Vol. 2.53, No 20, Issue of October 25, pp. 7393-7396, 1978 Printed in C! .%A

Sea Anemone Toxin and Scorpion Toxin Share a Common Receptor Site Associated with the Action Potential Sodium Ionophore*

(Received for publication, April 20, 1978)

William A. CatteralQ

From the Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98195

Laszlo Beressg

From the Institut fur Meereskunde an der Universitat Kiel, 23 Kiel, Dusternbrooker Weg 20, West Germany

Toxin II isolated from the sea anemone Anemonia sukata enhances activation of the action potential so- dium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This hetero- tropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a llO-fold higher concentration. Depolarization of the neuroblas- toma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scor- pion toxin binding is inhibited by sea anemone toxin with KD = 90 11~. These results show that the polypep- tides scorpion toxin and sea anemone toxin II share a common receptor site associated with action potential sodium ionophores.

Extracts of sea anemone nematocysts cause repetitive ac- tion potentials in crustacean giant axons (1) by inhibiting the inactivation process of the action potential sodium ionophore (2). Recently, three polypeptide toxins have been purified from the sea anemone Anemonia sulcata (3, 4). The most abundant of these (designated sea anemone toxin II) inhibits inactivation of sodium ionophores in crustacean giant axons and in frog myelinated nerve (5-7).

Venom of the scorpions Leiurus quinquestriatus and Bu- thus tam&us and a polypeptide toxin purified from L. quin- questriatus venom also inhibit inactivation of sodium iono- phores in nerve axons and muscle’ (8,9). Scorpion toxin binds to a specific receptor site associated with action potential sodium ionophores of electrically excitable neuroblastoma cells (10, 11). Binding to this site is highly dependent upon membrane potential (10, 11). Occupancy of this receptor site by scorpion toxin causes a striking enhancement of activation of sodium ionophores by veratridine, batrachotoxin, aconitine, and grayanotoxin (12, 13). This heterotropic cooperative in- teraction can be described by a simple allosteric model, which assumes that the activating toxins bind with high affinity to the active state of the sodium ionophore and that scorpion toxin reduces the energy required for the transition from the inactive to the active state (13).

* 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.

f Sunnorted bv Grant HL 22239-01 from the National Heart, Lung, , .- and Blood Institute and by a grant from the University of Washing& Graduate Research Fund. To whom reprint requests should be ad- dressed.

0 Supported by the Deutsche Forschungsgemeinschaft Grant BE 55418.

’ W. Schwan, P. Palade, and W. A. Catterall, unpublished results.

Since both sea anemone and scorpion toxins are polypep- tides and inhibit inactivation of sodium ionophores, it seemed likely that these two toxins might act by a common mecha- nism. In these experiments, we have compared the interaction of sea anemone and scorpion toxins with specific receptor sites associated with sodium channels in electrically excitable neu- roblastoma cells.

EXPERIMENTAL PROCEDURES

Materials-Sea anemone toxin II was purified from A. sulcata as described previously (3, 4). Scorpion toxin was purified from the venom of L. quinquestriatus (13) and iodinated (11) as described previously. Batrachotoxin was kindly provided by Drs. J. Daly and B. Witkop (Laboratorv of Chemistrv. National Institute of Arthritis. Metabolism, and Digestive Diseases, National Institutes of Health): Other materials were purchased commercially as previously described (11).

Cell Cultures-Clone N18 of mouse neuroblastoma Cl300 was used for all studies. Cell cultures were prepared as described previously (12).

Sodium Permeability Measurements-Sodium permeability was determined by measuring the initial rate of **Na* influx from medium of low sodium concentration (10 mm) after inhibition of (Na+,K+)- ATPase by ouabain. The details of these procedures have been described previously (13, 14). Control experiments have shown that 22Na+ influx (&) is linearly related to sodium permeability (PN*) under these conditions (14).

Scorpion Toxin Binding Measurements-Scorpion toxin binding was measured exactly as described previously using pure scorpion mono[““I]iodotoxin as the binding ligand (11).

