Hallaq Haifa, Yang Zhenjiang, Viswanathan Prakash C, Fukuda Koji, Shen Wangzhen, Wang Dao W, Wells K Sam, Zhou Jingsong, Yi Jianxun, Murray Katherine T
Department of Medicine, Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA.
Cardiovasc Res. 2006 Nov 1;72(2):250-61. doi: 10.1016/j.cardiores.2006.08.007. Epub 2006 Aug 16.
Na(+) current derived from expression of the principal cardiac Na(+) channel, Na(v)1.5, is increased by activation of protein kinase A (PKA). This effect is blocked by inhibitors of cell membrane recycling, or removal of a cytoplasmic endoplasmic reticulum (ER) retention motif, suggesting that PKA stimulation increases trafficking of cardiac Na(+) channels to the plasma membrane.
To test this hypothesis, green fluorescent protein (GFP) was fused to Na(v)1.5 (Na(v)1.5-GFP), and the effects of PKA activation were investigated in intact, living cells that stably expressed the fusion protein. Using confocal microscopy, the spatial relationship of GFP-tagged channels relative to the plasma membrane was quantitated using a measurement that could control for variables present during live-cell imaging, and permit an unbiased analysis for all cells in a given field.
In the absence of kinase stimulation, intracellular fluorescence representing Na(v)1.5-GFP channels was greatest in the perinuclear area, with additional concentration of channels beneath the cell surface. Activation of PKA promoted trafficking of Na(+) channels from both regions to the plasma membrane. Experimental results using a chemiluminescence-based assay further confirmed that PKA stimulation increased expression of Na(v)1.5 channels at the cell membrane.
Our results provide direct evidence for PKA-mediated trafficking of cardiac Na(+) channels into the plasma membrane in living, mammalian cells, and they support the existence of multiple intracellular storage pools of channel protein that can be mobilized following a physiologic stimulus.
由主要心脏钠通道Na(v)1.5表达产生的钠电流可通过蛋白激酶A（PKA）激活而增加。细胞膜再循环抑制剂或去除细胞质内质网（ER）保留基序可阻断这种效应，这表明PKA刺激可增加心脏钠通道向质膜的转运。
为验证这一假设，将绿色荧光蛋白（GFP）与Na(v)1.5融合（Na(v)1.5-GFP），并在稳定表达融合蛋白的完整活细胞中研究PKA激活的效应。使用共聚焦显微镜，通过一种能够控制活细胞成像过程中存在的变量并允许对给定视野中的所有细胞进行无偏分析的测量方法，对GFP标记通道与质膜的空间关系进行定量。
在无激酶刺激的情况下，代表Na(v)1.5-GFP通道的细胞内荧光在核周区域最强，细胞表面下方也有额外的通道聚集。PKA激活促进了钠通道从这两个区域向质膜的转运。使用基于化学发光的检测方法的实验结果进一步证实，PKA刺激增加了质膜上Na(v)1.5通道的表达。
我们的结果为PKA介导的心脏钠通道在活的哺乳动物细胞中转运到质膜提供了直接证据，并且支持存在多个可在生理刺激后被动员的通道蛋白细胞内储存池。
