J. Biol. Chem. 1977 Catterall 8669 76

THE JOI,RNAL OF BKXOGICAL CHEM~~RY Vol. 252, No. 23, Issue of December 10, pp. 8669-8676, 1977

Prmfed m U S A.

Activation of the Action Potential Na+ Ionophore by Neurotoxins AN ALLOSTERIC MODEL*

(Received for publication, May 9, 1977, and in revised form, August 22, 1977)

WILLIAM A. CATTERALL~

From the Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

The alkaloid neurotoxins aconitine, veratridine, grayanotoxin, and batrachotoxin activate the action potential Na+ ionophore by interaction with a common binding site. Con- centration-response curves are fit by simple Langmuir iso- therms. The fraction of Na+ ionophores activated at saturating concentrations of neurotoxin are: aconitine, 0.02; veratridine, 0.08; grayanotoxin, 0.51; and batrachotoxin, 0.95. The concentrations required to obtain 50% of the maximum activation are: aconitine, 3.6 x 10m6 M; veratridine, 2.9 X 10ms M; grayanotoxin, 1.2 x 1O-3 M; and batrachotoxin, 7.0 X It7 M.

Incubation of cells with scorpion toxin causes an increase in the fraction of Na+ ionophores activated at saturating concentrations of alkaloid toxins (P,) and a decrease in the concentration required to obtain 50% of maximum activation (K,,,): aconitine, P, = 0.21, Ko.5 = 2.7 x 10m6 M; veratridine, P, = 0.56, Ko.s = 1.5 x 1O-5 M; grayanotoxin, P, = 0.96, K,,, = 9.2 x 10m5 M; batrachotoxin, P, = 1.0, K,., = 5.2 x 10-S M. For the partial activators, the primary effect of scorpion toxin is to increase P,. For the full activators, the primary effect of scorpion toxin is to decrease K,,.5. In each case the concentration response curves are fit by a simple Langmuir isotherm with a Hill coefficient

of 1.0. An allosteric mode1 is presented which describes the

activation of the Na+ ionophore by alkaloid toxins and the heterotropic cooperative effect of scorpion toxin. In this model, the Na+ ionophore is assumed to exist in two states, active and inactive. Alkaloid toxins activate the ionophore by binding preferentially to the active state. Scorpion toxin enhances activation by alkaloid toxins by altering the equilibrium constant (allosteric constant) for the transition between the active and inactive states.

The alkaloid neurotoxins batrachotoxin, veratridine, acon-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked &~~rtisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Department of Pharmacology, University of Washington, Seattle, Wash. 98105.

1 For brevity, the term alkaloid neurotoxins is used to refer to aconitine, veratridine, batrachotoxin, and grayanotoxin as a group.

itine, and grayanotoxin and the polypeptide toxins of scorpion venom cause repetitive action potentials and persistent depolarization of nerve axons (l-8). Tetrodotoxin, a specific inhibitor of the action potential Na+ current (91, blocks the effect of these toxins, suggesting that they act by causing a persistent ion transport activity of the action potential Na+ ionophore.

Clonal lines of neuroblastoma cells grown in vitro are electrically excitable (10, 11). The Nat-dependent portion of the action potential is inhibited by tetrodotoxin at low concentration, suggesting that an action potential Na+ ionophore identical with that in nerve axons is present in these cells (11, 12). Veratridine, batrachotoxin, and aconitine increase the passive Na+ permeability of electrically excitable neuroblastoma cells (13, 14). Two kinds of evidence indicate that this increase in Na+ permeability reflects activation of the action potential Na+ ionophore: (a) the increase is completely inhibited by low concentrations of tetrodotoxin (13, 141, and (b) variant neuroblastoma clones specifically lacking the depolarizing phase of the action potential (13) do not respond to veratridine.

Equilibrium concentration-response relationships indicate that veratridine, aconitine, and batrachotoxin interact competitively with a single class of binding sites in activating the action potential Nat ionophore of electrically excitable neuroblastoma cells (14, 15). Venom of the scorpion Leiurus quinquestriatus, and a polypeptide toxin purified from that venom, act cooperatively with veratridine, batrachotoxin, and aconitine to activate the action potential Na+ ionophore (15, 16). Batrachotoxin enhances the binding of [Y]monoiodo scorpion toxin to the action potential Na+ ionophore (17). These observations suggest that the alkaloid toxins and scorpion toxin bind to two functionally separate regulatory components that interact allosterically in controlling the ion transport activity of the action potential Na+ ionophore. The binding of scorpion toxin to the action potential Na+ ionophore is examined in detail in the preceding report (17). In this report, I present more extensive data analyzing the interactions among the alkaloid neurotoxins and scorpion toxin in activating the action potential Na+ ionophore and describe a two-state allosteric model which quantitatively accounts for the observations.

In fact, grayanotoxin contains no mcrogen and thus, strictly speak- ing, is not an alkaloid.

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8670 Activation of Na+ Ionophores by Neurotoxins

EXPERIMENTAL PROCEDURES

Materials -The sources of materials and the growth of neuroblastoma cells is described in the preceding manuscript (17). Grayano- toxin I was kindly provided by Dr. T. Narahashi (Department of Physiology and Pharmacology, Duke University) and Dr. B. Witkop (Laboratory of Chemistry, National Institute of Arthritis, Metabo- lism, and Digestive Diseases, National Institutes of Health).

