Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Channels (Austin)
2018 Jan 01;121:34-44. doi: 10.1080/19336950.2017.1405196.
Show Gene links
Show Anatomy links
Regulation of Kv1.4 potassium channels by PKC and AMPK kinases.
Andersen MN
,
Skibsbye L
,
Saljic A
,
Larsen MZ
,
Rasmussen HB
,
Jespersen T
.
???displayArticle.abstract???
Over 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.
Figure 1. AMPK and PKC activation leads to removal of Kv1.4 channels from the cell surface. (A) MDCK cells transiently expressing Kv1.4 were subjected to a calcium switch assay (see materials and methods) to control the onset of MDCK polarization. At different time points after initiation of polarization, cells were fixed, stained with Kv1.4 antibody and subcellular localization of channels analyzed by confocal microscopy. (B) Quantification of Kv1.4 signals from (A). (C) MDCK cells transiently expressing Kv1.4 were kept unpolarized in low calcium medium. At confluency, the cells were treated with 100 nM PMA for 3 hr (to activate PKC) or 0.5 mM AICAR for 6 hr (to activate AMPK) and the subcellular localization of Kv1.4 analyzed by confocal microscopy. DAPI and phalloidin were used to stain the nucleus and actin, respectively. Scalebar for A and B, 10 µm. (D) Quantification of Kv1.4 signals from (C). The intensity of the fluorescent signal from Kv1.4 channels expressed at the plasma membrane was obtained and expressed as a percentage of the whole cell Kv1.4 fluorescent signal. All groups were statistically compared to the signal at 0 hr. Quantification was performed as described in the Materials and Methods section; *p < 0.05 and ***p < 0.001. 6 ≤ n ≤ 10 for each group. Bars represent means of each group ± SEM.
Figure 2. PKC activation promote reduced Kv1.4 currents. TEVC recordings of Xenopus laevis oocytes expressing Kv1.4 channels. Representative current traces of Kv1.4 currents following depolarization steps in control oocytes (A) and from oocytes treated with 100 nM of the PKC activator PMA (B). Insert: Step protocol. (C) Current-voltage relationship of Kv1.4 currents from control and PMA-treated oocytes. PMA significantly down-regulated the current level by 59%. Kv1.4 (n = 10) and Kv1.4+PMA (n = 7).
Figure 3. AMPK activation promote reduced Kv1.4 currents.TEVC recordings of Xenopus laevis oocytes expressing Kv1.4 channels. Representative current traces of Kv1.4 current following depolarization steps in control oocytes (A) and from oocytes injected with 100 ng ZMP to activate AMPK (B). Insert: Step protocol. (C) Current-voltage relationship of Kv1.4 currents in control oocytes or oocytes injected with ZMP. ZMP significantly down-regulated the current level by 73%. Kv1.4 (n = 10) and Kv1.4+ZMP (n = 7).
Figure 4. Inhibition of PI3K and SGK1 kinases do not affect Kv1.4 localization in polarized MDCK cells. (A) MDCK cells transiently expressing Kv1.4 were subjected to a calcium switch assay for 24 hr and following treated with inhibitors of either PI3K (10 µM LY-294.002) or SGK1 (1 µM GSK-650394) for 3 hr before fixation. Scalebar, 10 µm. (B) Quantification of Kv1.4 signals from (A). The Kv1.4 fluorescent cell surface Kv1.4 signal is expressed as a percentage of fluorescent Kv1.4 total cell signals. All groups were compared to the signal at 24 hr. Quantification was performed as described in the Materials and Methods section. 6 ≤ n ≤ 10 for each group. Bars represent means of each group ± SEM.
Figure 5. Inhibition of PI3K and SGK1 kinases selectively impacts Kv7.1 localization in polarized MDCK cells. MDCK cells transiently co-expressing Kv1.4 and Kv7.1 were treated with inhibitors of either PI3K or SGK1 for 3 hours before fixation. No effect of the PI3K inhibitor LY-294.002 (10 µM) or the SGK1 inhibitor GSK-650394 (1 µM) was observed upon the subcellular localization of Kv1.4, while Kv7.1 was endocytosed upon treatment with both. Scalebar, 10 µm.
Figure 6. Co-expression of Kv1.4 channels with different ratios of Nedd4-x. TEVC recordings of Xenopus laevis oocytes expressing Kv1.4 channels with either Nedd4-1 or Nedd4-2. The current levels were measured at peak amplitude at the +30 mV pulse and normalized to the peak current for Kv1.4 expressed alone. (A) Kv1.4 and Nedd4-1 or Nedd4-2 in a molar ratio of 1:1. (B) Kv1.4 and Nedd4-1 or Nedd4-2 in a molar ratio of 1:2. (C) Kv1.4 and Nedd4-1 or Nedd4-2 in a molar ratio of 1:5. 7 ≤ n ≤ 17 for each group. Noteworthy, no change was observed on current kinetics following Nedd4-x co-expression (data not shown).
Figure 7. Two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing Kv1.4 channels and Nedd4-x. The current levels were measured at peak amplitude at +30 mV for Kv1.4 and +60 mV for Kv7.1 and normalized to the peak current for Kv1.4 or Kv7.1 expressed alone. (A) Kv1.4 and Nedd4-1 or Nedd4-2 in a molar ratio of 1:10. Nedd4-x had no significant effect on Kv1.4 current levels when comparing oocytes co-expressing Kv1.4 and Nedd4-x to oocytes only expressing Kv1.4 (B) Kv7.1 and Nedd4-1 or Nedd4-2 in a molar ratio of 20:1. Nedd4-x had significant effects on Kv7.1 current levels when comparing oocytes co-expressing Kv7.1 and Nedd4-x to oocytes only expressing Kv7.1. Presented is a summary of three individual two-electrode voltage clamp experiments. 16 ≤ n ≤ 27 for each group. Noteworthy, no change was observed on current kinetics of either Kv1.4 or Kv7.1 following Nedd4-x co-expression (data not shown).
