Intracellular free Ca2+ concentration ([Ca2+]i) and ATP play important roles in the regulation of K- channels in pulmonary artery (PA) myocytes. Previous studies have demonstrated that hypoxia and the metabolic inhibitor, 2-deoxy-D-glucose, decrease voltage-gated K+ (KV) currents [IK(V)] and thereby depolarize PA myocytes; these effects lead to a rise in [Ca2+]i. Here, we used carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a protonophore that uncouples mitochondrial respiration from ATP production, to test whether the inhibition of oxidative phosphorylation affects K+ channel activities in rat PA myocytes. Patch-clamp and fluorescent-imaging microscopy techniques were used to measure K+ currents (IK) and [Ca2+]i, respectively. FCCP (3-5 microM) reversibly raised [Ca2-]i in the presence and absence of external Ca2+. This effect was prevented by pretreating the cells with the membrane-permeable Ca2+ chelator, 1,2-bis(2-amino-phenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). This suggests that much of the FCCP-evoked rise in [Ca2-]i was due to Ca2+ release from intracellular stores. Brief exposure to FCCP (approximately 2 min) reversibly enhanced Ik. This augmentation was not influenced by glibenclamide, an ATP-sensitive K channel blocker, but was eliminated by pretreatment with BAPTA-AM. This implies that the FCCP-evoked rise in [Ca2+]i activated Ca(2+)-activated K- (Kca) channels. Furthermore, in BAPTA-treated cells, longer application (> or = 6 min) of FCCP reversibly decreased IK(V) in PA cells bathed in Ca(2+)-free solution. These results demonstrate that FCCP affects KCa and Kv channels by different mechanisms. FCCP increases IK[Ca] by raising [Ca2+]i primarily as a result of Ca2+ release, but decreases IK(V) by a Ca(2+)-independent mechanism, presumably the inhibition of oxidative ATP production.
Voltage-dependent Gating Rearrangements in the Intracellular T1–T1 Interface of a K+ Channel