Measurements of 22Na+ Uptake -Unless otherwise indicated, the following procedure was used. Cell cultures were incubated with the concentrations of toxins indicated in the figure legends for 30 min at 36 in 0.25 ml of medium consisting of 135.4 rnM KCl, 50 rnM Hepes2 (adjusted to pH 7.4 with Tris base), 5.5 rnM glucose, 0.8 rnM MgSO,, and 1 mg/ml of bovine serum albumin. After 30 min, this medium was removed and the cells were rinsed twice in 15 s with 1 ml of medium consisting of 5.4 mM KCl, 130 mM choline chloride, 50 mM Hepes (adjusted to pH 7.4 with Tris base), 5.5 mM glucose, 0.8 mM MgSO,, and 1 mglml of bovine serum albumin. The cells were then incubated for 30 s in medium containing the same concentrations of neurotoxins and 5.4 mM KCl, 125 mM choline chloride, 5 mM NaCl, 5 mM ouabain, 50 mM Hepes (adjusted to pH 7.4 with Tris base), 5.5 mM glucose, 0.8 mM MgSO,, and 1.0 @X/ml of Z2NaCl. Finally the cells were washed three times with 3 ml of medium consisting of 163 mM choline chloride, 5 mM Hepes (adjusted to pH 7.4 with Tris base), 5.5 mM glucose, 0.8 mM MgSO,, and 1.8 mM CaCl,. Under these washing conditions, extracellular 22Na+ is effectively removed, whereas intracellular zzNa+ is retained (16). The cells were suspended and radioactivity was determined as described previously (15).

Cells were labeled with 4,5-c3H]leucine by growth for 24 h in growth medium containing 0.2 &i/ml of L3H]leucine. The concentration of [3H]leucine in individual cultures after the experiment was used to normalize the results from different cultures. Protein was determined by a modification of the method of Lowry et al. (20).

Data were analyzed and fit to possible mathematical models using the MLAB curve fitting procedure (Division of Computer Research and Technology, National Institutes of Health). In experiments where the data are expressed as fractional activation of the Na+ ionophore, the maximum rate of uptake of Na+ estimated from a computed fit of a batrachotoxin titration curve in the presence of 100 nM scorpion toxin was assigned a value of 1.0.

RESULTS

Relationship between Measured 22Na+Znflux and P,,-The goal of these experiments is to make detailed measurements of the concentration dependence of activation of the action potential Na+ ionophore by neurotoxins. Since the fraction of ionophores activated is proportional to Na+ permeability (P& it is important to establish that the measured zzNa+ influx varies linearly with P,,. Goldman (21) and Hodgkin and Katz (22) have derived a relationship between ionic flux and ion permeability which is obeyed by many excitable cells.

F JNB = &JNa+l,,, RT & (1)

This relationship predicts that *2Na+ influx (J,,) is linearly proportional to Na+ permeability only if the membrane potential (V) is constant. However, V depends on ionic permeabilities and concentrations according to Equation 2 (20).

v = RT ln JKIK+lout + PNJNa+lout + pc&-lh F P,[K+l,, + P,,[Na+l,, + P,,ICl-I,,

C-3

Thus, in general, when cells are treated with toxins which increase PNa, the relationship between JNa and P,, is nonlinear. The conditions used in these experiments are designed to eliminate these difficulties.

The toxins studied in these experiments require up to 60 min to equilibrate with their sites of action (14). During this time Na+ permeability is dramatically increased. In order to prevent entry of Na+ into cells, the incubations are carried

* The abbreviation used is: Hepes, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid.

out in Na+-free medium. In addition, it was noted during previous experiments (16) that some loss of intracellular K+ occurred during these incubations because the Na+ ionophore is not absolutely specific for Na +. Since loss of intracellular K+ depolarizes the cells (Equation 21, it alters the relationship between measured flux and PNa. To prevent loss of intracellular K+, cells were incubated with toxins in medium containing 135 mM K+. The experimental protocol therefore involves incubation of the cells with neurotoxins for 30 to 60 min under conditiops ([K+but = 135, [Na+but = 0, V = 0) where there are no ionic gradients. Therefore no ionic rearrange- ments occur despite the change in ion permeabilities.

After incubation with the toxins, 2*Na+ influx is measured in a choline-substituted medium containing ouabain to inhibit (Na+,K+)-ATPase and having [K+&,, = 5 mM and [Na+but = 5 mM. Under these conditions the membrane potential is approximately -41 mV (23). A low concentration of Na+ is chosen so that the membrane potential is unaffected by the increased Na+ permeability caused by neurotoxin treatment and therefore JNa remains directly proportional to P,, as the concentration of neurotoxin is varied. The untake of 22Na+

I

remains linear with time for at least 2 min. Membrane potential measurements with microelectrodes

indicate that batrachotoxin (the most effective toxin studied) causes at most a small depolarization of neuroblastoma cells in medium containing 10 mM Na+. A more quantitative test of this point is suggested by Equation 1. Thus, at low Na+ concentrations where V is constant, JNa should increase linearly with extracellular Na+ concentration. At higher concentrations the relationship should become nonlinear. An experiment testing this point is illustrated in Fig. IA. After incubation with batrachotoxin, JNa varies linearly with [Na+but up to 10 mM in this experiment. In some experiments, linearity was maintained up to 15 mM Na+. Therefore, in this concen- tratlon range, JNa is proportional to P,,. Subsequent experiments were carried out in medium containing 5 mM Na+.

