Regulation of Ca2+ current in frog ventricular myocytes by the holding potential, c-AMP and frequency

The whole-cell patch-clamp technique was used to study the effects of holding potential and frequency on the Ca²⁺ current in frog ventricular myocytes. I_Na was blocked by TTX, and i_ca was activated with depolarizing clamps from different holding potentials. Variation of the holding potential revealed three new effects on i_Ca: (1) At -40 mV i_Ca declined with a time constant of 15 min, while at-90 mV, this irreversible decline (run down) in i_Ca did not occur. (2) The decline of i_Ca at -40 mV was biphasic: run down was preceeded by a slow inactivation with a time constant of 40 s, which was reversible upon returning the holding potential to -90 mV. (3) Increasing the frequency of the clamp pulses from 0.1 to 1 Hz led to a rapid decline of i_Ca when the holding potential was positive to -60 mV, but at -90 mV had either no effect or increased i_Ca by 35%, if c-AMP was included in the dialyzing solution. On the other hand, c-AMP did not alter the time course of the run down and the slow inactivation. Replacement of extracellular Ca²⁺ by Ba²⁺ markedly slowed i_Ca kinetics, but did not change the very slow inactivation or the frequency-induced enhancement of i_Ca. Injection of c-AMP led to a transient increase of i_Ca. The phosphodiesterase inhibitor theophylline enhanced the amplitude of the transient and slowed its decay. This effect was mimicked by increased frequency. It is concluded that frequency-induced enhancement of i_Ca is highly dependent on the holding potential, independent of Ca²⁺, and may involve elevation of the intracellular level of c-AMP via inhibition of phosphodiesterase activity. The new type of very slow inactivation is probably under direct voltage control and independent of Ca²⁺ and c-AMP.