AMP‐activated protein kinase inhibits Kv1.5 channel currents of pulmonary arterial myocytes in response to hypoxia and inhibition of mitochondrial oxidative phosphorylation

KEY POINTS Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage-gated potassium channels (Kv ) in pulmonary arterial smooth muscle by hypoxia, although the precise molecular mechanisms have been unclear. AMP-activated protein kinase (AMPK) has been proposed to couple inhibition of mitochondrial metabolism by hypoxia to acute hypoxic pulmonary vasoconstriction and progression of pulmonary hypertension. Inhibition of complex I of the mitochondrial electron transport chain activated AMPK and inhibited Kv 1.5 channels in pulmonary arterial myocytes. AMPK activation by 5-aminoimidazole-4-carboxamide riboside, A769662 or C13 attenuated Kv 1.5 currents in pulmonary arterial myocytes, and this effect was non-additive with respect to Kv 1.5 inhibition by hypoxia and mitochondrial poisons. Recombinant AMPK phosphorylated recombinant human Kv 1.5 channels in cell-free assays, and inhibited K(+) currents when introduced into HEK 293 cells stably expressing Kv 1.5. These results suggest that AMPK is the primary mediator of reductions in Kv 1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. ABSTRACT Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage-gated potassium channels (Kv ) in pulmonary arterial smooth muscle cells that is mediated by the inhibition of mitochondrial oxidative phosphorylation. We sought to determine the role in this process of the AMP-activated protein kinase (AMPK), which is intimately coupled to mitochondrial function due to its activation by LKB1-dependent phosphorylation in response to increases in the cellular AMP:ATP and/or ADP:ATP ratios. Inhibition of complex I of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited Kv currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors. Myocyte Kv currents were also markedly inhibited upon AMPK activation by A769662, 5-aminoimidazole-4-carboxamide riboside and C13 and by intracellular dialysis from a patch-pipette of activated (thiophosphorylated) recombinant AMPK heterotrimers (α2β2γ1 or α1β1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK-sensitive K(+) currents, which were also blocked by the selective Kv 1.5 channel inhibitor diphenyl phosphine oxide-1 but unaffected by the presence of the BKCa channel blocker paxilline. Moreover, recombinant human Kv 1.5 channels were phosphorylated by AMPK in cell-free assays, and K(+) currents carried by Kv 1.5 stably expressed in HEK 293 cells were inhibited by intracellular dialysis of AMPK heterotrimers and by A769662, the effects of which were blocked by compound C. We conclude that AMPK mediates Kv channel inhibition by hypoxia in pulmonary arterial myocytes, at least in part, through phosphorylation of Kv 1.5 and/or an associated protein.