Copyright © 2006, European Society of Cardiology
Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells
aDepartment of Medicine, Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, United States
bDepartment of Anesthesia, Vanderbilt University School of Medicine, Nashville, TN, United States
cDepartment of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, United States
* Corresponding author. Division of Clinical Pharmacology, Room 559 Preston Research Building, Vanderbilt University School of Medicine, 23rd Avenue South at Pierce Avenue, Nashville, TN 37232-6602, United States. Tel.: +1 615 322 3304; fax: +1 615 343 6334. Email address: kathy.murray{at}vanderbilt.edu
Objective: Na+ current derived from expression of the principal cardiac Na+ channel, Nav1.5, 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 Nav1.5 (Nav1.5–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 Nav1.5–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 Nav1.5 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.
KEYWORDS Ion channels; Membrane currents; Protein kinase A; Sodium current; Nav1.5
1 Current address: Cardiovascular Research Institute, University of Pittsburg Medical Center, 1704 Biomedical Science Tower, 200 Lothrop Street, Pittsburg, PA 15213, United States.
2 Current address: Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-Machi, Aoba-ku, Sendai, Japan.
3 Current address: Department of Molecular Biophysics and Physiology, Rush University School of Medicine, 1750 West Harrison, Chicago, IL 60612, United States.
Time for primary review 27 days
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Casini, H. L. Tan, I. Demirayak, C. A. Remme, A. S. Amin, B. P. Scicluna, H. Chatyan, J. M. Ruijter, C. R. Bezzina, A. C.G. van Ginneken, et al. Tubulin polymerization modifies cardiac sodium channel expression and gating Cardiovasc Res, November 23, 2009; (2009) cvp352v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Liu, S. Sanyal, G. Gao, I. S. Gurung, X. Zhu, G. Gaconnet, L. J. Kerchner, L. L. Shang, C. L.-H. Huang, A. Grace, et al. Cardiac Na+ Current Regulation by Pyridine Nucleotides Circ. Res., October 9, 2009; 105(8): 737 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Dominguez, R. Felix, and E. Monjaraz Upregulation of voltage-gated Na+ channels by long-term activation of the ghrelin-growth hormone secretagogue receptor in clonal GC somatotropes Am J Physiol Endocrinol Metab, May 1, 2009; 296(5): E1148 - E1156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Nicolas, K.-H. Park, A. El Harchi, J. Camonis, R. S. Kass, D. Escande, J. Merot, G. Loussouarn, F. Le Bouffant, and I. Baro IKs response to protein kinase A-dependent KCNQ1 phosphorylation requires direct interaction with microtubules Cardiovasc Res, August 1, 2008; 79(3): 427 - 435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. Van Norstrand, C. R. Valdivia, D. J. Tester, K. Ueda, B. London, J. C. Makielski, and M. J. Ackerman Molecular and Functional Characterization of Novel Glycerol-3-Phosphate Dehydrogenase 1-Like Gene (GPD1-L) Mutations in Sudden Infant Death Syndrome Circulation, November 13, 2007; 116(20): 2253 - 2259. [Abstract] [Full Text] [PDF]
|
|