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J. Biol. Chem., Vol. 279, Issue 25, 26588-26596, June 18, 2004

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Activation of Skeletal Ryanodine Receptors by Two Novel Scorpion Toxins from Buthotus judaicus^*

Xinsheng Zhu, Fernando Z. Zamudio¶, Beth A. Olbinski, Lourival D. Possani¶, and Héctor H. Valdivia||

From the Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706 and ^¶Department of Molecular Medicine and Bioprocesses, Biotechnology Institute, National Autonomous University of Mexico, Cuernavaca, Morelos 62271, Mexico

Buthotus judaicus toxin 1 (BjTx-1) and toxin 2 (BjTx-2), two novel peptide activators of ryanodine receptors (RyR), were purified from the venom of the scorpion B. judaicus. Their amino acid sequences differ only in 1 residue out of 28 (residue 16 corresponds to Lys in BjTx-1 and Ile in BjTx-2). Despite a slight difference in EC₅₀, both toxins increased binding of [³H]ryanodine to skeletal sarcoplasmic reticulum at micromolar concentrations but had no effect on cardiac or liver microsomes. Their activating effect was Ca²⁺-dependent and was synergized by caffeine. B. judaicus toxins also increased binding of [³H]ryanodine to the purified RyR1, suggesting that a direct protein-protein interaction mediates the effect of the peptides. BjTx-1 and BjTx-2 induced Ca²⁺ release from Ca²⁺-loaded sarcoplasmic reticulum vesicles in a dose-dependent manner and induced the appearance of long lived subconductance states in skeletal RyRs reconstituted into lipid bilayers. Three-dimensional structural modeling reveals that a cluster of positively charged residues (Lys¹¹ to Lys¹⁶) is a prominent structural motif of both toxins. A similar structural motif is believed to be important for activation of RyRs by imperatoxin A (IpTx_a), another RyR-activating peptide (Gurrola, G. B., Arevalo, C., Sreekumar, R., Lokuta, A. J., Walker, J. W., and Valdivia, H. H. (1999) J. Biol. Chem. 274, 7879-7886). Thus, it is likely that B. judaicus toxins and imperatoxin A bind to RyRs by means of electrostatic interactions that lead to massive conformational changes in the channel protein. The different affinity and structural diversity of this family of scorpion peptides makes them excellent peptide probes to identify RyR domains that trigger the channel to open.

Received for publication, March 24, 2004

The protein sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) P83405 and P83406.

^* This work was supported by National Institutes of Health Grant RO1HL55438 and by a grant-in-aid from the American Heart Association (to H. H. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Established Investigator of the American Heart Association.

To whom correspondence should be addressed: Dept. of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, 149 13th St., CYN-4, CVRC, 4201, Charlestown, MA 02129. Tel.: 617-726-0945; Fax: 617-726-5806; E-mail: xzhu2{at}bics.bwh.harvard.edu.

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