Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 218-222, January 1978 Biochemistry Binding of saxitoxin to electrically excitable neuroblastoma cells (ion transport/scorpion toxin/batrachotoxin) WILLIAM A. CATTERALL* AND CYNTHIA S. MORROW* Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Bernhard Witkop, November 1, 1977

ABSTRACT Saxitoxin inhibits the action potential Na+ permeability by 22Na+ influx (9), and measurement of scorpion ionophore of electrically excitable neuroblastoma cells with a toxin binding (11) have been described in detail. Scorpion toxin KI of 3.7 nM. Binding experiments detect a single class of sat- was purified by ion exchange chromatography and appeared urable binding sites with KD = 3.9 nM and a binding capacity of 156 fmol/mg of cell protein (78 sites per ,.m2 of cell surface). to be homogeneous in gel electrophoresis and isoelectric fo- Saturable binding is completely inhibited by tetrodotoxin but cusing experiments (9). The purified toxin was iodinated in a is unaffected by scorpion toxin or batrachotoxin. No saturable lactoperoxidase-catalyzed reaction and the labeled derivatives binding is observed in cultures of clone N103, a variant neuro- were separated by ion exchange chromatography (11). Unla- blastoma clone lacking the action potential Na+ response. Thus, beled, monoiodo and diiodo derivatives are separated by this saxitoxin binds specifically to the action potential Na+ iono- phore in neuroblastoma cells. Comparison of saxitoxin and technique. Only diiodo scorpion toxin was used in these studies. scorpion toxin binding reveals that there are three saxitoxin Because the preparations used contained only diiodo scorpion receptor sites for each scorpion toxin receptor site. The impli- toxin and no contaminating species, the specific radioactivity cations of this stoichiometry are considered. was exactly twice that of the sodium iodide (125I + 127I) used in the reaction and therefore was known with good accura- The heterocyclic neurotoxins, saxitoxin and tetrodotoxin, are cy. specific inhibitors of the action potential Na+ ionophore of Preparation and Characterization of [3HJSaxitoxin. Saxi- nerve axons (1, 2) and cultured neuroblastoma cells (3). These toxin was kindly supplied by E. J. Schantz, University of Wis- toxins bind specifically to axonal membranes at sites associated consin. [3H]Saxitoxin was prepared by the specific [3H]H20 with the action potential Na+ ionophore (4-6). The density and exchange procedure of Ritchie et al. (4). Dry saxitoxin (1.85 mg) some of the properties of these receptor sites have been de- was dissolved in [3H]H20 (25 Ci) and incubated at 500 for 3 hr. scribed. Labile 3H was removed in vacuo with H20 rinses at 0°. (These Purified polypeptide toxins from scorpion venom inhibit operations were performed by New England Nuclear Corp.) inactivation of the action potential Na+ ionophore in voltage Aliquots of this material were then purified by high-voltage clamp experiments on nerve axons (7, 8)t and act cooperatively paper electrophoresis as described by Ritchie et al. (4). Fractions with grayanotoxin and the alkaloids batrachotoxin, veratridine, from the electrophoretogram were tested for saxitoxin biologic and aconitine to cause persistent activation of the Na+ iono- activity and for [3H]saxitoxin. The concentration of saxitoxin phore (9). lodinated derivatives of scorpion toxin retain full in each fraction was determined by comparing the inhibition biologic activity (10, 11). These derivatives bind specifically of the action potential Na+ ionophore by [3H]saxitoxin fractions to action potential Na+ ionophores in electrically excitable and by standard saxitoxin solutions of known concentration as neuroblastoma cells (10, 11). Studies of scorpion toxin binding, illustrated in Fig. 1. A standard saxitoxin solution obtained from activation of the Na+ ionophore by scorpion toxin and alkaloid the U.S. Food and Drug Administration and a standard saxitoxin toxins, and inhibition of the Na+ ionophore by tetrodotoxin and solution prepared from dry saxitoxin in our laboratory gave saxitoxin have led to the hypothesis that the action potential Na+ identical results. ionophore contains three distinct receptor sites for neurotoxins The radiochemical purity of [3H]saxitoxin was determined (11, 12). One of these sites is specific for the polypeptide scor- by using the method of Levinson and Meves (5) and of Ritchie pion toxin