Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells.
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.
Hallaq
Haifa
H
Department of Medicine, Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA.
Yang
Zhenjiang
Z
Viswanathan
Prakash C
PC
Fukuda
Koji
K
Shen
Wangzhen
W
Wang
Dao W
DW
Wells
K Sam
KS
Zhou
Jingsong
J
Yi
Jianxun
J
Murray
Katherine T
KT
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D049449:Luminescence; D018613:Microscopy, Confocal; D009206:Myocardium / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021381:Protein Transport; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014981:Xenopus
D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; D015222:Sodium Channels; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases