Protein kinase C downregulates IKs by stimulating KCNQ1-KCNE1 potassium channel endocytosis

BackgroundThe slow-activating cardiac repolarization K+ current (IKs), generated by the KCNQ1-KCNE1 potassium channel complex, is controlled via sympathetic and parasympathetic regulation in vivo. Inherited KCNQ1 and KCNE1 mutations predispose to ventricular fibrillation and sudden death, often triggered by exercise or emotional stress. Protein kinase C (PKC), which is activated by α1 adrenergic receptor stimulation, is known to downregulate IKs via phosphorylation of KCNE1 serine 102, but the underlying mechanism has remained enigmatic. We previously showed that KCNE1 mediates dynamin-dependent endocytosis of KCNQ1-KCNE1 complexes.ObjectiveThis study sought to determine the potential role of endocytosis in IKs downregulation by PKC.MethodsWe utilized patch clamping and fluorescence microscopy to study Chinese hamster ovary (CHO) cells coexpressing KCNQ1, KCNE1, and wild-type or dominant-negative mutant (K44A) dynamin 2, and neonatal mouse ventricular myocytes.ResultsThe PKC activator phorbol 12-myristate 13-acetate (PMA) decreased IKs density by >60% (P < .05) when coexpressed with wild-type dynamin 2 in CHO cells, but had no effect when coexpressed with K44A-dynamin 2. Thus, functional dynamin was required for downregulation of IKs by PKC activation. PMA increased KCNQ1-KCNE1 endocytosis in CHO cells expressing wild-type dynamin 2, but had no effect on KCNQ1-KCNE1 endocytosis in CHO cells expressing K44A-dynamin 2, determined using the Pearson correlation coefficient to quantify endosomal colocalization of KCNQ1 and KCNE1 with internalized fluorescent transferrin. KCNE1-S102A abolished the effect of PMA on IKs currents and endocytosis. Importantly, PMA similarly stimulated endocytosis of endogenous KCNQ1 and KCNE1 in neonatal mouse myocytes.ConclusionPKC activation downregulates IKs by stimulating KCNQ1-KCNE1 channel endocytosis.