and is involved in inactivation of the Na+ ionophore. et al. (4). This method depends on the capacity of excitable A second receptor site interacts specifically with the alkaloid tissue to bind native saxitoxin specifically. Freshly dissected rat toxins to cause persistent activation of the Na+ ionophore. Te- brains were homogenized in 0.25 M sucrose (10 ml/g wet trodotoxin and saxitoxin bind at a third receptor site which may weight). A membrane fraction was obtained by centrifugation be located within the transmembrane Na+ channel itself at 40,000 X gfor 15 min and was resuspended at a concentra- (13). tion of 0.4 g original wet weight/ml in the medium used for In this report, we describe the density, specificity, and saxitoxin binding assays (see below). Purified [3H]saxitoxin binding characteristics of the receptor sites for saxitoxin in (approximately 3 nM) was incubated at 00 for 30 min with in- electrically excitable neuroblastoma cells and compare these creasing concentrations (up to 150 mg original brain wet properties to those of the scorpion toxin receptor site described weight/ml) of the membrane fraction. These samples were then previously (10, 11). subjected to centrifugation for 5 min in a Beckman model B Microfuge and the [3H]saxitoxin remaining in the supernatant EXPERIMENTAL PROCEDURE was determined by scintillation assay and by measuring the The methods for growth of neuroblastoma cells (9), purification inhibition of the action potential Na+ ionophore as described and iodination of scorpion toxin (9, 11), measurement of Na+ (9). If the [3H]saxitoxin is pure, as suggested by the paper electrophoresis patterns, then the binding of 3H radioactivity The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked * Present address: Department of Pharmacology, University of "advertiement" in accordance with 18 U. S. C. §1734 solely to indicate Washington, Seattle, WA 98105. this fact. t Schwarz, W, Palade, P., and W. A. Catterall, unpublished data. 218 Downloaded by guest on September 26, 2021 Biochemistry: Catterall and Morrow Proc. Natl. Acad. Sci. USA 75 (1978) 219 and the saxitoxin biologic activity should be identical-In fact, as found previously by Ritchie et al. (4), a significant fraction of the 3H remains in the supernatant after 100% of the saxitoxin X /0 biologic activity is bound. This fraction ranged from 20 to 45% 6 ~~~~~~~~~~~~0 of the 3H in different purified fractions and must represent impurity in the [3H]saxitoxin preparations. This procedure provides a sensitive test of the purity of the labeled toxin. z -6 To verify that the 3H bound by the brain membranes in these 0 experiments represented [3H]saxitoxin, parallel experiments 0 were carried out in the presence of 10,uM tetrodotoxin to sat- `0 40 urate all saxitoxin receptor sites. Under these conditions, only 5% as much 3H was bound. Therefore, the bound 3H was almost 0 entirely [3H]saxitoxin. We conclude that our [3H]saxitoxin 20 - preparations have radiochemical purities ranging from 55 to 0 80%. Experiments with these different preparations gave quantitatively identical results, suggesting that the impurities 0D/ 0. 1 present had little influence on the binding measurements. At Saxcitoxin, nM (0) _ .,, I , , , ,~~,I~ ~ ~ ~, ~ , , least a portion of the impurity is [3H]H20 produced by back 10 100 1000 exchange from [3H]saxitoxin during electrophoresis and sub- 13H]Saxitoxin, ,sCilliter (a) sequent manipulations (4). Our initial attempts to remove the FIG. 1. Inhibition of the action potential Na+ ionophore by other impurities by thin-layer chromatography have not been [3Hjsaxitoxin and native saxitoxin. N18 cells were incubated with 100 successful. nM scorpion toxin for 30 min at 360 and then the initial rate of 22Na+ [3H]Saxitoxin Binding Assay. Saxitoxin binding was mea- influx was measured in the presence of 200 ,M veratridine and the sured in a medium consisting of 50 mM N-2-hydroxyethylpi- indicated concentrations of saxitoxin or [3H]saxitoxin as described perazine-N'-2-ethanesulfonic acid (adjusted to pH 7.4 with Tris (9). base), 130 mM choline chloride, 5.4 mM KC1, 0.8mM MgSO4, this is an incorrect assumption. Using their method (see Ex- and 5.5 mM glucose. At 36°, saxitoxin inhibition of the action perimental Procedure), we found that 55% of the 3H inthis potential Na+ ionophore of neuroblastoma cells is complete in preparation (Fig. 1) was associated with saxitoxin and therefore 10 min and binding