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 Ca2+ current (ICa) 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 Ca2+ chelator, BAPTA, was included in the patch pipettes, respectively. The same double-pulse protocol was applied under conditions where the stored Ca2+ was depleted by using caffeine or the stored Ca2+ release function was blocked by using ryanodine or procaine and heparin. No significant difference in the maximum ICa inactivation before (45%) and after (50%) application of 10 mM caffeine was observed. The maximum ICa inactivations of 48 +/- 3.2% (n = 4) and 52 +/- 8.4% (n = 6) were still observed after treatment of the cell with ryanodine (20 microM) or loading 10 mM procaine and 1 mg/mL heparin in the patch pipettes, respectively. These results suggest that Ca2+ mobilization from an internal Ca2+ store is not essential for the Ca(2+)-mediated inactivation observed in the double-pulse experiment, rather influx of Ca2+ through a voltage-dependent Ca channel seems to be important for ICa inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)