RESULTS

Cooperative Activation of Sodium Ionophores by Sea Ane- mone Toxin and Veratridine or Batrachotoxin-Sea ane- mone toxin has no effect on sodium permeability when added to neuroblastoma cells alone (Fig. 1, 0). When electrically excitable neuroblastoma cells are incubated with increasing concentrations of sea anemone toxin for 30 min and rinsed, and then sodium permeability is measured in the presence of 200 pM veratridine, sea anemone toxin causes a large increase in veratridine-dependent sodium permeability (Fig. 1, A). Both the increase in sodium permeability caused by veratri- dine alone (Fig. 1,points on ordinate) and the increase caused by veratridine plus sea anemone toxin are completely in- hibited by 1 pM tetrodotoxin, a specific inhibitor of action potential sodium ionophores (Fig. 1, 0). These results are similar to those described previously for scorpion toxin (13) and suggest a heterotropic cooperative interaction between veratridine and sea anemone toxin in activating the action potential sodium ionophore.

In order to study this interaction in more detail, complete concentration-effect curves were determined for veratridine

7393

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7394 Common Receptor Site for Sea Anemone and Scorpion Toxins

[SEA ANEMONE TOXIN], pM

FIG. 1. Effect of sea anemone toxin on “Na+ uptake. N18 cells were incubated for 30 min at 36’C in sodium-free choline-substituted medium (13) containing the indicated concentrations of sea anemone toxin. 22Na+ uptake was then measured in choline-substituted medium with no additions (a), 200 pM veratridine (A), or 200 pM veratridine plus 1 PM tetrodotoxin (0).

and batrachotoxin in the presence of different fixed sea ane- mone toxin concentrations (Fig. 2). Under the conditions of these experiments, veratridine is a partial agonist activating

approximately 8% of the sodium ionophores (14). Sea anemone toxin increases the fraction of sodium ionophores activated at saturating concentrations of veratridine and reduces the con- centration of veratridine required to give 50% maximum acti- vation (Fig. 2, left). Batracbotoxin is a full agonist activating greater than 95% of the sodium ionophores (14). Sea anemone toxin reduces KM for batrachotoxin (Fig. 2, right) but has little effect on P, since nearly all the sodium ionophores are activated by batrachotoxin alone. The curves in Fig. 2 are computed fits to a simple hyperbolic saturation function of the form P(A) = P,A/(Ko.5 + A) where A is the concentration of veratridine or batrachotoxin. The data points conform closely to these theoretical curves, indicating that no homo- tropic cooperativity is involved in activation of sodium iono- phores by veratridine and batrachotoxin in the presence or absence of sea anemone toxin.

An allosteric model has been described previously which successfully fits the data on the heterotropic cooperative interaction between scorpion toxin and the activating toxins (14). In this model, the sodium ionophore is assumed to exist in (at least) two states, active (R) and inactive (T). There are reversible transitions between the two states characterized by an equilibrium constant (Mm-). Veratridine and batrachotoxin cause activation by binding more tightly to the active (R) state; that is, KR < KT where KR and KT are equilibrium dissociation constants for binding of veratridine and batrach- otoxin to the R and T states. Scorpion toxin and sea anemone toxin reduce the value of MRT and thereby enhance. the activation by veratridine and batrachotoxin. The fraction of ionophores activated (PR) as a function of activator concen- tration (A) is given by:

PI<(A) = 1

1 + A/K., 1 + A4II.I. ~

1 + A/Kn

The values of these parameters derived from least squares fits the data are presented in Table I. As found previously for scorpion toxin (14), the assumption that sea anemone toxin reduces the allosteric equilibrium constant MRT allows an

’ The abbreviations used are: Kor,, the concentration of toxin caus- ing 50% maximal effect on 2*Na+ uptake or scorpion ‘2”I-toxin binding; P,, the maximum Na’ permeability at saturating concentrations of toxin.

adequate fit of the data. Thus, the effects of both sea anemone toxin and scorpion toxin can be accommodated by this allo- steric model.

