Cooperative Activation of Action Potential Na+ lonophore by Neurotoxins (neuroblastoma cells/passive 22Na+ influx/batrachotoxin/veratridine/aconitine/scorpion toxin)

Four neurotoxins that activate the action potential Na+ ionophore of electrically excitable neuroblastoma cells interact with two distinct classes of sites, one specific for the alkaloids veratridine, batrachotoxin, and aconitine, and the second specific for scorpion toxin. Positive heterotropic cooperativity is observed between toxins bound at these two classes of sites. Tetrodotoxin is a noncompetitive inhibitor of activation by each of these toxins (K1 = 4-8 nM). These results suggest the existence of three functionally separable components of the action potential Na+ ionophore: two regulatory components, which bind activating neurotoxins and interact allosterically in controlling the activity of a third ion-transport component, which binds tetrodotoxin. Several neurotoxins cause repetitive action potentials and, in some cases, persistent depolarization of nerves. This group of toxins includes the alkaloids veratridine (1), batrachotoxin (2, 3), grayanotoxin (4), and aconitine (5), and the polypeptide neurotoxins of scorpion (6) and coelenterate (7) venoms (see Fig. 1 for structures). Since the action of these toxins is blocked by tetrodotoxin, a specific inhibitor of the action potential Na+ current (8, 9), their effects have been ascribed to activation of the action potential Na+ ionophore.* These toxins, therefore, are potentially important tools in studying the mechanism of action potential generation. Cultured neuroblastoma cells provide a useful experimental system in which to study the action potential Na+ ionophore by ion transport methods. Previous work has shown that treatment of electrically excitable neuroblastoma cells with veratridine results in a marked increase in passive Na+ permeability detectable by measurements of 2INa+ uptake (10). Two kinds of evidence indicate that this increase in Na+ permeability reflects ion transport activity of the action potential Na+ ionophore: (i) the increase is completely inhibited by low concentrations of tetrodotoxin (10) and (ii) variant neuroblastoma clones specifically lacking the depolarizing phase of the action potential spike do not respond to veratridine (10). Batrachotoxin has some structural features in common with veratridine (Fig. 1). Equilibrium dose-response relationships indicate that veratridine and batrachotoxin compete for a Abbreviation: HEPES, N-2-hydroxyethyl piperazine N'-2ethanesulfonic acid. * The term "action potential Na+ ionophore" refers to the macromolecular structure responsible for the increase in Na+ permeability during the depolarizing phase of the nerve action potential. The term "ionophore" is taken to refer in general to ion-transporting molecules without implying a specific mechanism of action. single class of binding sites in activating the action potential Na+ ionophore (11) and that activation by both toxins is inhibited competitively by divalent cations (11) and noncompetitively by tetrodotoxin (K1 = 8-11 nM) (11). Those results and the results of Albuquerque et al. (23, 24) suggest two kinds of toxin binding sites associated with the action potential Na+ ionophore: an activation or regulatory site with which veratridine and batrachotoxin interact (11) and an ion transport site with which the inhibitors tetrodotoxin and saxitoxin interact (9, 12). In the experiments described in this report, I have examined the interactions among four neurotoxins (veratridine, batrachotoxin, aconitine, and scorpion toxin) during activation of the action potential Na+ ionophore. EXPERIMENTAL PROCEDURE Materials. Chemicals were obtained from the following sources: veratridine from Aldrich; aconitine from K and K; tetrodotoxin from Calbiochem; ouabain and scorpion venom (Leiurus quinquestriatus) from Sigma; 22NaCl and [3H]leucine from New England Nuclear; Dulbecco-Vogt modification of Eagle's minimal essential medium from Gibco; fetal bovine serum from Colorado Serum Co.; and recrystallized trypsin from Worthington. Batrachotoxin was kindly provided by Drs. John Daly and Bernhard Witkop. Stock solutions of batrachotoxin and aconitine were prepared in ethanol at 100 times the final concentration and diluted into assay medium at 360 immediately prior to use. Scorpion venom was dissolved at 1 mg (dry weight)/ml in distilled water at 00, incubated for 1 hr, and centrifuged for 10 min at 12000 X g. The resulting supernatant, which contained all the activity, showed only low-molecular-weight polypeptides (3000-7000) on gel electrophoresis in sodium dodecyl sulfate. Cell Cultures. Clone N18 of mouse neuroblastoma C1300 was used for all studies. The cells were propagated in 100 mm petri dishes (Falcon) containing 10 ml of growth medium consisting of 5% fetal bovine serum and 95% Dulbecco's modified Eagle's medium in a water-saturated atmosphere of 10% C02/90% air. For experiments, cells from stock cultures were suspended after treatment with 0.02% (w/v) trypsin in Ca++and Mg++-free Dulbecco's phosphate-buffered saline, sedimented, resuspended in growth medium, and seeded at a density of 20,000 cells per cm2 in multi-well plates (1.6 cm diameter, Linbro Chemical Co.). Growth medium was replaced on day 3 with fresh growth medium and on day 5 with fresh growth medium containing 0.2 uCi/ml of [8H]leucine. Cultures were used on day 6.