method of Merril et al. (23).