Evidence That Tetrodotoxin and Saxitoxin Act at a Metal Cation Binding Site in the Sodium Channels of Nerve Membrane (Solubilized Membrane/Receptors/Surface Charge) R
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Proc. Nat. Acad. Sci. USA 71 Vol. 71, No. 10, pp. 3936-3940, October 1974 Evidence That Tetrodotoxin and Saxitoxin Act at a Metal Cation Binding Site in the Sodium Channels of Nerve Membrane (solubilized membrane/receptors/surface charge) R. HENDERSON*, J. M. RITCHIE, AND G. R. STRICHARTZt Departments of Molecular Biophysics and Biochemistry, and of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Frederic M. Richards, June 17, 1974 ABSTRACT The effects of monovalent, divalent, and STX. These experiments suggest how the toxins exert their trivalent cations on the binding of tetrodotoxin and saxi- toxin to intact nerves and to a preparation of solubilized action, and provide a unifying explanation of how several nerve membranes have been examined. All eight divalent cations affect nerve membrane permeability. and trivalent cations tested, and the monovalent ions MATERIALS AND LiW, Tl+, and H+ appear to compete reversibly with the METHODS toxins for their binding site. The ability of lithium to Tritium-labeled TTX and STX were prepared and purified reduce toxin binding is paralleled by its ability to reduce (3, 5). Olfactory nerves from garfish, obtained from the Gulf tetrodotoxin-sensitive ion fluxes through the nerve mem- brane. We conclude that the toxins act at a metal cation Specimen Co., Florida, were dissected by the method of binding site in the sodium channel and suggest that this Easton (12). A detergent-solubilized extract of the nerves site is the principal coordination site for cations (normally was prepared by the method of Henderson and Wang (4). Na+ ions) as they pass through the membrane during an Binding experiments were also carried out on intact garfish action potential. The dissociation constant for Li+ is olfactory nerves and rabbit vagus nerves (3, 5). Where pos- 0.1-0.2 M and for Na+ > 0.6 M, reflecting the weak binding necessary for rapid passage of sodium ions through the sible, means ± standard errors of the mean are given. channel. Binding Assays. Most of the experiments described below consisted of the measurement of tritium-labeled toxin bound Tetrodotoxin (TTX) specifically blocks the early inward at equilibrium to nerves in the presence of different cations. sodium current that underlies the excitability of nerve and The method depended on whether intact nerve or the deter- muscle plasma membranes (1). Since the determination of its gent-solubilized extract was being used; the intact nerve was structure in 1964 (2), its relatively small size (molecular soaked in a solution containing the toxin, and the radioactivity weight 319), single positively charged guanidinium group, taken up by the nerve was determined after tissue solubiliza- and numerous hydroxyl groups have provoked much specula- tion (3), whereas the detergent extract was assayed for bind- tion about how the toxin acts. ing activity by a gel filtration equilibration technique (4). Recent studies have shown that tritium-labeled tetrodo- Corrections for nonsaturable uptake (determined largely toxin (3, 4, 6) and saxitoxin (STX) (5), a compound with some from measurements at high toxin concentrations) and for similar structural features and identical physiological action toxin present in the extracellular volume (determined with (1), can be used both to estimate the number of binding sites ['4C]mannitol) were always applied in determining the inhibi- present in nerve and to characterize the properties of the tion of saturable binding in intact nerves. The experiments toxin binding sites. The equilibrium dissociation constants with intact nerves were limited by the fact that a 6-hr soak and the kinetic rate constants for the toxin-receptor inter- was required for full equilibration of the toxin with nerve (3). action so obtained (3-7) agree well with those measured The binding to the detergent extract, by contrast, took less electrophysiologically (8-11). The labeled toxins thus promise than 5 min (4), but was limited to the pH range 6.5-8.5 by to be powerful tools in the purification of the receptor, which the lack of stability of the binding activity outside this range. must form at least part of the sodium channel (4, 7). In the detergent extract there is no detectable nonsaturable Here, we report evidence that TTX and STX act at a uptake at physiological ionic strength. Most results were specific metal cation coordination site. All the divalent and checked on both preparations. All assays were carried out