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

Objective: Na+ cur-rent 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. (c) 2006 European Society of Cardiology. Published by Elsevier B.V.All rights reserved.