Biochemistry zyxwvu 1984, 23, zyxwvu 1147-1 152 1147 Acknowledgments The assistance and valuable advice of Dr. Martha Vaughan in preparation of the manuscript is gratefully acknowledged. I also thank Dr. Hideo Hidaka for kindly providing a gift of W-7, D. Marie Sherwood and Barbara Mihalko for expert secretarial help, and Carol Coulson for excellent technical assistance in some of the experiments. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Registry zyxwvutsrqpo No. cGMP, 7665-99-8; W-7,65595-90-6; Ca, 7440-70-2; trifluoperazine, 11 7-89-5; dithiothreitol, 3483- 12-3. References Andreasen, T. J., Keller, C. H., LaPorte, D. C., Edelman, A. M., zyxwvutsrqpo & Storm, D. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2782-2785. Davies, G. E., & Stark, G. R. (1970) Proc. Natl. Acad. Sci. Kincaid, R. L., & Vaughan, M. (1983) Biochemistry 22, Kincaid, R. L., Manganiello, V. C., & Vaughan, M. (1981) Kincaid, R. L., Osborne, J. C., Jr., Vaughan, M., & Tkachuk, LaPorte, D. C., Toscano, W. A., Jr., & Storm, D. R. (1979) Peters, K., & Richards, F. M. (1977) Annu. Reu. Biochem. Quiocho, F. A., & Richards, F. M. (1966) Biochemistry zy 5, U.S.A. 66, 651-656. 826-830. J. Biol. Chem. 256, 11345-11350. V. A. (1982) J. Biol. Chem. 257, 10638-10643. Biochemistry 18, 2820-2825. 46, 523-551. 4062-4076. Binding of ,&Scorpion Toxin: A Physicochemical Study? Emmanuel Jover,* Joseph Bablito, and Fransois Couraud ABSTRACT: The binding to rat brain synaptosomes of a 0- scorpion toxin, i.e., toxin I1 of Centruroides zyxwvut suffusus suffusus (Css 11), was studied as a function of pH, temperature, and concentration of some monovalent and divalent cations. At 10 “C and pH 6.0, the specific binding of 1251-labeled Css I1 corresponds to a single class of noninteracting high-affinity binding sites (KD = 0.18 nM) with a capacity (4.2 pmol/mg of protein) that is almost identical with that generally accepted for saxitoxin. The equilibrium dissociation constant of p- scorpion toxin is pH independent, but the maximum binding capacity is reduced with increasing pH. Li+, guanidinium, Ca2+, Mg2+, and Mn2+ modified the apparent KD of the A large number of neurotoxins of animal or plant origin modify the properties of the sodium channel involved in the action potential of excitable cells. Some have been radiolabeled and their binding properties compared to their pharmacological effect. These investigations lead Catterall (1980, 1982) to suggest the existence of three distinct neurotoxin binding sites associated with the sodium channel. Site “1” binds saxitoxin and tetrodotoxin, which are heterocyclic molecules with a guanidium group. Sodium transport is inhibited when this site, located on the outer side of the membrane, is occupied. Site “2” binds several liposoluble toxins, e.g., grayanotoxin and the alkaloids batrachotoxin, veratridine, and aconitine. These toxins cause persistent activation of sodium channels at the resting membrane potential by blocking channel inactivation and by shifting the voltage dependence of sodium-channel activation to more negative membrane potentials. Site “3” recognizes the scorpion neurotoxins that we have called CY- scorpion toxins (Jover et al., 1980) and sea anemone neuro- toxins. These neurotoxins slow or block the sodium-channel inactivation phase. We have identified a fourth site linked to the sodium channel that recognizes neurotoxin I1 of Centruroides suffusus suf- From the Labratoire de Biochimie and U.172 de l’I.N.S.E.R.M., Facultt de Madecine, 13326 Marseille Cedex 15, France. Received Muy 16,1983. This work was supported in part by the CNRS (ERA 070617, ATP Pharmacologie des REcepteurs Neuromtdiateurs). lZ51-labeled Css I1 toxin. The equilibrium dissociation constant varies markedly with the temperature. The van’t Hoff plot of the data is curvilinear, corresponding to a standard free- energy change associated with an entropy-driven process. The association rate constant also varies considerably with the temperature whereas the Arrhenius plot is linear between 1 and 30 OC. The energy of activation determined from these data is 17.6 kcal/mol. These results support the hypothesis that a cluster of nonpolar amino acid residues present on one face of the molecule is involved in the toxin-receptor inter- action. fusus venom (Css 11), a @-scorpion toxin (Jover et al., 1980). Contrary to a-scorpion toxins, the affinity of p-scorpion toxins for their receptor site does not vary with the membrane po- tential. Css I1 binding to site 4 modifies sodium-channel activation, as shown by the Occurrence of an abnormal sodium current after repolarization of the myelinated frog nerve (Couraud et al., 1982) and by the blocking of sodium con- ductance activation in the frog skeletal muscle (Jaimovich et al., 1982). Css I1 toxin inhibits the uptake and stimulates the release of y-aminobutyric acid by rat brain synaptosomes, an effect that is abolished by tetrodotoxin (Couraud et al., 1982). Two toxins from the venom of the scorpion Centruroides sculpturatus Ewing showed similar effects to those of Css I1 on the node of Ranvier of the myelinated frog nerve (Meves et al., 1982), and a toxin from Tityus serrulatus venom com- petes with Css I1 at the same binding site (Wheeler et al., 1982). The present paper describes a physicochemical study of a 0-scorpion toxin (Css 11) interaction with site 4. The association and dissociation kinetic constants of this toxin have been determined and compared with those of saxitoxin as defined by Weigele & Barchi (1978a) and those of toxin I1 of Androctonus australis Hector, an a-scorpion toxin, which we have measured in the same biological system (Jover et al., 1978). In addition, the thermodynamic param- eters in the ligand-receptor reaction were estimated. The results obtained suggest that the interaction of Css I1 with its receptor involves polar attraction, which may be followed by 0006-2960/84/0423-1147$01.50/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1984 American Chemical Society