of [3H]saxitoxin is complete in 10 min. In the specific radioactivity of the saxitoxin was 22.7 Ci/mmol. these experiments, cells in multiwell plates were incubated with Other preparations ranged up to 80% radiochemical purity with [3H]saxitoxin for 20 min at 36° to allow completion of the similar specific radioactivities. binding reaction. The inhibition of action potential Na+ iono- Binding of [3HJSaxitoxin to the Action Potential Na+ lo- phores in nerve axons and neuroblastoma cells by saxitoxin is nophore. Inhibition of the action potential Na+ ionophore of rapidly reversible. Bound [3H]saxitoxin dissociated from the neuroblastoma cells by saxitoxin is reversible and saturable and cells with a half-time of approximately 75 sec at 00. In the has an inhibition constant (KI) of 3.5 nM. Therefore, binding binding experiments, unbound [3H]saxitoxin was removed by experiments were designed to detect a saturable component of washing three times at 00 with 3 ml of wash medium consisting [3H]saxitoxin binding having a KD of approximately 3.5 nM. of 163 mM choline chloride, 5 mM N-2-hydroxyethylpipera- Fig. 2 illustrates the results of binding competition experiments zine-N'-2-ethanesulfonic acid (adjusted to pH 7.4 with Tris with [3H]saxitoxin. In these experiments, cells were incubated base), 1.8 mM CaC12, and 0.8 mM MgSO4. These washes re- with 2 nM [3H]saxitoxin and progressively increasing concen- quired 6-8 sec. During this time, less than 10% of the bound trations of unlabeled saxitoxin. If there is a saturable class of saxitoxin was lost. After completion of the experiment, cells receptor sites for saxitoxin; it is expected that the binding of were dissolved in 0.4 M NaOH and the bound radioactivity was [3H]saxitoxin will be progressively decreased by unlabeled determined by scintillation assay. saxitoxin as the unlabeled toxin saturates the binding sites. We observe that unlabeled saxitoxin inhibited 90% of the binding RESULTS of [3H]saxitoxin, with 50% inhibition obtained at 8 nM. These Inhibition of the Action Potential Na+ Ionophore by Na- results show that neuroblastoma cells contain a class-of saturable tive Saxitoxin and [3H]Saxitoxin. The increase in the Na+ binding sites for saxitoxin with a KD similar to the KI for inhi- permeability of neuroblastoma cells caused by persistent acti- bition of-the action. potential Na+ ionophore. vation of the action potential Na+ ionophore by alkaloid toxins The specificity of these binding sites was tested by studying and scorpion toxin is inhibited by tetrodotoxin and saxitoxin (9, the inhibition of [3Htsaxitoxin binding by other neurotoxins that 12). Fig. 1 illustrates inhibition curves for [3H]saxitoxin and have been shown to bind to the action potential Na+ ionophore. native saxitoxin. Like native saxitoxin, [3H]saxitoxin inhibited Tetrodotoxin and saxitoxin bind to a common receptor site in the Na+ permeability increase completely and the concentra- nerve axons with approximately equivalent affinity (4). We tion-response curve was described by a simple hyperbola, found (Figs 2) that tetrodotoxin competes for [3H]saxitoxin consistent with binding of the toxin to a single class of receptor binding sites as effectively as saxitoxin, indicating that these two sites. Because [3H]saxitoxin and native saxitoxin must have es- toxins also bind to a common receptor site on action potential sentially identical binding constants at this site, the concen- Na+ ionophores in neuroblastoma cells. tration of saxitoxin in the [3H]saxitoxin preparation can be de- Scorpion toxin also binds to specific receptor sites associated termined by superimposing the two inhibition curves as in Fig. with the action potential Na+ ionophore of neuroblastoma cells 1. Thus, 50% inhibition was obtained at 3.4 nM saxitoxin or at (10, 11). Saxitoxin and tetrodotoxin have no effect on scorpion a [3H]saxitoxin concentration of 140 ,uCi/liter. The specific toxin binding (11). As expected from these results, scorpion toxin radioactivity of this [3H]saxitoxin preparation therefore was 41.2 had no effect on saxitoxin binding (Fig. 2). Batrachotoxin and Ci/mmol, if it is assumed that all of the 3H was associated with other neurotoxic alkaloids activate Na+ ionophores in neuro- biologically active saxitoxin. Ritchie et al. (4) have shown that blastoma cells by interaction with a specific receptor site sep- Downloaded by guest on September 26, 2021 220 Biochemistry: Catterall and Morrow Proc. Nati. Acad. Sci. USA 75 (1978)

