Abstract
Ca currents (ICa) were measured by whole-cell patch clamp in single cells isolated from frog ventricle in which K currents were blocked with intracellular (120 mM) and extracellular (20 mM) Cs. Inward currents elicited by depolarizing voltage steps from a holding potential of -80 mV were blocked completely by 0.5 mM-Cd. The quality of the voltage clamp was assessed using two patch electrodes on a single cell. One electrode was used in the voltage-clamp mode to measure membrane currents and the other in current-clamp to measure membrane potential. Ca currents as large as 2 nA were well clamped in cells as long as 210 micron. Acetylcholine (ACh) had no effect on ICa in the absence of beta-adrenergic stimulation but reduced to control levels ICa elevated by isoprenaline. Nanomolar concentrations of ACh were able to reduce significantly ICa elevated by 2 microM-isoprenaline. ACh had no effect on the shape of the I-V curve, on the reactivation (recovery from inactivation), or on the inactivation of ICa. Although isoprenaline increased ICa by an average of 6.5-fold, it had no effect on the shape of the I-V curve or on the inactivation at test potentials negative to +40 mV. However, isoprenaline slowed the half-reactivation time from a control value of 120 +/- 10 ms (mean +/- S.D.) to 153 +/- 12 ms at -80 mV. The effect of cyclic AMP on ICa was investigated using two patch electrodes, one filled with cyclic AMP. Maximal effects of cyclic AMP were observed with 5 microM-cyclic AMP in the pipette. Maximal ICa was recorded several minutes after breaking the patch with the second electrode. After removing the cyclic-AMP-containing electrode, ICa declined to control levels after approximately 10 min. 5 microM-cyclic AMP in the patch electrode increased ICa by an average of 6.9-fold, but had no effect on the shape of the I-V curve or on inactivation. Cyclic AMP had a slowing effect on reactivation (half-reactivation time = 155 +/- 24 ms) similar to that of isoprenaline. ACh (1-10 microM) did not reduce ICa elevated with cyclic AMP (0.1-20 microM-cyclic AMP in the pipette). With low concentrations of cyclic AMP in the pipette (0.1 microM), isoprenaline augmented ICa, but with 5 microM-cyclic AMP in the pipette, isoprenaline was incapable of increasing ICa further. These results suggest that the decrease of ICa produced by ACh can be explained solely by decreases in cyclic AMP levels.