Rapid regulation of the 'second inward current' by intracellular calcium in isolated rat and ferret ventricular myocytes.

1. Single cells were isolated from the ventricles of ferret and rat hearts. Cells were voltage clamped using a single conventional microelectrode. Membrane voltage, membrane currents and cell length were monitored. 2. The current elicited by decreasing the membrane potential from a holding potential of -40 or -45 mV to potentials more positive than -20 mV was abolished by D600, by Cd2+ and by removal of Ca2+ from the cell superfusate. This current activated within 20 ms and inactivated over several hundred milliseconds; it had a bell-shaped current-voltage relation, and was maximal at about +10 mV. It is concluded that this is the fast Ca2+ current ICa. 3. Increasing bathing [Ca2+] (Ca2+o) led to the appearance of transient inward currents (Iti). If ICa was triggered during Iti, it was reduced in magnitude, and inactivated more slowly. 4. The sarcoplasmic reticulum inhibitor ryanodine (1 mumol/l) abolished Iti, and reduced twitch contraction, but had no direct effect on the magnitude of ICa, although its rate of inactivation was slowed. 5. Iti produced by depolarization of the holding potential, or by lowering bathing [K+] or [Na+], led to similar changes to those described in paragraph 3. 6. Gradually increasing diastolic cytoplasmic [Ca2+] (Ca2+i) by rapid stimulation in the presence of ryanodine, by lowering bathing [K+], or lowering bathing [Na+], led to a parallel decrease of ICa. 7. The effects of lowering bathing [Na+] could be abolished by using an electrode-filling solution containing EGTA. 8. In some ferret cells a slow component of the second inward current was observed. The size of this current was directly related to the size of the twitch: changes in the size of the twitch produced by changing the pattern of stimulation or application of ryanodine were paralleled by changes in the size of this current, but had no effect on the size of ICa. 9. It is concluded that the magnitude of ICa can be decreased by an increase of either resting Ca2+i, or the spontaneous increase of Ca2+ which underlies Iti, but it is not affected by the size of the stimulated calcium transient (although the time course of inactivation is dependent on the calcium transient). The size of the slow component of the second inward current, however, is directly related to the size of the twitch and may, therefore, be activated by Ca2+.