The membrane potential of N18 cells can be varied between -41 and 0 mV by increasing extracellular K+ (23). To further test Equation 1, JNa was measured at different membrane potentials and the results were plotted according to.Equation 1 (Fig. IB). The linear relationship observed between JNa and VFI(RT exp(VFlRT) - 1) confirms the validity of Equation 1

u- ol 10 20 30 40 1.0 1.5 2.0

[Na], mM VF ATexpWFlRTl-,

FIG. 1. The dependence of 22Na+ uptake on Na+ concentration and membrane potential. A, N18 cells were incubated for 30 min as described under Experimental Procedures either with (0) or without (0) 1 PM batrachotoxin. 22Na+ uptake was then measured for 30 s as described under Experimental Procedures in medium with the indicated Na+ concentration plus choline so that Na+ + choline+ = 130 mm. B, N18 cells were incubated with or without batrachotoxin as in A. 2*Na+ uptake was then measured in medium with 5 mM Na+ plus choline chloride and KC1 so that choline + K+ = 130 mM. The membrane potentials under these conditions are: IK+l = 5.4 mM, V = -41 mV; [K+] = 10 mM, V = -37 mV; [K+] = 25 mM, V = -28 mV; [K+] = 60 mM, V = -13 mV; [K+] = 135 mM, V = 0 mV (23).


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Activation of Na+ Ionophores by Neurotoxins 8671

and indicates a linear relationship between JK, and PY,. I conclude then that measurements of 22Na+ influx under these conditions provide an accurate measure of P,, and thus of activation of the action potential Na+ ionophore.

Specificity of Neurotoxin Action -The depolarization of nerve axons by aconitine, batrachotoxin, grayanotoxin, and veratridine is inhibited by low concentrations of tetrodotoxin (1, 3, 5, 6). The increase in Na+ permeability of cultured neuroblastoma cells caused by these toxins is inhibited non- competitively by tetrodotoxin with K, values of 5 to 10 nM3 (13-16). In voltage clamp experiments in frog node of Ranvier, batrachotoxin at high concentration alters the properties of all the voltage-sensitive Na+ ionophores (24). In denervated rat muscle (25) and in cultured rat muscle (26) both action potentials and the increase in Na+ permeability caused by veratridine and batrachotoxin become relatively resistant to tetrodotoxin inhibition. These observations are all most consistent with the view that aconitine, batrachotoxin, grayanotoxin, and veratridine act specifically on the Na+ ionophore involved in action potential generation.

Additional evidence in favor of that view has been derived from studies of neuroblastoma and hybrid cell clones having heritable defects in action potential generation. Previous experiments have shown that veratridine increases the Na+ permeability of electrically excitable neuroblastoma and hybrid clones but has no effect on inexcitable clones (13). Table I summarizes the results of experiments testing the effect of veratridine, batrachotoxin, aconitine, and scorpion toxin on clonal lines differing in electrical excitability. The electrically excitable clone N18 responds to all four toxin treatments with large increases in Na+ permeability, whereas clone N103 which lacks the action potential Na+ response but retains the action potential K+ response (27) has a 20-fold smaller response to all four treatments. Hybrid cell clones formed between neuroblastoma clone NlSTG2 (electrical excitable) and L cell clone B82 (inexcitable) have inheritable differences in electrical excitability (28). The electrically excitable clones NL308 and NL309 respond to all four toxin treatments. In contrast the inexcitable clones NL304 and NL30.5 have lo-fold smaller responses to all four treatments.

In addition to these experiments, Stallcup and Cohn (29) have analyzed 19 clones derived from rat brain tumors and

TABLE I

Effect ofueratridine, batrachotozin, scorpion toxin, and aconitine on clonal lines differing in electrical excitability

Cells of each clonal line were cultured, incubated with neurotoxins, and assayed for Na+ permeability as described under Experi- mental Procedures. The concentrations of neurotoxins used were: veratridine, 200 PM; batrachotoxin, 1 PM; scorpion toxin, 100 nhi; and aconitine, 100 PM. The electrophysiologic properties are taken from Refs. 27 and 28.

Cell line

Electrophysio- logic properties

Nat re- K+ re- BpXltX S.plXW2

Neurotoxin-stimulated 22Na+ uptake

Verat- Ba- Aconi- ridine tracho-

v.?; tine + toxin + s&e 6&x

nmollminlmg

N18 + + 12 90 82.5 22.3 N103 - + 0.7 3.4 2.4 0.7 NL304 - + CO.5 3.1 1.6 CO.5 NL305 - CO.5 3.5 1.5


permeability increase caused by grayanotoxin almost completely. In the presence of scorpion toxin (Fig. 3B), aconitine activates a substantial fraction of the Na+ ionophores and reduces the Na+ permeability increase caused by grayanotoxin only to the value observed with aconitine. The concentration dependence of these effects is consistent with a competitive interaction between the two toxins. These results are identical to those described earlier with veratridine, batrachotoxin, and aconitine and support the view expressed previously (15) that all four of these toxins bind to a common binding site.

