MUTUALLY EXCLUSIVE ACTION OF CATIONIC VERATRIDINE AND CEVADINE AT AN INTRACELLULAR SITE OF THE CARDIAC SODIUM-CHANNEL

Veratridine modification of Na current was examined in single dissociated ventricular myocytes from late-fetal rats by applying pulses to -30 mV for 50 ms every 2 or 5 s from a holding potential of - 100 mV (20-degrees-C) and measuring amplitude, I(tail), and time constant, tau(tail). Of the post-repolarization inward tail current induced by the alkaloid. Increasing the pH of a 30-mu-M veratridine superfusate from 7.3 to 8.3 (which increases the fraction of uncharged veratridine molecules from 0. 5 to 5% while decreasing that of protonated molecules from 99.5 to 95%) increased I(tail) by a factor of 2.5 +/- 0.5 (mean +/- SEM; n = 3). Switching from 100-mu-M veratridine superfusate at pH 7.3 to 10-mu-M at pH 8.3 did not affect the size of I(tail) (n = 4). Intracellular (pipette) application of 100-mu-M veratridine at pH 7.3 or 8.3 produced small I(tail)'s suggesting transmembrane loss of alkaloid. If this was compensated for by simultaneous extracellular application of 100-mu-M veratridine at a pH identical to intracellular pH, I(tail) (measured relative to the maximum amplitude induced by a combination of 100-mu-M veratridine and 1-mu-M BDF 9145 in the same cell) at pH(i) 7.3 did not significantly differ from that at pH(i) 8.3 (84 +/- 4 vs. 70 +/- 6%; n = 3 each). Results from six control cells and five cells subjected to extra-and /or intracellularly increased viscosity by the addition of 0.5 or 1 molal sucrose showed that increasing intracellular viscosity 1.6- and 2.5-fold increased tau(tail) 1.5- and 2.3-fold, respectively, while a selective 2.5-fold increase of extracellular viscosity did not significantly affect tau(tail). Superfusion with the related ceveratrum alkaloid cevadine (1-100-mu-M), in cells additionally treated with 1-mu-M BDF 9145 to remove inactivation, induced a tail current whose amplitude was half-maximal at 4-mu-M and saturating at 30-mu-M, while tau(tail) (mean value 49 ms; eight cells) was not affected by concentration. The addition of 100 or 30-mu-M cevadine to the superfusate containing a saturating concentration of veratridine (30-mu-M) had little effect on the initial amplitude of the veratridine-induced tail current, but converted its monoexponential decay into a biexponential decay characterized by time constants corresponding to cevadine and veratridine, respectively. The veratridine-induced component of I(tail) was reduced to 25% by 100-mu-M cevadine (n = 1) and to 60 +/- 7% (n = 3) by 30-mu-M cevadine. We conclude on the basis of the extracellular and intracellular pH and viscosity effects that extracellularly applied veratridine diffuses in its free base form through the sarcolemma and activates the Na channel at an intracellular site in its protonated form. This action is mutually exclusive with that of cevadine, the C-3 angelic acid ester analogue of veratridine, suggesting a competitive interaction. Chemical complementarity of veratridine to its binding site suggests the intracellular presence of a negative charge and adjacent hydrophobic amino acid residue as essential components of the veratridine recognition site at the Na channel macromolecule.