[Ca 2+ ] i Inhibition of K + Channels in Canine Renal Artery

Abstract

Abstract The patch-clamp technique was used to examine the inhibition of delayed rectifier K + channels by agents that release intracellular Ca 2+ . During voltage-clamp experiments on isolated myocytes with 4-aminopyridine (4-AP, 10 mmol/L) and niflumic acid (100 μmol/L) present to inhibit delayed rectifier K + current (I K(dr) ) and Ca 2+ -activated Cl − current (I Cl(Ca) ), angiotensin II (Ang II) and caffeine increased Ca 2+ -activated K + current (I K(Ca) ) between −25 and 80 mV (n=5). Conversely, with charybdotoxin (ChTX, 100 nmol/L) and niflumic acid (100 μmol/L) present to inhibit I K(Ca) and I Cl(Ca) , Ang II and caffeine only caused inhibition of I K(dr) . Block was achieved within 15 seconds of drug application and was reversible upon washout (n=5). The effects of Ang II on I K(Ca) and I K(dr) were inhibited by the specific Ang II receptor antagonist losartan (1 mmol/L, n=3). Intracellular BAPTA (10 mmol/L) also abolished the effects of Ang II and caffeine on both I K(Ca) and I K(dr) . In current-clamp experiments, the application of ChTX (100 nmol/L) and niflumic acid (100 μmol/L) caused little change in resting membrane potential; however, subsequent application of caffeine (10 mmol/L) caused a 26±2.9 mV depolarization from −54±3.1 to −28±1.7 mV (n=6). 4-AP (10 mmol/L) blocked the caffeine-induced depolarization. When isolated cells were loaded with the Ca 2+ indicator indo 1 (100 μmol/L), Ang II, caffeine, and 4-AP increased [Ca 2+ ] i and depolarized the cells. Both Ang II and caffeine caused an increase in [Ca 2+ ] i that preceded membrane depolarization, whereas 4-AP depolarized the cell first and then caused an increase in [Ca 2+ ] i (n=4). In inside-out patches, with 200 nmol/L ChTX in the patch pipette to block large-conductance Ca 2+ -activated K + channels, a 45±7-picosiemen 4-AP–sensitive K + channel was identified that was sensitive to cytoplasmic Ca 2+ (n=6). Increasing intracellular Ca 2+ decreased channel opening probability [N×P(open), where N is the number of functional channels in a patch and P(open) is the opening probability] at all membrane potentials examined. At 0 mV, increasing Ca 2+ from <5 to 200 and 600 nmol/L free Ca 2+ decreased N×P(open) by 52±3% and 73±7%, respectively (n=6). The decrease in opening probability of the delayed rectifier K + channel resulted from a concentration- and voltage-dependent decrease in mean open time. The decrease in mean open time reflected significant decreases and increases in open and closed time constants, respectively. These results suggest that agonist-induced changes in intracellular Ca 2+ can alter vascular smooth muscle membrane potential through regulation of delayed rectifier K + channels.