Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na + channels

Haifa Hallaq, Dao W. Wang, Jennifer D. Kunic, Alfred L. George, K. Sam Wells, Katherine T. Murray

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

Na + current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na + channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na + currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na + channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca 2+-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser 1503 or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleach-ing demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na + channels in living cells.

Original languageEnglish (US)
Pages (from-to)H782-H789
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume302
Issue number3
DOIs
StatePublished - Feb 2012

Keywords

  • Membrane trafficking
  • Protein kinase C
  • Sodium channels

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

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