Cooperative Effect of Scorpion Toxin -Previous results have shown that the venom of the scorpion Leiurus quinquestriatus (15) and a toxin purified from that venom (16) act cooperatively with aconitine, veratridine, and batrachotoxin to activate the action potential Na+ ionophore. In the present experiments, I have made a detailed analysis of the concentration dependence of this cooperative interaction using the more rigorous experimental conditions described in this report. In each experiment the concentration of one alkaloid toxin was varied over a wide range in the presence of different fixed scorpion toxin concentrations. The greatest increase in Na+ permeability was observed in the presence of saturating concentrations of batrachotoxin plus scorpion toxin. The value of V,,, (extrapolated to [batrachotoxinl = 03) was determined under these conditions in each experiment and was assigned a value of 1.0. All other data were normalized to this value.

The data from these experiments are presented in Figs. 4 to 7. The curves drawn are least squares fits to a model described below. In general, the data also can be fit well by a simple Langmuir isotherm as described previously (14, 16). The curves are essentially superimposable on those illustrated. The Hill coefficients are approximately 1.0 for all the data. The parameters for fits of each curve individually to a simple Langmuir isotherm are given in Table II. K0.5 is the concentration of toxin required to give 50% maximum effect and P, is the fractional activation at infinite toxin concentration. The apparent dissociation constants (K,.,) for the poor activators, veratridine and aconitine, are relatively little affected by scorpion toxin. In contrast, the values of K0.5 for the good activators, batrachotoxin and grayanotoxin, are decreased more than lo-fold by scorpion toxin. The maximum uptake velocities (P,) for veratridine and aconitine are substantially increased by scorpion toxin. In contrast, the values ofP, for batrachotoxin and grayanotoxin are relatively little affected by scorpion toxin.

I I r I

8.

%I

20

10

Ii?4 0 25 50 75 loo

FIG. 3. Competitive interaction between grayanotoxin and aconitine. A, N18 cells were incubated with the indicated concentrations of aconitine plus 1 rn~ grayanotoxin as described under Experimen- tal Procedures. The initial rate of 22Na+ uptake was then determined as described under Experimental Procedures. B, N18 cells were incubated with the indicated concentrations of aconitine plus 100 n&f scorpion toxin either with (0) or without (0) 400 PM grayanotoxin. The initial rate of 22Na+ uptake was then determined as described under Experimental Procedures.

These results are similar to those obtained previously in experiments using whole scorpion venom (15) under other experimental conditions. In the present experiments, the difference between P, in the presence of the poor activators, veratridine and aconitine, and that in the presence of batrachotoxin is more pronounced. This is due to underestimation of PNa in the presence of scorpion toxin plus batrachotoxin in the earlier experiments (15) because relatively high concentrations of Na+ were used (50 mM, compare with Fig. 1) and incubations with toxins were carried out in medium with [K+k,, = 5 mM causing some loss of intracellular K+ (16).

Two-state Conformational Change Model -The four alkaloid toxins act competitively at a common binding site in activating the action potential Nat ionophore but each causes

FIG. 4 (top left). Cooperative interaction between aconitine and scorpion toxin. N18 cells were incubated with the indicated concentrations of aconitine and 0 (A), 3 (a), 10 (01, 100 (01, and 300 (A) nM scorpion toxin as described under Experimental Procedures. The initial rate of 2*Na+ uptake was then determined as described under Experimental Procedures in medium containing the same aconitine concentrations.

FIG. 5 (top right). Cooperative interaction between veratridine and scorpion toxin. N18 cells were incubated with the indicated concentrations of veratridine and 0 (A), 3 (01, 10 (01, 30 (O), and 100 (A) nM scorpion toxin as described under Experimental Proce- dures. The initial rate of 2*Na+ uptake was then determined as described under Experimental Procedures in medium containing the same veratridine concentrations.

FIG. 6 (bottom left). Cooperative interaction between grayanotoxin and scorpion toxin. N18 cells were incubated with the indicated concentrations of grayanotoxin and 0 (A), 10 (01, 30 (01, and 100 (0) nM scorpion toxin as described under Experimental Proce- dures. The initial rate of =Na+ uptake was then determined as described under Experimental Procedures in medium containing the same grayanotoxin concentrations.

FIG. 7 (bottom right). Cooperative interaction between batrachotoxin and scorpion toxin. N18 cells were incubated with the indicated concentrations of batrachotoxin and 0 (A), 3 (01, 10 (01, 30 (01, and 100 (A) nM scorpion toxin as described under Experimental Proce- dures. The initial rate of *2Na+ uptake was then determined as described under Experimental Procedures in medium containing the same batrachotoxin concentrations.


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TABLE II

Parameters derived from fits to Langmuir isotherm

The data of Figs. 4 to 7 were fit to a modified Michaelis-Menten equation of the form P(A) = PJ/(K,, + A) where P, is the fraction of Na+ ionophores activated at saturation, K,,, is the concentration of toxin required to obtain 50% maximum activation, and A is toxin concentration.

