Abstract
ABSTRACTOver the last years extensive kinase-mediated regulation of a number of voltage-gated potassium (Kv) channels important in cardiac electrophysiology has been reported. This includes regulation of Kv1.5, Kv7.1 and Kv11.1 cell surface expression, where the kinase-mediated regulation appears to center around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further demonstrate that unlike related Kv channels, Kv1.4 current levels in Xenopus laevis oocytes are not reduced by co-expression of Nedd4-2, or the related Nedd4-1 ubiquitin ligase. In conclusion, we demonstrate that the surface expression of Kv1.4 is downregulated by the two kinases AMPK and PKC, but is unaffected by PI3K-SGK1 signaling, as well as Nedd4-1/Nedd4-2 activity. In the light of previous reports, our results demonstrate an impressive heterogeneity in the molecular pathways controlling the surface expression of highly related potassium channel subunits.
Highlights
The voltage-gated potassium channel Kv1.4 is part of the Shaker Kv-channel family and contributes to the transient outward potassium current Ito,s involvement in the repolarization of the cardiac action potential [1]
In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK)
We further demonstrate that unlike related Kv channels, Kv1.4 current levels in Xenopus laevis oocytes are not reduced by co-expression of Nedd4-2, or the related Nedd4-1 ubiquitin ligase
Summary
The voltage-gated potassium channel Kv1.4 is part of the Shaker Kv-channel family and contributes to the transient outward potassium current Ito,s involvement in the repolarization of the cardiac action potential [1]. Studies suggest that the Kv1.4 channel is endocytosed in response to Protein Kinase A (PKA) activation [3,7,8,9,10,11]. Several studies suggest that Protein Kinase C (PKC) exerts an inhibitory effect upon Kv1.4. In a study by Walsh et al it was demonstrated that activation of PKC inhibits the Ito current in cardiac fibroblasts [12]. In line with this observation, Murray et al reported that PKC inhibits Kv1.4 currents in a slow timedependent manner in Xenopus laevis oocytes. The underlying mechanism for the PKC-mediated downregulation of Kv1.4 currents has not been investigated [9]
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