William A. Catterall

Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600.

Abstract During Days 4 to 7 in ovo, beating of embryonic chick hearts becomes progressively more sensitive to inhibition by tetrodotoxin, an inhibitor of fast Na+ channels, and progressively less sensitive to inhibition by D600, an inhibitor of slow Ca2+/Na+ channels. The developmental change in tetrodotoxin sensitivity is not retained in heart cells cultured in monolayer. In contrast, the developmental change in D600 sensitivity is retained. Veratridine-stimulated 22Na+ influx mediated by fast Na+ channels is inhibited by tetrodotoxin (Ki = 1.6 nM) in cells prepared from either 3-day or 12-day embryos. These results suggest that young embryonic hearts contain physiologically inactive Na+ channels. 22Na+ influx mediated by slow Ca2+/Na+ channels is inhibited by D600 with a Ki of 40 nM for cells from 3-day hearts and 8 μM for cells from 12-day hearts. Beating of heart cells in aggregate cultures is also inhibited by D600. Aggregates which have reactivated after inhibition by tetrodotoxin are 10-fold more sensitive to inhibition by D600 than untreated controls. The results suggest that the primary developmental event is a change in slow Ca2+/Na+ channels which reduces their sensitivity to D600 and diminishes their ability to support beating without the activity of the fast Na+ channel.

Activation and inhibition of the action potential Na+ ionophore of cultured rat muscle cells by neurotoxins

Abstract Both myoblasts and myotubes in cultures of clonal rat muscle cells have action potential Na + ionophore activity. The ionophore is activated by batrachotoxin (K 0.5 = 3 to 5 × 10 −7 M) and veratridine (K 0.5 = 4 to 6 × 10 −5 M) which compete for the same activation site. As in denervated rat muscle, the ionophore of cultured muscle is 100 fold more resistant to inhibition by tetrodotoxin (K 0.5 = 1.5 to 3 × 10 −6 M) and 20 fold more resistant to inhibition by saxitoxin (K 0.5 = 1.5 to 3 × 10 −7 M) than in nerve, innervated muscle, or cultured neuroblastoma cells.

Interaction between batrachotoxin and yohimbine.

The neurotoxins, batrachotoxin and veratridine, are specific activators of sodium channels and cause an increase in the rate of 22Na uptake in neuroblastoma cells. Yohimbine, an indolakylamine alkaloid, inhibits this batrachotoxin-induced 22Na uptake. The dose-response curve of yohimbine suggest that the inhibitor acts reversibly on a single class of binding sites with dissociation constant of 3--4 x 10(-5) M. The dissociation constant is not affected by depolarization from--41 to 0 mV. Kinetic and equilibrium experiments indicate that yohimbine is a competitive inhibitor of the action of batrachotoxin. These results support the conclusion that yohimbine inhibitis the sodium flux by acting on the channel gating mechanism rather than by occluding the channels.

MEMBRANE POTENTIAL DEPENDENT BINDING OF SCORPION TOXIN TO THE ACTION POTENTIAL SODIUM IONOPHORE. STUDIES WITH A 3-(4-HYDROXY 3-[125I] IODOPHENYL) PROPIONYL DERIVATIVE

Abstract— A polypeptide toxin purified 80‐fold from the venom of the scorpion Leiurus quinquestriatus enhances activation of the action potential Na+ ionophore by the alkaloid neurotoxins veratridine, batrachotoxin and aconitine in electrically excitable neuroblastoma cells. The purified toxin can be labelled with [125I] by reaction with N‐succinimidyl 3‐(4‐hydroxy 3‐[125I] iodophenyl) propionate. The [125I] labelled toxin obtained from carboxymethyl Sephadex ion exchange chromatography appears homogeneous by gel electrophoresis and isoelectric focusing. The [125I] labelled toxin binds to a single class of saturable binding sites and also activates the action potential Na+ ionophore in electrically excitable neuroblastoma cells showing identical concentration dependence for both the binding and the activation effects. The labelled toxin does not show any saturable binding or activation of the action potential Na+ ionophore in variant neuroblastoma clones that specifically lack the action potential Na+ ionophore. The results indicate that scorpion toxin binds specifically to the action potential Na+ ionophore. The binding sites have a mean equilibrium dissociation constant of 3 IIH, a mean binding capacity of 46fmol toxin per mg cell protein and a mean density of 24 sites per μm2 of cell surface membrane. A single action potential Na+ ionophore transports 1 × 108 ions per min and has a conductance of 3 psiemens at physiologic ion concentrations. Depolarization of cells by elevated K+ concentration inhibits the saturable binding. Depolarization of cells by incubation in high Na+ medium (130mm‐Na+, 5mm‐K+) with gramicidin A or batrachotoxin also inhibits the saturable toxin binding. These results suggest that scorpion toxin binds specifically to a regulatory component (gate) of the Na+ ionophore. whose conformation is dependent on membrane potential.