Competition between Sea Anemone and Scorpion Toxins-Since the binding of scorpion toxin to its receptor site on the action potential sodium ionophore can be measured directly, it is possible to test whether sea anemone toxin competes with scorpion toxin for that receptor site. In these experiments, cells were incubated with 0.5 nM ‘““I-labeled scorpion toxin and increasing concentrations of either unla- beled scorpion toxin or sea anemone toxin and specifically bound ““I-labeled scorpion toxin was measured. Bound scor- pion toxin was displaced by unlabeled scorpion toxin with a KO,S of approximately 1.4 nM (Fig. 3). Bound scorpion toxin was also displaced by sea anemone toxin but with a KOA of approximately 150 nM. Assuming simple competitive inhibi- tion, the Kjj for sea anemone toxin at this receptor site is 90 nM. This concentration of sea anemone toxin is similar to the Ko,s observed for enhancement of veratridine activation of sodium ionophores (Fig. 1). These results indicate that sea anemone toxin binds to the same receptor site as scorpion toxin and has similar effects on the sodium ionophore.

Data from a more extensive experiment like that in Fig. 3 are replotted in Fig. 4 using coordinates analogous to those of a Scatchard plot. A straight line is obtained consistent with the presence of a single class of independent binding sites for sea anemone toxin with K/j = 78 nM.

Tetrodotoxin and saxitoxin inhibit sodium ionophores in neuroblastoma cells by binding to a different receptor site from scorpion toxin (11, 12). Tetrodotoxin (1 ,uM) has no effect

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FIG. 2. Effect of sea anemone toxin on activation of sodium iono- phores by veratridine and batrachotoxin. NM cells were incubated for 30 min at 36°C in sodium-free potassium-substituted medium (14) containing the indicated concentrations of veratridine (left) or bat- rachotoxin (right) and 0 pM (O), 0.03 pM (01, 0.3 PM (0, n ), or 3 pM (A) sea anemone toxin. “Na+ uptake was then measured in choline substituted medium containing the same toxin concentrations.

TABLE I

Computedparameters of allosteric model

The data of Fig. 2 were fitted to Equation I as described previously (14). The best fit parameters are presented.

Computed dissociation constants

K, K/f KY,/ K, M M

Veratridine 3.3 x 10 + 5.3 x lo-* 6.2 x 10’ Batrachotoxin 1.5 x lo-” 1.9 x lo-“’ 1.4 x 10”

Computed values of allosteric constant Sea anemone toxin

Veratridine Batrachotoxin

M

0 7.2 x 10” 6.9 x lo” 3 x wR 5.5 x lo” 5.5 x lo:’ 3 x lo-’ 2.2 x lo” 2.4 x IO:' 3 x lo-” 1.5 x lo? 0.7 x 10"

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Common Receptor Site for Sea Anemone and Scorpion Toxins

on displacement of bound scorpion toxin by sea anemone toxin (Fig. 3, A), indicating that tetrodotoxin and sea anemone toxin also bind to separate receptor sites.

Effect of Membrane Potential on Sea Anemone Toxin Action-The heterotropic cooperative effect of scorpion toxin on activation of sodium ionophores by veratridine and

!$ , ‘,b’,o’ , ,,, ,l, , O>\j lo9 lo* 10’ 106

TOXIN CONCENTRATION (MI

FIG. 3. Inhibition of scorpion toxin binding by sea anemone toxin. N18 cells were incubated for 60 min at 36°C in sodium-free choline- substituted medium (11) containing 0.5 nru scorpion mono[““I]iodo- toxin (SCTX) and the indicated concentrations of unlabeled scorpion toxin (0) or sea anemone toxin (0, A). Tetrodotoxin (1 pM) was added to one group of sea anemone toxin samples (a). Specifically bound scorpion toxin was measured as describe previously (11). The amount of scorpion mono[““I]iodotoxin bound in the presence of a saturating concentration (200 nM) of unlabeled scorpion toxin was considered nonspecific and was subtracted from the results.

