Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels
Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels
复制标题
DOI：
10.1152/ajpheart.00817.2010
复制
发表时间：
2012-02-01
影响因子：
4.8
通讯作者：
Murray, Katherine T.
中科院分区：
文献类型：
--
作者：
Hallaq, Haifa;Wang, Dao W.;Murray, Katherine T.
Hallaq H，Wang DW，Kunic JD，乔治AL，J，威尔斯KS，Murray KT.蛋白激酶C的激活改变了心肌Na+通道的细胞内分布和流动性。Am J Physiol Heart Circ Physiol 302：H782-H789，2012.首次发表于2011年11月18日; doi：10.1152/ajpheart.00817.2010. -源自心脏亚型SCN 5A表达的Na+电流通过蛋白激酶C（PKC）的受体介导或直接激活而降低。以前的工作表明，在这种影响中质膜上的Na+通道的损失可能起作用，但结果是有争议的。在这项研究中，我们测试的假设，PKC激活急性调节SCN 5A通道的细胞内分布，这种效果可以在活细胞中可视化。在稳定表达SCN 5A与绿色荧光蛋白（GFP）融合到通道COOH-末端（SCN 5A-GFP）的人胚肾细胞中，暴露于PKC激活抑制Na+电流。使用共聚焦显微镜，在对照和刺激条件下定量SCN 5A-GFP通道与质膜的共定位。对含有细胞外表位的SCN 5A通道的单独群体进行免疫标记以允许质膜的暂时稳定标记。我们的研究结果表明，Na+通道被优先贩运远离质膜的PKC激活，与Ca2+敏感或传统的PKC亚型的主要贡献，而蛋白激酶A（PKA）的刺激有相反的效果。去除保守的PKC位点Ser（1503）或暴露于NADPH氧化酶抑制剂夹竹桃苷消除了PKC介导的改变通道运输的作用，表明通道磷酸化和ROS都是必需的。使用荧光恢复光漂白后的实验表明，PKC和PKA也修改通道流动性的方式与通道分布的动态一致。这些结果表明，蛋白激酶的激活可以急性调节细胞内分布和分子流动性的心脏Na+通道在活细胞中。
Hallaq H, Wang DW, Kunic JD, George AL, J, Wells KS, Murray KT. Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels. Am J Physiol Heart Circ Physiol 302: H782-H789, 2012. First published November 18, 2011; doi:10.1152/ajpheart.00817.2010.-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 Ca2+ 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 photobleaching 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.