Aconitine Veratridine Grayanotoxin Batrachotcxin [s&i]=

K 0.1 P, K 0.1 P, K.5 P, K 0.1 P, lLM M M M M

0 3.6 x 10m6 0.02 2.9 x 10-S 0.08 1.2 x 10-S 0.51 7.0 x 10-1 0.95 3 3.4 x 10-e 0.04 2.7 x lo- 0.17 4.4 x 10-T 0.97

10 3.0 x IO-6 0.07 2.2 x 10-S 0.31 2.7 x lo- 0.89 1.2 x 10-T 0.99 30 1.9 x 10-S 0.43 1.6 x 1O-4 0.94 7.6 x 10-H 0.99

100 2.7 x 10m6 0.21 1.5 x 10-S 0.56 9.2 x 10-S 0.96 5.2 x lo- 1.0

300 2.3 x 1O-6 0.31

a Sctx, scorpion toxin.

a different level of Na+ permeability at saturation. Scorpion toxin enhances the effect of each of the alkaloids but causes different changes in P, and K0.5 for each. I have previously suggested that this behavior can be accommodated in terms of a two-state model with the following characteristics: (a) the action potential Na+ ionophore exists in (at least) two conformational states, active and inactive; (b) there are reversible transitions between states characterized by an equilibrium constant; (c) depolarization changes the equilibrium constant transiently, shifting more of the ionophore to the active conformation; (d) interaction with neurotoxins changes the equilibrium constant chronically, shifting more of the ionophore to the active conformation. The ability of a toxin to shift the conformational equilibrium depends on its relative affinities for the active and inactive states (15).

This model predicts that the effects of the toxins can be completely accounted for in terms of the laws of mass action. Models of this kind have been considered previously by several authors with respect to conformational changes in allosteric enzymes (33,341 and receptors (35-37). Adopting the approach of Monod et al. (331, the equilibrium between two states, R (active) and T (inactive), is characterized by an equilibrium constant MRT , the allosteric constant.

MllT A +T.-A +R

Krll 11Ks (3) TA RA

A 1igandA binds to the two states with dissociation constants KT and KH.

None of the ligands tested in this system exhibits homotropic cooperativity. Thus, it is necessary to consider only binding of 1 ligand molecule. Following the derivation of Monod et al. (331, the following equilibrium equations are obtained

T = MErR

RA=R& H

TA=T& T

The fraction of ionophores in the R state is given by

Pd-4 = R+RA

RiRAiTiTA-

Using the equilibrium equations,

This relationship defines the dependence of the fraction of active Na+ ionophores on the activator concentration, A, and three constants, the conformational equilibrium constant, MHT, and the dissociation constants, KT and KH It is identical to the function of state of Monod et al. (331 for the special case of n, the number of interacting protomers, equal to 1.

From Equation 6, in the absence of ligand,

PJO) = l/(1 + M/J (7,

Thus, in general, some fraction of the Na+ ionophores must be in the R state in the absence of toxins. At infinite activator concentration,

(8)

Thus, the fraction of Na+ ionophores activated by saturating concentrations of toxin (P,) can take on any vahre between 0 and 1 depending on the values of the parameters MH7., KH, and KT.

Equation 6 also defines the relationship between K,,.5 and the parameters MHT, KH , and KT, if M,Sr is restricted to values ~100. By definition, P, (K0.5)/P,(=) = 0.5. Therefore,

1 + Ko.,IK, 1 + &,IK,

(9)

For MKr > 100, K0.5 is always much greater than KH and thus 1 + Ko.sI& - KO.JKH. Making this approximation and rear- ranging terms yields

(10)

Thus, as a function of MHT, K,, varies from a maxium of K, at MET = = to a minimum of MRTKH as MHI. becomes smaller.

In fitting this model to the experimental data, KT and KH are taken as constants for each of the alkaloid toxins. When KH < KT, alkaloid toxins activate some fraction of the Na+ ionophores according to Equation 6. The effect of scorpion toxin is to reduce MHT. Thus, scorpion toxin alters the energy required to cause the T f R transition.

It is not possible to define all three parameters uniquely unless MRT can be defined from Equation 7. Tetrodotoxin has no effect on steady state Na+ permeability of N18 cells unless the Na+ ionophores are activated by treatment with alkaloid toxins. Therefore, the fraction of ionophores active in the


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absence of an alkaloid toxin is too small to measure. Since our experiments can detect activation of 0.5% of the Na+ ionophores, PR (0) 5 0.005 (Equation 7) and M,tT > 200. In the terms of the model, the effect of scorpion toxin is to reduce MET. However, purified scorpion toxin does not activate a detectable fraction of the action potential Na+ ionophores (16). Thus MH7. in the presence of saturating concentrations of scorpion toxin must also be greater than 200. In fitting the data, MH7. was set at 500 in the presence of 100 nM scorpion toxin. Values of KT and Ks were then derived by fitting the data at 100 nM scorpion toxin to Equation 6 (Table III). The ratio of dissociation constants for the T and R states (KJ,.lK,) range from 1.4 x lo2 for aconitine to 1.4 x 10 for batrachotoxin.