0 20 40 60 SO 100

% INHlEilTlON

FIG. 4. Analysis of sea anemone toxin binding. Binding competi- tion data from a more extensive experiment carried out as in Fig. 3 is plotted in a form analogous to the Scatchard plot. Per cent inhibition of specific scorpion toxin binding by sea anemone toxin (ordinate and abscissa) is proportional to fractional saturation of the receptor sites by sea anemone toxin. Therefore, plot of per cent inhibition divided by free sea anemone toxin (AZ'X ZZ) concentration versus per cent inhibition is formally analogous to a Scatchard or Eadie-Hofstee plot where the slope is -I/&A.

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Time (mln)

FIG. 6. Effect of depolarization on reversal of scorpion toxin action. N18 cells were incubated 30 min at 36°C with 0.3 pM sea anemone toxin in medium containing 5 mM K’. The cells were then rinsed and incubated for the indicated times in medium containing 5 mru K’ (0) or 135 mM K’ (0). “Na+ uptake was then measured in medium with 5 mM K’ and 200 pM veratridine as described previously (10).

batrachotoxin is dependent on membrane potential (10). De- polarization of the neuroblastoma cells by K+ or other agents causes a large increase in Ko., for scorpion toxin action and in K,, measured in direct binding experiments (11). Similar ex- periments were carried out to test the effect of membrane potential on KOs for sea anemone toxin. In these experiments, cells were incubated with increasing concentrations of sea anemone toxin for 30 min in 5 mM K+ (membrane potential = -41 mV (10)) or in 135 mM K’ (membrane potential = 0 mV (10)). The cells were then briefly rinsed and sodium permeability was measured in 5 IIIM K’ and 200 pM veratri- dine. The results of these experiments show that depolariza- tion increases Kos for sea anemone toxin approximately 5-fold (Fig. 5, left). These data, when plotted on a double reciprocal plot, do not yield straight lines (Fig. 5, right). The curves obtained are most consistent with a common ordinate inter- cept, indicating no effect of membrane potential on P,, and different slopes, indicating an increase in Ko.5 due to depolar- ization.

The effect of membrane potential on scorpion toxin binding is due mainly to an increase in the rate of dissociation of the scorpion toxin receptor complex (10, 11). In order to test the effect of membrane potential on the rate of reversal of sea anemone toxin action, cells were incubated for 30 min with 0.3 pM sea anemone toxin in 5 ITIM K+, rinsed, and incubated

I I

0 60 Id0 Ii0 2bo 240 360 [SEA ANEMONE TOXIN1 -’ , PM-’

Fm. 5. Effect of depolarization on the action of sea anemone toxin. N18 cells were incubated for 30 min at 36°C in medium containing 5 mrvr K+ (0) or 135 mM K’ (0) and the indicated concentrations of sea anemone toxin. 22Na+ uptake was measured in medium containing 5 mM K’ and 200 pM veratridine as described previously (10). The data are presented as a velocity ( u) uerws concentration plot (left) and a double- reciprocal plot ( ri&t).

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7396 Common Receptor Site for Sea Anemone and Scorpion Toxins

for a variable period of time in the absence of toxin in either 5 mM K’ or 135 mM K’, and then sodium permeability was measured in the presence of 200 pM veratridine and 5 mM K+. In experiments of this kind, the rate of reversal of sea anemone toxin action is substantially increased by depolarization (Fig. 6). The semilogarithmic plots are consistently nonlinear pre- venting precise determination of rate constants. The initial rate of reversal is increased almost 5-fold by depolarization suggesting that most of the observed increase in Ko,, is due to an increase in the rate constant for dissociation of the toxin. receptor complex.

DISCUSSION

The effects of sea anemone toxin II and scorpion toxin on sodium ionophores in neuroblastoma cells are similar in sev- eral respects. Neither toxin has a measurable effect on sodium permeability when tested alone. Each toxin enhances the activation of action potential sodium ionophores by veratri- dine and batrachotoxin. In both cases, this heterotropic co- operative effect is inhibited by depolarization of the cells. The effect of membrane depolarization is primarily to increase the rate of dissociation of the toxin.receptor complex. These results are consistent with the conclusion that sea anemone toxin and scorpion toxin alter the properties of the action potential sodium ionophore by a common mechanism. In direct binding assays using ‘*“I-labeled scorpion toxin, sea anemone toxin II inhibits specific binding of scorpion toxin to its receptor site associated with action potential sodium ion- ophores. Considered together, these results provide strong evidence that scorpion toxin and sea anemone toxin share a common receptor site. Scorpion toxin ( KD = 0.8 nM) has llO- fold greater affinity for this site than sea anemone toxin (Ko = 90 nM).

