title: Protein kinase C downregulates I(Ks) by stimulating KCNQ1-KCNE1 potassium channel endocytosis.
abstract: BACKGROUND
  The slow-activating cardiac repolarization K(+) current (I(Ks)), 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 I(Ks) 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.
  
  
  OBJECTIVE
  This study sought to determine the potential role of endocytosis in I(Ks) downregulation by PKC.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased I(Ks) 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 I(Ks) 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 I(Ks) currents and endocytosis. Importantly, PMA similarly stimulated endocytosis of endogenous KCNQ1 and KCNE1 in neonatal mouse myocytes.
  
  
  CONCLUSIONS
  PKC activation downregulates I(Ks) by stimulating KCNQ1-KCNE1 channel endocytosis.
authors.affiliation: Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021, USA.
authors.forename: Vikram A
authors.initials: VA
authors.lastname: Kanda
authors.forename: Kerry
authors.initials: K
authors.lastname: Purtell
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Abbott
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D004705:Endocytosis; D051657:KCNQ1 Potassium Channel / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D014162:Transfection
chemical_list: D002966:Clathrin; C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; D024642:Potassium Channels, Voltage-Gated; D011493:Protein Kinase C