Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells.

@article{citeulike:853232, abstract = {OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.}, address = {Department of Medicine, Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, United States.}, author = {Hallaq, Haifa and Yang, Zhenjiang and Viswanathan, Prakash C C. and Fukuda, Koji and Shen, Wangzhen and Wang, Dao W W. and Wells, K Sam S. and Zhou, Jingsong and Yi, Jianxun and Murray, Katherine T T. }, citeulike-article-id = {853232}, comment = {full length Nav1.5-eGFP / membrane red Di-8-ANEPPS / R/G ratio /}, doi = {10.1016/j.cardiores.2006.08.007}, issn = {0008-6363}, journal = {Cardiovasc Res}, keywords = {fluorescent\_proteins, live\_cell\_imaging, nav, trafficking}, month = {August}, posted-at = {2006-09-21 17:03:36}, priority = {0}, title = {Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells.}, url = {http://dx.doi.org/10.1016/j.cardiores.2006.08.007}, year = {2006} }

TY - JOUR ID - citeulike:853232 L3 - citeulike-article-id:853232 TI - Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells. JF - Cardiovasc Res AD - Department of Medicine, Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, United States. SN - 0008-6363 N2 - OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. KW - fluorescent_proteins KW - live_cell_imaging KW - nav KW - trafficking N1 - full length Nav1.5-eGFP / membrane red Di-8-ANEPPS / R/G ratio / AU - Hallaq, Haifa AU - Yang, Zhenjiang AU - Viswanathan, Prakash C AU - Fukuda, Koji AU - Shen, Wangzhen AU - Wang, Dao W AU - Wells, K Sam AU - Zhou, Jingsong AU - Yi, Jianxun AU - Murray, Katherine T PY - 2006/08/16/ UR - http://dx.doi.org/10.1016/j.cardiores.2006.08.007 ER -