Although our results strongly support the conclusion that the site and mechanism of action of scorpion and sea anemone toxins are identical, some differences between the action of the two toxins have been observed. Depolarization of the cells causes a 70-fold increase in Ko.~ for scorpion toxin action (10) but only a 5-fold increase in Ko.~ for sea anemone toxin action. Since it is likely that the change in Kc).5 represents a difference in toxin binding to two different conformational states of the receptor site (ll), these results imply that sea anemone toxin discriminates less effectively between these two receptor site conformations. In addition, concentration effect curves for scorpion toxin are consistently hyperbolic (10, 13) whereas curves for sea anemone toxin deviate from simple hyperbolae as illustrated by the curved double-reciprocal plots of Fig. 5. In contrast, binding curves for sea anemone toxin were con- sistently hyperbolic as illustrated by the linear Scatchard plot in Fig. 4. Comparison of the data from these two different types of experiments suggests that the deviation from hyper- bolic behavior observed in the ion flux experiments resulted from dissociation of sea anemone toxin during the ion flux measurements which must be made in the absence of toxin and did not reflect complexity in the interaction between toxin and receptor site. To test this possibility, flux experiments were carried out in two ways. In the first, cells were incubated with anemone toxin for 30 min and then uptake was measured in the presence of 200 pM veratridine but no anemone toxin. This experimental design is required for the experiment of Fig. 5. In the second experimental design, sea anemone toxin was present in both preincubation and assay. Results from experiments of this second type showed smaller deviation from hyperbolic behavior. Thus, it seems likely that at least part of the deviation from hyperbolic behavior is due to the manipulations required to test for membrane potential de- pendence as in Fig. 5. The more rapid rate of dissociation of

TABLE II Properties of neurotoxin receptor sites

Toxin re- ceptor site Ligands Physiologic effect

1 Tetrodotoxin Inhibit ion transport Saxitoxin

2 Veratridine Alter activation and Batrachotoxin inactivation Aconitine Cause persistent acti- Grayanotoxin vation

3 Scorpion toxin Inhibit inactivation Sea anemone toxin Enhance persistent

activation by verat- ridine, batracho- toxin, aconitine, and grayanotoxin

the sea anemone toxin.receptor complex compared to the scorpion toxin receptor complex is probably responsible for the different behavior observed under these conditions. We conclude from the binding competition data that sea anemone toxin, like scorpion toxin, binds to a single class of independent sites associated with the action potential sodium ionophore.

Previous studies (10-14) have defined three toxin receptor sites associated with sodium ionophores in neuroblastoma cells: one specific for the inhibitors, tetrodotoxin and saxitoxin; a second specific for veratridine, batrachotoxin, aconitine, and grayanotoxin; and a third specific for scorpion toxin. The results presented here show that sea anemone toxin acts at the third of these receptor sites. Binding of sea anemone toxin to its site of action enhances the action of veratridine and batrachotoxin, showing that their site of action is distinct. Tetrodotoxin has no effect on binding of sea anemone toxin, showing that its site of action is distinct. The properties of these three toxin receptor sites are summarized in Table II. The results of this and previous investigations (12-14) show that receptor sites 2 and 3 interact allosterically in regulating the ion transport activity of the action potential sodium ionophore. The toxins which bind specifically to these receptor sites provide important tools for analysis of the mechanisms of activation and inactivation of the sodium ionophore and for solubilization and purification of the sodium ionophore.

Acknowledgments-We thank Mrs. Cynthia Morrow for excellent technical assistance and Drs. J. Daly and B. Witkop for supplying batrachotoxin.

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W A Catterall and L Beresswith the action potential sodium ionophore.

Sea anemone toxin and scorpion toxin share a common receptor site associated

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