' 30

20 C

C :60 210 x

E 40 co, I I 20 0 5 10 15 20 [3H]Saxiloxin, nM

20 FIG. 4. Binding of [3H]saxitoxin (STX) to N103 cells. N103 cells were incubated for 20 min with the indicated concentrations of 0',i} 111 ii|l , ,1 [3H]saxitoxin alone (a) or with 1 ,M tetrodotoxin (0). Bound saxi- 10-10 10-9 10-8 10-7 10-6 toxin was then determined. Unlabeled toxin, nM RG. 2. Inhibition of [3H]saxitoxin binding by neurotoxins. N18 periment. The mean (+SEM) values for 10 experiments were cells were incubated for 20 min at 360 with 2 nM [3H]saxitoxin and 3.9 ± 0.8 nM for KD and 156 fmol/mg of cell protein for the the indicated concentrations of unlabeled saxitoxin (M), tetrodotoxin binding capacity. (0), batrachotoxin (0), and scorpion toxin (o). Bound saxitoxin was then determined. To further test the specificity of saxitoxin binding, neuro- blastoma clone N103 was studied. This clone lacks the action arate from the scorpion toxin binding site (14). Tetrodotoxin potential Na+ response (15) and does not bind scorpion toxin (14) and saxitoxin inhibited this activation noncompetitively. (11). We found only a small saturable component of saxitoxin As expected from these results, batrachotoxin had no effect on binding to N103 cells (Fig. 4), with a KD of approximately 4 nM saxitoxin binding (Fig. 2). These observations provide further and a binding capacity of 8 fmol/mg of cell protein. Thus, N103 support for the conclusion (11, 12) that there are three specific cells have only 5% as many [3H]saxitoxin receptor sites as N18 neurotoxin receptor sites associated with the action potential cells. These results provide further support for the conclusion Na+ ionophore: one that binds the inhibitors tetrodotoxin and that the saturable component of [3H]saxitoxin binding repre- saxitoxin, a second that binds scorpion toxin, and a third with sents binding to the action potential Na+ ionophore. which batrachotoxin, veratridine, aconitine, and grayanotoxin Comparison of [3H]Saxitoxin Binding and 125I-Labeled interact. Scorpion Toxin Binding. Previous work has shown that 125I- Saturable binding of saxitoxin also was observed in experi- labeled scorpion toxin binds specifically to action potential Na+ ments in which neuroblastoma cells were incubated with in- ionophores in electrically excitable neuroblastoma cells (10, 11). creasing concentrations of [3H]saxitoxin. The concentration A single class of saturable binding sites was observed with a profile of binding suggests the presence of both saturable and mean KD (for diiodo scorpion toxin) of 2.3 nM and a mean nonsaturable components. When the experiments were carried binding capacity of 47 fmol/mg of cell protein (11). Previous out in the presence of excess tetrodotoxin (1 ,uM) only a linear, results (11) and the data of Fig. 2 show that scorpion toxin and nonsaturable binding component was observed (Fig. 3 left). saxitoxin bind to separate receptor sites. Because it is likely that Because the receptor sites associated with the Na+ ionophores both of these receptor sites are associated with the action po- are saturated by this concentration of tetrodotoxin, this binding tential Na+ ionophore, it was of interest to compare their must represent nonspecific adsorbtion of [3H]saxitoxin. The density in electrically excitable cells. -We found 156 fmol of difference between the total binding and nonspecific binding saxitoxin sites per mg of cell protein in. these experiments represents specific binding to action potential Na+ ionophores. whereas we found only 47 fmol of scorpion toxin binding sites The specific binding component gave a straight line on a per mg of cell protein in our previous work. Scatchard plot (Fig. 3 right), indicating the presence of a single In order to confirm this large difference in receptor site class of saturable saxitoxin binding sites with a KD of 2.8 nM and density, we carried out several experiments in which both a binding capacity of 178 fmol/mg of cell protein in this ex- saxitoxin binding and scorpion toxin binding were measured in parallel in identical cell cultures. In addition, two different preparations of [3H]saxitoxin and of 125I-labeled scorpion toxin 0E were used. Typical results are illustrated in Fig. 5. In each ex- l- Total periment, we found approximately 3 times as many saxitoxin *0' binding sites. The data from eight experiments on clone N18 + 40 are summarized in Table 1.