at trivalent cations tested, and the three monovalent cations, 200. Li+, Tl+, and H+ reversibly block the binding of TTX and Flux Measurements were carried out on intact garfish olfactory nerves. The method used was to measure the efflux of tracer amounts of 22Na in the presence of both ouabain Abbreviations: TTX, tetrodotoxin; STX, saxitoxin; TMA, (0.2 mM), to abolish the pumped efflux of 22Na, and veratrine tetramethyl ammonium. (5.0 Ag/ml), to keep the channels "open." The specific flux of * Present address: MRC Laboratory of Molecular Biology, Hills 22Na through the sodium channels was measured by comparing Road, Cambridge CB2 2QH, England. of efflux in the and absence TTX. The t Present address: Department of Physiology and Biophysics, the rate presence of State University of New York, Stony Brook, L. I., N.Y. 11790. efflux in the absence of TTX was normally 50-100% Reprints should be requested from: Prof. J. M. Ritchie, De- higher than with TTX present. Measurements in hypertonic partment of Pharmacology, Yale University School of Medicine, solutions of both NaCl and LiCl could be made easily since the New Haven, Conn. 06510. fibers had a mean diameter of only 0.25 Am (13), allowing a 3936 Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Cationic Binding Site in Sodium Channels of Nerve 3937 TABLE 1. Binding of the toxins to solubilized membrane 1.41 in the presence of added divalent and trivalent cations 1.2 Choline Toxin bound -6 (fraction of K.pp -S. Cation (mM) Toxin control) (mM) c 1.0 0 0 La' ++ nitrate, 25 STX -0.03 <1 C .2 0.8 Cs 5 STX 0.02 <1 TMA 0 0.5 STX 0.56 <1 K Sm3+ acetate, 1 STX 0.16 <1 la 0.6 1 STX -0.10 <1 C 0 2.5 STX -0.03 <1 .0c Er'+ bromide, 1 STX 0.33 <1 04 x Be2+ chloride, 50 STX 0.02 <1 10 TTX 0.02 <1 ).2 100 STX 0.25 49 [- Mg2+ chloride, .'1 Tl b 20 STX 0.53 15 4 Ca2+ chloride, 100 STX 0.19 15 0.1 0.2 0.3 0.4 0.5 100 STX -0.10 (<1) 0 Molority of added cation 50 STX 0.14 5 50 STX 0.34 17 FIG. 1. Binding of TTX (2 nM) in the presence of monovalent 50 TTX 0.28 13 cations to solubilized membrane extract. All solutions contained 25 STX 0.28 6 10 mM Tris buffer (pH 7.2) in addition to the indicated concen- 10 STX 0.41 5 tration of the added cation. Chloride was the anion in all cases 8 STX 0.73 14 except with Tl+, where the nitrate salt was used. Furthermore, 8 TTX 0.67 11 0.15 M NaCl was also present in the experiments with Tl+. Sr2+ chloride, 160 STX 0.08 6 Each point is the mean of two or three experiments. 20 STX 0.36 7 Ba2+ chloride, 100 STX 0.29 27 20 STX 0.35 7 range 5-50 mM. The corresponding values for the trivalent ions were less than 1 mM. The inhibition was reversed on Toxin concentration was 2 nM. The binding in presence of removal of the ions. It also greatly exceeded that due to the added cation is expressed as fraction (5) of corresponding binding increased ionic strength alone; addition of 0.5 M choline in the absence of added cation. All binding measurements were chloride had only a minimal effect on toxin binding (Fig. 1). carried out on the solubilized nerve preparation by addition of The possible contribution of surface potential changes to the the appropriate concentration of ions to normal bathing solution observed inhibition is considered below. [0.15 M NaCI, 10 mM Tris-HCl (pH 7.2)]. K... for each cation, of TTX binding experiments with C, was calculated from the relation: a = {2 + KXTX(1 + [Na]/ Fig. 1 shows the results KN)}/{2 + KXTX(1 + [Na]/KN. + [C]/Kapp)I. Krrx was seven monovalent cations; similar experiments with STX gave taken as 9 nM (4, 5), KSTX as 7 nM (4, 5), KNa as 0.6 M (Fig. 1). similar results. With Tl+ and Li+ there was an appreciable inhibition of toxin binding that was clearly greater than the rapid equilibration of the internal ionic composition of the effects of any of the other monovalent cations; this inhibition nerve after the presumed initial shrinkage (see refs. 11 and was reversible. The inhibition can again be explained by a 14 for a more complete description of the methods). competitive inhibition with dissociation constants of about 10 mM for Tl+ (9.9 i 0.6 mM for the observations in Fig. 1) and RESULTS 0.13 i 0.01 M (four determinations) for Li+. The inhibition inhibition by Cations of Toxin Binding to Solubilized Mlem- by the other monovalent ions was smaller, and the differences branes. Table 1 gives the results of toxin binding assays in the between them were only slightly greater than the standard presence of eight different divalent and trivalent cations, the errors. However, the sequence Li+ > Na+ > K+ > Cs+, tetra- cation being added in the appropriate concentration to a methyl ammonium (TMA+), choline+ was clearly discerned.