Departments of Medicine and Pharmacology Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Am J Physiol Heart Circ Physiol. 2012 Feb 1;302(3):H782-9. doi: 10.1152/ajpheart.00817.2010. Epub 2011 Nov 18.
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 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.
Na(+) 电流来源于心脏 SCN5A 同工型的表达，可被受体介导或蛋白激酶 C（PKC）的直接激活所抑制。先前的研究表明，在这种效应中，质膜上 Na(+) 通道的丢失可能起作用，但结果存在争议。在这项研究中，我们检验了这样一个假设，即 PKC 的激活可急性调节 SCN5A 通道的细胞内分布，并且这种效应可以在活细胞中观察到。在稳定表达与 GFP 融合的 SCN5A 的人胚肾细胞（SCN5A-GFP）中，Na(+) 电流在 PKC 激活的情况下被抑制。使用共聚焦显微镜，在对照和刺激条件下对 SCN5A-GFP 通道与质膜的共定位进行了量化。用免疫标记分离出含有细胞外表位的 SCN5A 通道，以允许对质膜进行时间稳定的标记。我们的结果表明，Na(+) 通道优先通过 PKC 激活从质膜转运，其中 Ca(2+) 敏感或常规 PKC 同工型起主要作用，而蛋白激酶 A（PKA）的刺激则产生相反的效果。去除保守的 PKC 位点 Ser(1503)或暴露于 NADPH 氧化酶抑制剂 apocynin 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。使用荧光恢复后光漂白实验表明，PKC 和 PKA 都可以以与通道分布动力学一致的方式修饰通道的流动性。这些结果表明，蛋白激酶的激活可以在活细胞中急性调节心脏 Na(+) 通道的细胞内分布和分子流动性。
