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Purification, Sequence, and Model Structure of Charybdotoxin, a Potent

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Purification, Sequence, and Model Structure of Charybdotoxin, a Potent Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3329-3333, May 1988 Biochemistry Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels (ion-channel blocker/scorpion toxin/sequence homologies/snake neurotoxin) GUILLERMO GIMENEZ-GALLEGO*t, MANUEL A. NAVIAt, JOHN P. REUBEN§, GEORGE M. KATZ§, GREGORY J. KACZOROWSKI§, AND MARIA L. GARCIA§¶ Departments of *Growth Factor Research, tBiophysics, and Membrane Biochemistry, Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065 Communicated by Edward M. Scolnick, January 6, 1988 ABSTRACT Charybdotoxin (ChTX), a protein present in crepancies were noted in the determination of the molecular the venom of the scorpion Leiurus quinquestriatus var. he- mass of ChTX based on amino acid composition and electro- braeus, has been purified to homogeneity by a combination of phoretic mobility of the protein, which could be explained by ion-exchange and reversed-phase chromatography. Polyacryl- inhomogeneity of the preparation. amide gel electrophoresis, amino acid analysis, and complete The present study describes the purification of ChTX to amino acid sequence determination of the pure protein reveal homogeneity, the biological activity ofthe pure toxin, and the that it consists ofa single polypeptide chain of4.3 kDa. Purified chemical characterization of this peptide in terms of amino ChTX is a potent and selective inhibitor of the -220-pS acid composition and sequence. The primary structure Ca21 -activated K+ channel present in GH3 anterior pituitary uniquely identifies this molecule and reveals its similarity to cells and primary bovine aortic smooth muscle cells. The toxin other toxins of species as phylogenetically distant as snakes reversibly blocks channel activity by interacting at the external and marine worms. Based on these similarities, a tertiary pore ofthe channel protein with an apparentKd of2.1 nM. The structural model of ChTX has been generated from the primary structure of ChTX is similar to a number of neuro- published (7) x-ray coordinates of a-bungarotoxin deposited toxins ofdiverse origin, which suggests that ChTX is a member in the Brookhaven Protein Data Bank (8). At present, ChTX of a superfamily of proteins that modify ion-channel activities. is the only agent that has been identified to cause potent On the basis of this similarity, the three-dimensional structure selectiveblockofapamin-insensitiveCa2 + -activated K + chan- of ChTX has been modeled from the known crystal structure nels, and hence it should be useful as a probe for studying the of a-bungarotoxin. These studies indicate that ChTX is useful properties of these channels. A preliminary report of these as a probe of Ca2+-activated K+ -channel function and suggest findings has been made in abstract form (9). that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block. MATERIALS AND METHODS High-conductance Ca2+-activated K+ channels have been described in a variety of electrically excitable and nonexcit- Materials. Lyophilized venom of the scorpion Leiurus able cells (1). These channels provide a pathway by which quinquestriatus var. hebraeus was obtained from Latoxan cell repolarization can occur after membrane depolarization, Scorpion Farm (Rosans, France). GH3 cells were purchased and consequently they have been implicated in the regulation from the American Type Culture Collection, while primary of neuroendocrine secretion, in the control of muscle con- cultures of bovine aortic smooth muscle were obtained as tractility, and in a number of other cellular processes. described (10). + Electrophysiological Analysis. Both GH3 and aortic smooth However, to assess the physiological role of Ca2 -activated muscle cells were grown as described (11) and cultured for K+ channels and attempt their purification, potent specific 2-4 days on 25-mm glass coverslips before use in electro- inhibitors of these channels are required. physiological experiments. Single Ca2l -activated K +-chan- The venom of the scorpion Leiurus quinquestriatus var. nel currents were monitored in outside-out excised mem- hebraeus is known to inhibit a number ofdifferent K +-channel brane patches by conventional patch-clamp procedures (12). pathways (2, 3). A minor component of the crude venom, The samples to be assayed were added directly to an termed charybdotoxin (ChTX), was discovered to block re- experimental chamber in which the microelectrode contain- versibly a large-conductance (-200 pS) Ca2"-activated K+ ing the excised patch was suspended. channel from rat skeletal muscle plasma membrane vesicles Purification of ChTX. Lyophilized scorpion venom (480 that had been reconstituted into planar lipid bilayers (4). mg) was gently homogenized in 20 mM sodium borate (pH Subsequently, ChTX was also found