To fit the data at other scorpion toxin concentrations, Ks and Kr were held constant and MnT was allowed to vary. Good fits of all the data were obtained (Figs. 4 to 7) using the

TABLE III

Computed dwsociation constants for alkaloid neurotoxins Data at 100 no scorpion toxin were fit to Equation 6 taking MRT

= 500 and allowing K, and K, to vary. The best fit parameters are presented.

Toxin KT KR K,IK. M M

Aconitine 3.4 x 10-S 2.5 x 10-H 1.4 x 102 Veratridine 3.3 x 10-j 5.3 x 10-B 6.2 x lo* Grayanotoxin 2.5 x 10-a 1.9 x lo- 1.3 x 10 Batrachotoxin 1.5 x 10-S 1.1 x lo-0 1.4 x 105

TABLE IV

Computed values of allosteric constant The data of Figs. 4 to 7 were fit to Equation 6 using the values of

K, and K, from Table III and allowing M,, to vary. The best fit values of M,, are presented.

rsctxl Aconitine Veratridine Grayanotoxin Batrachotoxin ILM

0 6.8 x 103 7.2 x 103 (1.2 x 104) 7.3 x 103 3 3.0 x 103 3.1 x 103 4.2 x lo3

10 1.5 x 103 1.4 x 103 1.6 x lo3 1.2 x 103 30 8.3 x lo* 8.9 x lo2 7.3 x 102

100 5.0 x 102 5.0 x 102 5.0 x 102 5.0 x 102 300 2.7 x lo2

a S&x, scorpion toxin.

-I-

-II II It II 1 10 100

[Scorpion Toxin], nM

FIG. 8. Dependence of the allosteric constant on scorpion toxin concentration. The values of MST from Table IV are plotted as a function of scorpion toxin concentration for batrachotoxin (O), veratridine (O), aconitine (0) and grayanotoxin (A). The assumed value of MRT at 100 rm scorpion toxin is indicated by 0.

values of MHT collected in Table IV. The model requires that the values of MET change only as a function of scorpion toxin concentration and remain constant for different alkaloid toxins. The results in Table IV show that values of MHT are similar for all four alkaloid toxins. The values are plotted as a function of scorpion toxin concentration in Fig. 8. The agreement, while not perfect, is satisfactory since experiments with each different toxin were necessarily carried out on separate groups of cell cultures. The results which agree least well are those for grayanotoxin in the absence of scorpion toxin (enclosed in parentheses in Table IV). These values are the least reliable because limited supplies of grayanotoxin did not allow experiments at saturating concentrations in the absence of scorpion toxin. The model thus fits all the data with a single assumption, namely, that scorpion toxin causes a reduction in the value of MRI..

This model predicts quite clearly that scorpion toxin should have its major effect on P, for poor activators and on Ko.5 for good activators. Thus, from equation (lo), MRrKE > KT for poor activators and Ko.5 approximates KT, even in the presence of scorpion toxin (compare values of KO.5 and KT in Tables I and III). P, for poor activators is increased dramatically as MKT is decreased (Equation 5) as long as P, is substantially less than 1. In contrast, for good activators, MRTKti < KT and K 0.5 varies approximately as MHTKE (Table III) and thus is decreased dramatically by scorpion toxin. P, for good activators is near 1 and thus cannot be increased greatly by scorpion toxin.

The experimental data cannot be fit by holding MRT constant and allowing KT to vary as a function of scorpion toxin concentration. The experimental data can however be fit if MHT is assumed to remain constant and KH is allowed to vary as a function of scorpion toxin concentration. The curves derived from these two different assumptions are virtually superimposable. One would expect, however, that, if scorpion toxin affected KR , the extent of the scorpion toxin effect would vary significantly for the four different alkaloid toxins since they differ substantially in structure and binding constant. In fact, however, the increase in KR (or in MHT, Table IV) is similar for each alkaloid toxin. This result supports the more restrictive assumption that scorpion toxin affects MRT only. This treatment of the heterotropic cooperative interaction between scorpion toxin and the alkaloid toxins is analogous to the treatment of heterotropic interactions between substrates, activators, and inhibitors of allosteric enzymes by Monod et al. (33).

In the model of Monod et al. the change in MET caused by heterotropic allosteric ligands is considered to be due to the preferential binding of the ligands to the R or T states. The mechanism by which scorpion toxin affects MET for activation of the Na+ ionophore by alkaloid toxins must be more complex because scorpion toxin binding is only slightly affected by batrachotoxin and thus scorpion toxin must bind well to both active and inactive states of the ionophore. This conclusion remains correct whether scorpion toxin affects KR or MET.

DISCUSSION

The results presented in this report confirm and extend conclusions reached in previous reports in four respects.

1. Neuroblastoma cell lines specifically lacking the Na+ response of the action potential are unaffected by veratridine, batrachotoxin, and aconitine, strengthening the conclusion (13) that these toxins act specifically on the Na+ ionophores involved in action potential generation.


