AMPK phosphorylation of KCa2.3 alleviates angiotensin II-induced endothelial dysfunction.

The endothelial small-conductance calcium-activated potassium channels (KCa2.3) are indispensable for endothelium-dependent hyperpolarization (EDH) response, mainly in resistance arteries. We recently demonstrated in diet-induced obese mice that adenosine monophosphate-activated protein kinase (AMPK) upregulates endothelial KCa2.3 expression and improves endothelial function. However, the molecular mechanism of regulation of KCa2.3 by AMPK remains less explored. Using techniques of bioinformatics, molecular biology and wire myograph system, we examined KCa2.3 phosphorylation by AMPK in human umbilical vein endothelial cells (HUVECs), human embryonic kidney 293 (HEK-293T) cells and second-order mesenteric resistance arteries from angiotensin II-induced hypertensive mice. In HUVECs, treatment with activators of AMPK (AICAR, metformin, and MK-8722) significantly increased phosphorylation of KCa2.3 Thr106 (human), which was antagonized by AMPK inhibitor compound C. In HEK-293T cells, KCa2.3 current was enhanced by AMPK activation or phosphomimetic mutant KCa2.3 (T106D), which was abolished after de-phosphomimetic mutant (T106A) or deletion of KCa2.3 of Thr106 site (T106Del). In mice with angiotensin II infusion, 2-week treatment with AICAR or overexpressing phosphomimetic mutant KCa2.3 Thr107D (mouse) restored KCa2.3-mediated EDH-dependent relaxation in mesenteric resistance arteries together with reversal of early phase hypertension. Our study demonstrates for the first time that AMPK activation mediates KCa2.3 phosphorylation in endothelial cells with enhanced channel activity. This effect ameliorates endothelial dysfunction of mesenteric resistance arteries and alleviates angiotensin II-induced early phase hypertension in mice.