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J. Biol. Chem., Vol. 281, Issue 17, 11769-11779, April 28, 2006

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Regulation of Surface Localization of the Small Conductance Ca²⁺-activated Potassium Channel, Sk2, through Direct Phosphorylation by cAMP-dependent Protein Kinase^*

Yajun Ren, Lyndon F. Barnwell, Jon C. Alexander^¶, Farah D. Lubin, John P. Adelman^||, Paul J. Pfaffinger^¶, Laura A. Schrader^¶, and Anne E. Anderson^¶1

From the Cain Foundation Laboratories, Department of Pediatrics, Department of Neurology, ^¶Department of Neuroscience, Baylor College of Medicine, Houston, Texas, 77030 and the ^||Vollum Institute, Oregon Health and Sciences University, Portland, Oregon 97239

Small conductance, Ca²⁺-activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin (50 µM) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit. PKA phosphorylation studies using the intracellular domains of the SK2 channel subunit expressed as glutathione S-transferase fusion protein constructs showed that both the amino-terminal and carboxyl-terminal regions are PKA substrates in vitro. Mutational analysis identified a single PKA phosphorylation site within the amino-terminal of the SK2 subunit at serine 136. Mutagenesis and mass spectrometry studies identified four PKA phosphorylation sites: Ser⁴⁶⁵ (minor site) and three amino acid residues Ser⁵⁶⁸, Ser⁵⁶⁹, and Ser⁵⁷⁰ (major sites) within the carboxyl-terminal region. A mutated SK2 channel subunit, with the three contiguous serines mutated to alanines to block phosphorylation at these sites, shows no decrease in surface expression after PKA stimulation. Thus, our findings suggest that PKA phosphorylation of these three sites is necessary for PKA-mediated reorganization of SK2 surface expression.

Received for publication, December 8, 2005 , and in revised form, February 28, 2006.

^* This work was supported by grants from the NINDS, National Institutes of Health (to A. E. A., L. F. B., P. J. P., J. P. A., and F. D. L.), the National Institute of Mental Health (to L. A. S.), the Child Neurology Foundation (to A. E. A. and L. A. S.), and the Epilepsy Foundation of America (to L. F. B. and F. D. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Baylor College of Medicine, Feigin Center 955, 1102 Bates St., MC 3-6365, Houston, TX 77030. Tel.: 832-824-3976; Fax: 832-825-4217; E-mail: annea{at}bcm.tmc.edu.

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