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2. All four of the alkaloid neurotoxins (aconitine, batrachotoxin, grayanotoxin, and veratridine), which modify the kinetic properties of the Na+ ionophore in electrophysiologic experiments, interact competitively in causing persistent activation of the Na+ ionophore. Aconitine is a linear competitive inhibitor of activation of the Na+ ionophore by batrachotoxin indicating that binding of these two toxins is mutually exclusive. These results provide strong support for the conclusion that all four toxins act at a specific chemically sensitive site associated with the action potential Nat ionophore (15). In general, competitive inhibition can also be caused by allosteric interactions of ligands binding at separate sites. In this case, the competitive interaction arises because the allosteric inhibitor favors a conformational state that binds the second ligand poorly. In my experiments, all four alkaloid toxins are activators of the Na+ ionophore. Therefore, they all must bind preferentially to the active state of the Na+ ionophore. Since each pair of alkaloid toxins interacts competitively, four separate active states of the Na+ ionophore, each having specific high affinity for one toxin and low affinity for the other three, are necessary to explain the results on the basis of indirect allosteric competitive inhibition. This seems very unlikely. I conclude, then, that all four alkaloid toxins act at a common binding site.

3. In previous reports (15) I have hypothesized that the alkaloid toxins, scorpion toxin, and tetrodotoxin interact with three functionally separable components of the action potential Na+ ionophore. This hypothesis is strengthened by observations in this and the accompanying report (17): (a) tetrodotoxin does not affect binding of scorpion toxin; (b) the alkaloid toxins have only small effects on binding of scorpion toxin; (c) scorpion toxin, while it affects activation of the action potential Na+ ionophore by alkaloid toxins, does not alter the dissociation constants (K,., K,) for alkaloid toxins.

4. Using experimental conditions under which PNa is proportional to the measured Na+ influx, I have confirmed my earlier results (15) showing that the effect of scorpion toxin is to increase the maximum fraction of Na+ ionophores activated by partial activators (veratridine, aconitine, and grayanotoxin) and to reduce the concentration required for 50% activation by both partial and full activators. These results support the earlier conclusion that there are heterotropic cooperative interactions between scorpion toxin and each alkaloid neurotoxin whereas there are no homotropic cooperative interactions observed in experiments with a single neurotoxin (15).

The main purpose of this report is to describe a simple allosteric model which accomodates this heterotropic cooperative interaction. The model described under Results makes only two assumptions, namely, that the alkaloid toxins bind better to the active state of the Na+ ionophore and that scorpion toxin alters the energy required for activation of the Na+ ionophore by alkaloid toxins. These assumptions are identical to those made by Monod et al. in describing heterotropic cooperativity between allosteric modifiers and enzyme substrates. The theoretical curves generated by this model match the experimental data precisely (Figs. 4 to 7). Not all the parameters of the model are defined uniquely, however. The value of MRT, the allosteric constant, must be chosen before computed fits of KT and K, can be carried out. The only constraint in choosing MRT is that it must be 2200. To illustrate the fit of the data, MRT was taken as 500 at 100 nM scorpion toxin. Equally good fits of the data can be achieved by choosing MRT at 100 nM scorpion toxin to be larger than

500. Under these conditions, the best fit values of KT are approximately those in Table III, whereas the best fit values of K,t are reduced in rough proportionality to the increase in the value of M,ST selected. This behavior results from the fact that, under most conditions studied in these experiments, the term 1 + A/K,, in equation (6) is approximately equal to A/K,{ causing M,,. and K,$ to behave as the product M,17K,t when the equation is fit by iterative procedures. Values derived for KK and the ratio KHIKT therefore depend on the value of M,


toxin binding component causes persistent activation of the Na+ ionophore. The conformational change in the scorpion toxin binding component alters the energy required for activation of the Na+ ionophore by alkaloid toxins and is opposed by depolarization. The conformational changes in these two components are partially coupled so that the energy required for the conformational change in each component is dependent upon the state of the other. The partial coupling is reflected in the enhancement of scorpion toxin binding by batrachotoxin (17) and in the enhancement of alkaloid toxin activation by scorpion toxin. Experiments defining the stoichiometry and physical relationship between these binding components are required to verify these suggestions.

Hodgkin and Huxley (38) were able to describe many of the kinetic and voltage-dependent properties of the action potential Na+ ionophore of squid giant axon in terms of two independent processes, one controlling activation of the ionophore and one controlling inactivation during a maintained depolarization. In voltage clamp experiments, the venom of the scorpion Leiurus quinquestriatus inhibits specifically the inactivation of the Na+ ionophore (6). The purified scorpion toxin used in these studies also affects inactivation specifically. The alkaloid toxins, on the other hand, affect both activation and inactivation of the Na+ ionophores (3, 24, 42). Since it appears that scorpion toxin and the alkaloid toxins interact with separate components of the Nat ionophore, it seems likely that the scorpion toxin binding component is specifically involved in the process of inactivation while the alkaloid toxin binding component is involved in both activation and inactivation. The partial coupling between these two binding sites observed in my experiments may be the basis for the coupling between the processes of activation and inactivation observed in recent voltage clamp (43) and gating current (44) experiments. Binding experiments in tissues with known voltage clamp parameters are required to verify this relationship.

Acknowledgments-I thank Mrs. Cynthia Morrow for ex- pert technical assistance, Drs. J. Daly and B. Witkop for providing samples of batrachotoxin, and Drs. T. Narahashi and B. Witkop for providing samples of grayanotoxin.

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REFERENCES

Albuquerque, E. X., Daly, J. W., and Witkop, B. (1971) Science 172, 99551002

Straub, R. (1956) Helu. Physiol. Pharmacol. Acta 14, l-28 Ulbricht, W. (1969) Ergeb. Physiol. Biol. Chem. Exp. Pharmakol.