Almilaji,
AMP-activated protein kinase regulates hERG potassium channel.
2013,
Pubmed
,
Xenbase
Alzamora,
AMP-activated protein kinase inhibits KCNQ1 channels through regulation of the ubiquitin ligase Nedd4-2 in renal epithelial cells.
2010,
Pubmed
,
Xenbase
Andersen,
Kv7.1 surface expression is regulated by epithelial cell polarization.
2011,
Pubmed
Andersen,
AMPK: A regulator of ion channels.
2012,
Pubmed
Andersen,
A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein.
2013,
Pubmed
Andersen,
AMP-activated protein kinase downregulates Kv7.1 cell surface expression.
2012,
Pubmed
,
Xenbase
Andersen,
PKC and AMPK regulation of Kv1.5 potassium channels.
2015,
Pubmed
,
Xenbase
Andersen,
Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking.
2015,
Pubmed
Boehmer,
Modulation of the voltage-gated potassium channel Kv1.5 by the SGK1 protein kinase involves inhibition of channel ubiquitination.
2008,
Pubmed
,
Xenbase
Citi,
Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells.
1992,
Pubmed
Di Diego,
Pinacidil-induced electrical heterogeneity and extrasystolic activity in canine ventricular tissues. Does activation of ATP-regulated potassium current promote phase 2 reentry?
1993,
Pubmed
El-Kholy,
The phosphatidylinositol 3-kinase inhibitor LY294002 potently blocks K(V) currents via a direct mechanism.
2003,
Pubmed
Gassama-Diagne,
Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells.
2006,
Pubmed
Grunnet,
KCNE4 is an inhibitory subunit to Kv1.1 and Kv1.3 potassium channels.
2003,
Pubmed
,
Xenbase
Gumbiner,
A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide.
1986,
Pubmed
Hagiwara,
Differential inhibition of transient outward currents of Kv1.4 and Kv4.3 by endothelin.
2003,
Pubmed
,
Xenbase
Hanwell,
Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin-Darby canine kidney cells.
2002,
Pubmed
,
Xenbase
Ikonen,
Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells.
1998,
Pubmed
Jespersen,
The KCNQ1 potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family.
2007,
Pubmed
Kim,
A novel mechanism for the suppression of a voltage-gated potassium channel by glucose-dependent insulinotropic polypeptide: protein kinase A-dependent endocytosis.
2005,
Pubmed
Lamothe,
The serum- and glucocorticoid-inducible kinases SGK1 and SGK3 regulate hERG channel expression via ubiquitin ligase Nedd4-2 and GTPase Rab11.
2013,
Pubmed
Leonoudakis,
A multiprotein trafficking complex composed of SAP97, CASK, Veli, and Mint1 is associated with inward rectifier Kir2 potassium channels.
2004,
Pubmed
MacDonald,
Members of the Kv1 and Kv2 voltage-dependent K(+) channel families regulate insulin secretion.
2001,
Pubmed
Manganas,
Subunit composition determines Kv1 potassium channel surface expression.
2000,
Pubmed
Matlin,
Infectious entry pathway of influenza virus in a canine kidney cell line.
1981,
Pubmed
Murray,
Modulation of an inactivating human cardiac K+ channel by protein kinase C.
1994,
Pubmed
,
Xenbase
Nejsum,
Epithelial cell surface polarity: the early steps.
2009,
Pubmed
Nishino,
Ischemic preconditioning activates AMPK in a PKC-dependent manner and induces GLUT4 up-regulation in the late phase of cardioprotection.
2004,
Pubmed
Niwa,
Molecular determinants of cardiac transient outward potassium current (I(to)) expression and regulation.
2010,
Pubmed
Qi,
AMPK: energy sensor and survival mechanism in the ischemic heart.
2015,
Pubmed
Rasmusson,
C-type inactivation controls recovery in a fast inactivating cardiac K+ channel (Kv1.4) expressed in Xenopus oocytes.
1995,
Pubmed
,
Xenbase
Roeper,
Frequency-dependent inactivation of mammalian A-type K+ channel KV1.4 regulated by Ca2+/calmodulin-dependent protein kinase.
1997,
Pubmed
Staub,
Role of ubiquitylation in cellular membrane transport.
2006,
Pubmed
Stuart,
Regulated assembly of tight junctions by protein kinase C.
1995,
Pubmed
Suzuki,
aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization.
2002,
Pubmed
Tao,
Neuronal transmission stimulates the phosphorylation of Kv1.4 channel at Ser229 through protein kinase A1.
2005,
Pubmed
Vacher,
Localization and targeting of voltage-dependent ion channels in mammalian central neurons.
2008,
Pubmed
Walsh,
Neonatal rat cardiac fibroblasts express three types of voltage-gated K+ channels: regulation of a transient outward current by protein kinase C.
2008,
Pubmed
Watanabe,
Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking.
2004,
Pubmed
Williams,
LKB1 and AMPK in cell polarity and division.
2008,
Pubmed
Wollner,
Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity.
1992,
Pubmed
Xie,
Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells.
2008,
Pubmed
Xie,
Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells.
2006,
Pubmed
Yamanaka,
PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex.
2001,
Pubmed
Yang,
Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions.
2010,
Pubmed