Properties of Ca²⁺-mediated inactivation of L-type Ca channel in smooth muscle cells of the guinea-pig urinary bladder

The properties of Ca 2+ -mediated inactivation as revealed by a conventional double-pulse protocol were examined by using the whole-cell patch clamp technique. A U-shaped relationship between the conditioning potential and the Ca 2+ current (I Ca ) inactivation was observed, with a maximum inactivation of 52 ± 4% (n = 5) at 10 mV with 0.5 mM EGTA in the patch pipettes. The maximum inactivation was reduced significantly, to 31 ± 5.7% (n = 12) and 32 ± 7.0% (n = 5), when a high concentration of EGTA (20 mM) or a more efficient Ca 2+ chelator, BAPTA, was included in the patch pipettes, respectively. The same double-pulse protocol was applied under conditions where the stored Ca 2+ was depleted by using caffeine or the stored Ca 2+ release function was blocked by using ryanodine or procaine and heparin. No significant difference in the maximum I Ca inactivation before (45%) and after (50%) application of 10 mM caffeine was observed. The maximum I Ca inactivations of 48 ± 3.2% (n = 4) and 52 ± 8.4% (n = 6) were still observed after treatment of the cell with ryanodine (20 μM) or loading 10 mM procaine and 1 mg/mL heparin in the patch pipettes, respectively. These results suggest that Ca 2+ mobilization from an internal Ca 2+ store is not essential for the Ca 2+ -mediated inactivation observed in the double-pulse experiment, rather influx of Ca 2+ through a voltage-dependent Ca channel seems to be important for I Ca inactivation. Recovery from Ca 2+ -mediated inactivation could be fitted by the sum of two exponentials; the time constants of the fast and slow components were 210 and 810 ms, respectively, at a holding potential of −80 mV. By lowering the holding potential to −30 from −80 mV, the time constants of both fast and slow components were increased to 740 and 4820 ms, respectively, suggesting that the recovery of Ca 2+ channels from Ca 2+ -mediated inactivation is voltage dependent.