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

Hallaq H, Yang Z, Viswanathan PC, Fukuda K, Shen W, Wang DW, Wells KS, Zhou J, Yi J, Murray KT
Cardiovasc Res. 2006 72 (2): 250-61

PMID: 16973141 · DOI:10.1016/j.cardiores.2006.08.007

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.

MeSH Terms (21)

Animals Blotting, Western Cell Line Cell Membrane Cells, Cultured Cyclic AMP-Dependent Protein Kinases Enzyme Activation Female Green Fluorescent Proteins Humans Kidney Luminescence Microscopy, Confocal Myocardium NAV1.5 Voltage-Gated Sodium Channel Oocytes Patch-Clamp Techniques Protein Transport Sodium Channels Transfection Xenopus

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