c As a first step in testing the generality of this observation, we Er 20 an D0 studied clone SB37B, electrically excitable hybrid cell clone 'a Nonspecific formed between neuroblastoma clone NS20TG3 and L cell X I clone B82. our toxin data were more scattered 4 8 12 10 20 30 40 Although binding [3HISaxitoxin, nM STX bound, fmol/culture than those illustrated for clone N18, we consistently observed approximately three saxitoxin binding sites for each scorpion FIG. 3. Binding of [3H]saxitoxin (STX) to N18 cells. (Left) N18 toxin site with this cell line also. cells were incubated for 20 min at 360 with the indicated concentra- binding tions of [3H]saxitoxin alone (0) or in the presence of 1 ,uM tetrodo- DISCUSSION toxin (0). Bound saxitoxin was then determined. (Right) Specific binding, determined as the difference between the two curves in the Our results show that saxitoxin binds to a specific receptor site Left panel, is presented as a Scatchard plot. in electrically excitable neuroblastorma cells. As in nerve axons Downloaded by guest on September 26, 2021 Biochemistry: Catterall and Morrow Proc. Natl. Acad. Sci. USA 75 (1978) 221

T0 I Table 1. Saxitoxin inhibition, saxitoxin binding, and scorpion

0 toxin binding 3 i- 30II- - *15 0 0 Saxitoxin Scorpion toxin 0 0 20I %- - 0 KI, nM 3.7 4 0.5 ~10 I ~0 0 *0 KD, nM 4.1 i 0.9 2.4 0.6 In5 10 Binding capacity, fmol/mg 146 52.8 Site ratio 2.85 ± 0.18

I 0 I I-cLx 0o 4 8 12 0 10 20 30 40 Measurements were made on companion cultures in eight experi- Toxin, nM Toxin bound, fmol/culture ments using two different preparations of [3H]saxitoxin (55% pure and 80% pure) and two different preparations of scorpion toxin. All FIG. 5. Comparison of binding of [3H]saxitoxin and 1251-labeled experiments yielded comparable results. The mean ±SEM of each diiodo scorpion toxin to N18 cells. (Left) N18 cells were incubated measurement is presented. for 20 min at 360 with the indicated concentrations of [3H]saxitoxin alone or in the presence of 1 1AM tetrodotoxin, and specific binding 1) and binding of one molecule of scorpion toxin is sufficient (0) was determined. Companion cultures of N18 cells were incubated for 60 min at 360 with the indicated concentrations of 125I-labeled to enhance activation of one Na+ ionophore (9, 11). The 3:1 diiodo scorpion toxin alone or in the presence of 200 nM unlabeled stoichiometry of binding of saxitoxin and scorpion toxin can be scorpion toxin, and specific binding (0) was determined as described interpreted in at least two ways: (i) each action potential Na+ (11). Only the specific binding component is plotted. (Right) Specific ionophore might have three saxitoxin receptor sites and only binding of saxitoxin (0) and scorpion toxin (0) presented as a Scat- one scorpion toxin receptor site, or (ii) there might be two classes chard plot. of saxitoxin receptor sites, one associated with scorpion toxin receptor sites and one not. Because our present results do not (4), only a single class of binding sites is observed. The binding distinguish between these two alternatives, we consider briefly to these sites is complete within 10 min. Bound toxin is released the implications of both. with a half-time of 75 sec. The receptor sites have a KD of 3.9 1. One action potential Na + ionophore may contain three nM and a binding capacity of 156 fmol/mg of cell protein. This saxitoxin receptor sites and only one scorpion toxin receptor corresponds to 68,000 sites per cell or 78 sites per IAm2 of surface site. In this case, binding of saxitoxin to each of the three sites membrane. This density of saxitoxin receptor sites is similar to must be sufficient to inhibit Na+ transport because concen- that observed in nerve axons (4). tration-response curves are simple hyperbolas (Fig. 1 and ref. Our results provide strong evidence that the receptor sites 14); however, all three sites must be able to bind saxitoxin with are associated with the action potential Na+ ionophore. (i) The the same affinity. If each Na+ ionophore complex contains