Action of tertiary phenylalkylamines on cardiac transient outward current from outside the cell membrane.

The effects of the phenylalkylamines verapamil (V), gallopamil (G), and devapamil (D) and their corresponding quaternary derivatives on the transient outward current (Ito) were examined in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The question was addressed, whether phenylalkylamines act on Ito from the inside or the outside or from both sides of the cell membrane. To this end, the myocytes were either superfused extracellularly or perfused intracellularly with drug-containing solutions. In addition, the effects of verapamil were investigated at different pH-values. V, G, and D (30 microM each), applied extracellularly, reduced the steady state current of Ito, Ito(150 ms), to 34 +/- 3.3, 33 +/- 6, and 30 +/- 5, respectively (% of control; means +/- SEM). The effects of V (30 microM) on Ito were similar at various external pH-values (reduction of Ito(150 ms) by 69 +/- 6 at pH 6.5, by 66 +/- 4 at pH 7.4, by 68 +/- 8 at pH 8.5, and by 58 +/- 10 at pH 9.5; % of control; means +/- SEM). In contrast, the effect of 4-aminopyridine (300 microM) on Ito was enhanced after alkalinisation: the peak current of Ito was reduced to 49 +/- 5 at pH 7.4 and to 5 +/- 2 at pH 9.2 (% of control; means +/- SEM). V, G, and D (300 microM) failed to produce any effect on Ito, when applied intracellularly (values of Ito(150 ms): 97 +/- 6, 105 +/- 4, and 94 +/- 4, respectively; % of control; means +/- SEM). In contrast, 4-aminopyridine (3 mM) depressed the peak current of Ito to 69 +/- 6% of control (mean +/- SEM), when applied intracellularly. The permanently charged quaternary derivatives of the phenylalkylamines q-V, q-G, and q-D (300 microM) did not significantly affect Ito, when applied extracellularly (values of Ito(150 ms): 94 +/- 2, 90 +/- 3, and 94 +/- 3, respectively; % of control; means +/- SEM) but diminished Ito, when applied intracellularly (reduction of Ito(150 ms) to 43 +/- 5, 56 +/- 7, and 63 +/- 4, respectively; % of control; means +/- SEM). Intracellularly applied V (300 microM) did not reduce Ito at pH 6.5 at which V is protonated to 99.4%. It is suggested that tertiary phenylalkylamines act on Ito by binding to a membrane site accessible from the outside, whereas their quaternary derivatives affect Ito by binding to a membrane site located at the inside of the cell membrane. In contrast, 4-aminopyridine is supposed to act on Ito from the inside of the cell membrane.