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(Circulation Research. 2002;91:540.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Phosphorylation and Putative ER Retention Signals Are Required for Protein Kinase A-Mediated Potentiation of Cardiac Sodium Current

Jingsong Zhou, Hyeon-Gyu Shin, Jianxun Yi, Wangzhen Shen, Christine P. Williams, Katherine T. Murray

From Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn. Present address for J. Zhou and J. Yi is the Department of Molecular Biophysics and Physiology, Rush University School of Medicine, Chicago, Ill; the present address for H.-G. Shin is the Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pa.

Correspondence to Dr Katherine T. Murray, Dept of Pharmacology, Rm 559 Preston Research Bldg, Vanderbilt University School of Medicine, 23rd and Pierce Avenues, Nashville, TN 37232-6602. E-mail kathy.murray{at}vanderbilt.edu

Activation of protein kinase A (PKA) increases Na⁺ current derived from the human cardiac Na⁺ channel, hH1, in a slow, nonsaturable manner. This effect is prevented by compounds that disrupt plasma membrane recycling, implying enhanced trafficking of channels to the cell membrane as the mechanism responsible for Na⁺ current potentiation. To investigate the molecular basis of this effect, preferred consensus sites (serines 483, 571, and 593) and alternative sites phosphorylated by PKA in the rat heart isoform (serines 525 and 528) were removed in the I-II interdomain linker, a region in the channel previously implicated in the PKA response. Our results demonstrate that the presence of either serine 525 or 528 is required for Na⁺ current potentiation. The role of amino acid sequences that can mediate channel-protein interactions was also examined. Removal of a PDZ domain-binding motif at the carboxy terminus of hH1 did not alter the PKA response. The I-II interdomain linker of the channel contains 3 sites (479RKR481, 533RRR535, and 659RQR661) with the sequence RXR, a motif known to mediate retention of proteins in the endoplasmic reticulum (ER). The PKA-mediated increase in Na⁺ current was abolished when all 3 sites were eliminated, with RRR at position 533 to 535 primarily responsible for this effect. These results demonstrate that both -subunit phosphorylation and the presence of putative ER retention signals are required for the PKA-mediated increase in cardiac Na⁺ current, an effect that likely involves interaction of the I-II interdomain linker with other proteins or regions of the channel.

Key Words: Na⁺ channel • protein kinase • heart • protein trafficking

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