a binding constant is identical with the K1 for saxitoxin inhibition single ionic channel, this interepretation suggests a physical basis of the action potential Na+ ionophore in these cells. (ii) The for m3h dependence of the Hodgkin-Huxley model describing binding of [3H]saxitoxin is inhibited by tetrodotoxin with a KD voltage-dependent activation and inactivation of the Na+ io- identical to its KI for inhibition of the action potential Na+ io- nophore (16). These authors showed that activation and inac- nophore. (iii) A variant neuroblastoma clone (clone N103), tivation could be modeled as separate processes controlled by which lacks the action potential Na+ response but retains the two separate variables, m and h, which vary from 0 to 1 de- action potential K+ response (15), has no specific receptor sites pending on time and membrane potential. The fraction of Na+ for saxitoxin. We conclude that saxitoxin binds to a specific ionophores activated by a given voltage pulse depends on the receptor site on the action potential Na+ ionophores of neuro- product m3h in squid giant axon and on the product m2h in blastoma cells. A similar conclusion has been reached in studies frog node of Ranvier. Other higher power dependencies are of nerve axons (4). observed in special experimental circumstances. Hodgkin and Batrachotoxin causes persistent activation of the action po- Huxley suggested that this power dependence might reflect the tential Na+ ionophore of neuroblastoma cells by interaction involvement of three charged "particles" in activation of a with a specific receptor site (14). However, batrachotoxin has single ionic channel and one "particle" in inactivation. no effect on [3H]saxitoxin binding. Therefore, the batrachotoxin Because scorpion toxin specifically inhibits inactivation (7),t receptor site must be separate from the saxitoxin receptor site the 3:1 binding stoichiometry we observe may indicate that and saxitoxin must bind equally well to the active and inactive each ionophore contains three identical components (subunits?) (resting) states of the Na+ ionophore. that bind saxitoxin and are involved in the process of activation Scorpion toxin binds to a specific receptor on the action po- and only one component that binds scorpion toxin and is in- tential Na+ ionophore (10, 11) and inhibits inactivation of the volved in inactivation. If this is correct, the power to which m ionophore during a prolonged depolarization.t Scorpion toxin is raised is analogous to the Hill coefficients used to describe also has no effect on [3H]saxitoxin binding. Therefore, the cooperativity in hemoglobin and allosteric enzymes. The Hill scorpion toxin receptor site must be separate from the saxitoxin coefficients observed in these systems are always greater than receptor site and saxitoxin must bind equally well to inactivated 1, but they are seldom equal to the number of interacting sub- and resting states of Na+ ionophore. These results strongly units because they are strongly influenced by the energy of suggest that the saxitoxin receptor site undergoes no confor- interaction of the subunits (17). Similarly, the range of powers mational change during activation and inactivation of the Na+ of m observed in voltage clamp experiments may arise from ionophore. interactions among three identical components of the Na+ io- Comparison of the number of scorpion toxin and saxitoxin nophore whose energy of interaction varies in different species receptor sites in electrically excitable neuroblastoma cells in- and under different experimental conditions. Other authors dicates that there are approximately three saxitoxin receptor have suggested on theoretical grounds that the power depen- sites for each scorpion toxin receptor site. This observation is dence of m might arise from the presence ofsubunits in a 3:1 surprising because concentration-response curves and binding stoichiometry (18). curves suggest that binding of one molecule of saxitoxin is Alternatively, the action potential Na+ ionophore