to inhibit low-con- 9.0), clarified by centrifugation at 27,000 x g for 15 min, ductance (=35 pS) Ca2"-activated K+ channels in neurons passed twice through Millex-GV filters (pore size, 0.2 ,um) from Aplysia californica, but not block Na+, Ca2+, transient (Millipore) and loaded onto a Mono-S column (HR5/5; K+, or delayed rectifying K + channels in this preparation (5). Pharmacia) equilibrated with the same buffer, at a flow rate The properties ofChTX have been preliminarily characterized of 0.5 ml/min. When the optical absorbance at 280 nm of the (6). The toxin was reported to be a protein with an apparent eluate decreased to within 0.06 optical density units above molecular mass of 10 kDa and to inhibit Ca2+-activated that of the elution buffer, the retained material was eluted K+-channel function with a Kd of3.5 nM. The same work also with a linear gradient of NaCl (0.75 M/hr). Fractions of this reports the amino acid composition of the protein and identi- column containing ChTX activity were loaded onto an fication of the N-terminal amino acid. However, some dis- Abbreviation: ChTX, charybdotoxin. The publication costs of this article were defrayed in part by page charge tPresent address: Centro de Investigaciones Biologicas, C.S.I.C., payment. This article must therefore be hereby marked "advertisement" Velazquez 144, 28006 Madrid, Spain. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. Downloaded by guest on September 25, 2021 3329 3330 Biochemistry: Gimenez-Gallego et al. Proc. Natl. Acad. Sci. USA 85 (1988) Ultrapore RPSC reversed-phase column (Beckman) equili- 0 brated with 10 mM trifluoroacetic acid, and eluted at a flow 1. 100 rate of 0.5 ml/min with a 0-20% linear gradient of isopro- c panol/acetonitrile (2:1) over 30 min. Fractions to be assayed from this chromatography were made either 350 mM in NaCl L or 0.5% in bovine serum albumin, lyophilized, and later L m reconstituted to their original volume with 20 mM sodium 4.) .0 borate (pH 9.0). L 0 . 50 U U Polyacrylamide Gel Electrophoresis. Samples were heated of (1000C, 2 min) with or without dithiothreitol (100 mM) in 3% mM of NaDodSO4/63 Tris*HCl, pH 6.2. Gels (1.6 mm thick) z a continuous concentration (25.7% polyacrylamide; acrylam- T- ide/bisacrylamide ratio, 37:1) were prepared and run for 24 m 0 hr at 3.3 V per cm of gel as described (13). Gels were fixed U, a 0 m as described (14) except that the first two steps were for 2 hr E: 5 10 15 5 10 15 each with hourly changes of solution and the glutaraldehyde EFFLUENT VOLUME (ml) treatment was also carried out for 2 hr. Fixed gels were silver stained (15). FIG. 1. Purification of ChTX. (A) Mono-S chromatography of 80 mg oflyophilized scorpion venom treated as described. Buffer B was Amino Acid Analysis and Extinction Coefficient Determina- 375 mM NaCl in 20 mM sodium borate (pH 9) and the gradient is tion. The optical absorbance spectrum of protein samples depicted as a dashed line. ChTX activity eluted where indicated by eluted from the reversed-phase column was digitized in a the horizontal bars. (B) Reversed-phase chromatography of an Hewlett-Packard 8450A UV/VIS spectrophotometer. Ali- aliquot of fraction A of the Mono-S chromatography. Buffer B is quots (o10-3 optical absorbance units at 280 nm) were isopropanol/acetonitrile (2:1). (Inset) A silver-stained NaDodSO4/ analyzed for their amino acid composition as described (16) polyacrylamide gel of reduced ChTX. Approximately 280 ng of the and the protein content was correlated with the recorded material obtained from the reversed-phase chromatography was absorbance. treated as described. Molecular mass standards are expressed in Amino Acid Sequence Determination. Pure ChTX was kDa. alkylated, digested with Staphylococcus aureus V8 protease, and sequenced as described (17, 18). Endoproteinase Lys-C column (51 mg). Reversed-phase chromatography of pool A (Boehringer Mannheim GmbH) digestion was carried out at (Fig. LA) resulted in a single major peak, which coelutes with 370C for 24 hr in 20 mM sodium phosphate (pH 7.6) at a ratio ChTX activity (Fig. 1B). In the case of pool B, two peaks of 55 pFg of ChTX per enzyme unit. Phenylthiohydantoin- were separated (data not shown); one with an identical derivatized amino acids were detected by using an on-line elution time, specific activity, and amino acid composition as phenylthiohydantoin analyzer (Applied Biosystems, Foster the one of Fig. 1B, while the other is an unrelated protein. City, CA; model 120A). Pyroglutamate aminopeptidase Together, both active fractions represent 60o of the total (Boehringer Mannheim GmbH) digestion was carried out protein content ofpools A and B (Fig. LA). Typically, 100 mg according to described procedures (19) with a molar ratio of of lyophilized venom yields 230 ug of ChTX. enzyme to ChTX of 1:10 and adding all enzyme at the Characterization of ChTX. NaDodSO4/polyacrylamide beginning of the reaction. gels of purified ChTX with (Fig.
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