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

Abstract

The patch-clamp technique was used to examine the inhibition of delayed rectifier K+ channels by agents that release intracellular Ca2+. During voltage-clamp experiments on isolated myocytes with 4-aminopyridine (4-AP, 10 mmol/L) and niflumic acid (100 mumol/L) present to inhibit delayed rectifier K+ current (IK(dr)) and Ca(2+)-activated Cl- current (ICl(Ca)), angiotensin II (Ang II) and caffeine increased Ca(2+)-activated K+ current (IK(Ca)) between -25 and 80 mV (n = 5). Conversely, with charybdotoxin (ChTX, 100 nmol/L) and niflumic acid (100 mumol/L) present to inhibit IK(Ca) and ICl(Ca), Ang II and caffeine only caused inhibition of IK(dr). Block was achieved within 15 seconds of drug application and was reversible upon washout (n = 5). The effects of Ang II on IK(Ca) and IK(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 IK(Ca) and IK(dr). In current-clamp experiments, the application of ChTX (100 nmol/L) and niflumic acid (100 mumol/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 Ca2+ indicator indo 1 (100 mumol/L), Ang II, caffeine, and 4-AP increased [Ca2+]i and depolarized the cells. Both Ang II and caffeine caused an increase in [Ca2+]i that preceded membrane depolarization, whereas 4-AP depolarized the cell first and then caused an increase in [Ca2+]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 Ca2+ (n = 6). Increasing intracellular Ca2+ decreased channel opening probability [NxP(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 Ca2+ from < 5 to 200 and 600 nmol/L free Ca2+ decreased NxP(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.(ABSTRACT TRUNCATED AT 400 WORDS)