may exist sufficient to inhibit one action potential Na+ ionophore (Fig. as a complex containing three identical ion channels that all Downloaded by guest on September 26, 2021 222 Biochemistry: Catterall and Morrow Proc. Nati. Acad. Sci. USA 75 (1978) bind saxitoxin and one component that is involved in inacti- whereas, if mechanism 2 is correct, considerable variability will vation and binds scorpion toxin. This structure is consistent with be observed. the hypothesis (13) that saxitoxin binds to a site in the lumen of We thank Drs. John Daly and Bernhard Witkop for supplying ba- the ion channel and physically occludes it. It is also consistent trachotoxin and Dr. Edward Schantz for supplying saxitoxin. with the simple hyperbolic inhibition curves observed for sax- itoxin. In addition, this structure might provide a physical basis 1. Narahashi, T., Moore, J. W. & Scott, W. R. (1964) J. Gen. Physiol. for the power dependence of activation of the Na+ ionophore 47,965-974. if there are allosteric interactions among the three ion chan- 2. Hille, B. (1968) J. Gen. Physiol. 51, 199-219. nels. 3. Nelson, P. G., Pe~cock, J. H., Amano, T. & Minna, J. (1971) J. sites in Cell. Phsiol. 77, 337-352. 2. There may be two classes of saxitoxin receptor 4. Ritchie, J. M., Rogart, R. B. & Strichartz, G. R. (1976) J. Physiol. neuroblastoma cells, one associated with a scorpion toxin re- (London) 261, 477-494. ceptor and one not. If there are two classes of saxitoxin receptor 5. Levinson, S. R. & Meves, H. (1975) Philos. Trans. R. Soc. London sites, they must have identical affinities because our Scatchard Ser. B: 270,349-352. plots are consistently linear. In addition, both classes must be 6. Benzer, T. I. & Raftery, M. (1972) Proc. Natl. Acad. Sci. USA 69, missing from clone N10O. Finally, if there is a class of saxitoxin 3634-3637. receptor sites not associated with scorpion toxin receptor sites, 7. Okamoto, H., Takahashi, K. & Yamashita, N. (1977) Nature 266, these sites must also be insensitive to veratridine, batrachotoxin, 465-468. aconitine, and grayanotoxin because all of the Na+ ionophores 8. Romey, G., Chickeportiche, R. & Lazdunski, M. (1975) Biochem. these toxins also interact with scorpion Biophys. Res. Commun. 64,115-121. that are activated by 9. Catterall, W. A. (1976) J. Biol. Chem. 251, 5528-5536. toxin (11). It seems likely that such saxitoxin receptor sites that 10. Catterall, W. A., Ray, R. & Morrow, C. S. (1976) Proc. Natl. Acad. are not associated with scorpion toxin or alkaloid toxin receptor Sci. USA 73,2682-2686. sites might represent biosynthetic precursors to functional action 11. Catterall, W. A. (1977) J. Biol. Chem., in press. potential Na+ ionophores or Na+ ionophores modified by cel- 12. Catterall, W. A. (1975) Proc. Natl. Acad. Sci. USA 72,1782- lular regulatory mechanisms. It seems unlikely that receptor 1786. sites with such similar properties could arise on entirely inde- 13. Henderson, R., Ritchie, J. M. & Strichartz, G. R. (1974) Proc. Nati. pendent and unrelated macromolecules. Acad. Sci. USA 74,3936-3940. In order to distinguish rigorously between these two alter- 14. Catterall, W. A. (1975) J. Biol. Chem. 250, 4053-4059. native interpretations of the 3:1 binding stoichiometry we have 15. Peacock, J., Minna, J., Nelson, P. G. & Nirenberg, M. (1972) Exp. Cell Res. 73, 367-377. observed, it will be necessary to solubilize and purify the re- 16. Hodgkin, A. J. & Huxley, A. F. (1952) J. Physiol. (London) 117, ceptor molecules. This is not feasible at present. However, some 500-544. information concerning this important point can be obtained 17. Monod, J., Wyman, J. & Changeux, J. P. (1965) J. Mol. Biol. 12, from studies of saxitoxin and scorpion toxin binding in a spec- 88-118. trum of excitable tissues because we expect that, if mechanism 18. Hill, T. L. (1972) in Perspectives in Membrane Biophysics, ed. 1 is correct, all excitable tissues will have a 3:1 stoichiometry Agin, D. (Gordon and Breach, New York), pp. 187-203. Downloaded by guest on September 26, 2021