61, 18-71 Herzog, W. H., Feibel, R. M., and Bryant, S. H. (1964) J. Gen.

Physiol. 47, 719-733 Seyama, I., and Narahashi, T. (1973) J. Pharmacol. Exp. Ther.

184, 299-307 Koppenhofer, E., and Schmidt, H. (1968) Pflugers Archiu. Ges-

amte Physiol. Menschen Tiere 303, 133-161 Narahashi, T., Shapiro, B. I., Deguchi, T., Scuka, M., and

8. 9.

10.

11.

12.

13.

14. 15.

16. 17. 18.

19. 20.

21. 22. 23.

24.

25.

26.

27.

28.

29. 30. 31.

32. 33.

34.

35.

36. 37.

38.

39.

40.

41.

42.

43.

Wana. C. M. (1972)Am. J. Phvsiol. 222. 850-857 Cahalan, M. D. (1975) J. Physiol. 244, 511-534 Narahashi, T., Moore, J. W., and Scott, W. R. (1964) J. Gen.

Physiol. 47, 965-974 Nelson, P. G., Ruffner, W., and Nirenberg, M. (1969) Proc.

Natl. Acad. Sci. U. S. A. 64, 1004-1010 Nelson, P. G., Peacock, J. H., Amano, T., and Minna, J. (1971)

J. Cell. Physiol. 77, 337-352 Spector, I., Kimhi, Y., and Nelson, P. G. (1973) Nature 246,

124-126 Catterall, W. A., and Nirenberg, M. (1973) Proc. Natl. Acad.

Sci. U. S. A. 70, 3759-3763 Catterall, W. A. (1975) J. Biol. Chem. 250, 4053-4059 Catterall, W. A. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1782-

1786 Catterall, W. A. (1976) J. Biol. Chem. 251, 5528-5536 Catterall, W. A. (1977) J. Biol. Chem. 252, 8660-8668 Henderson, R., Ritchie, J. M., and Strichartz, G. R. (1974) Proc.

Natl. Acad. Sci. U. S. A. 71, 3936-3940 Hille, B. (1975) Biophys. J. 15, 615-619 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R.

J. (1951) J. Biol. Chem. 193, 265-275 Goldman. D. E. (1943) J. Gen. PhvsioZ. 27. 37-60 Hodgkin,A. L., and Katz, B. (1949) J. Physiol. 108, 37-77 Catterall, W. A., Ray, R., and Morrow, C. S. (1976) Proc. Natl.

Acad. Sci. U. S. A. 73, 2682-2686 Khodorov, B. I., Peganov, E. M., Revenko, S. V., and Shishkova,

L. D. (1975) Brain Res. 84, 541-546 Albuquerque, E. X., and Warnick, J. E. (1972) J. Pharmacol.

Exp. Ther. 180, 683-697 Catterall, W. A. (1976) Biochem. Biophys. Res. Commun. 68,

136-142 Peacock, J., Minna, J., Nelson, P., and Nirenberg, M. (1972)

Exp. Cell Res. 73, 367-377 Minna. J.. Nelson. P. G.. Peacock. J.. Glazer. D.. and Niren-

berg; MI (1971) Z&oc. N&Z. Acad.&. U. S. A. 68, 234-239 Stallcup, W., and Cohn, M. (1976) Erp. Cell Res. 98, 285-297 Narahashi, T., and Seyama, I. (1974) J. Physiol. 242, 471-487 Albuquerque, E. X., Seyama, I., and Narahashi, T. (1973) J.

Pharmacol. Exp. Ther. 184, 308-314 Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 173-187 Monad. J.. Wvman. J.. and Chaneeux. J.-P. (1965) J. Mol. Biol.

12, ss-li8 Koshland. D. E.. Nemethv. G.. and Filmer. D. (1966) Biochem-

istry 5, 365-385 Colquhoun, D. (1973) in Drug Receptors (Rang, H. P., ed) pp.

149-182, MacMillan Ltd., London Karlin, A. (1967) J. Theoret. Biol. 16, 306 Changeux, J. P., and Podleski, T. (1968) Proc. Natl. Acad. Sci.

U. S. A. 59, 944-950 Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. 117, 500-

544 Campbell, D. T., and Hille, B. (1976) J. Gen. Physiol. 67, 309-

323 Hille, B. (1971) in Biophysics and Physiology ofExcitable Mem-

branes. (Adelman. W. J.. ed) DD. 230-246. Van Nostrand Reinhold Co., New York * 1

Khodorov, B. I. (1977) inlon Transport Across Membranes-The Proceedings of a Joint U.S.-USSR Conference, (Tosteson, D. C., Ovchinnikov, Y. A., and Latorre. R., eds) Raven Press, New York. in mess

Schmidt, H.; and Schmitt, 0. (1974) Pflugers Archiu. Gesamte Physiol. Menschen Tiere 349, 133-148

Goldman, L., and Schauf, C. L. (1972) J. Gen. Physiol. 59, 659- 675

j W. Schwarz, P. Palade, and W. A. Catterall, unpublished data. 44. Bezanilla, F., Armstrong, C. M. (1974) Science 183, 753-754


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