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J. Biol. Chem., Vol. 280, Issue 34, 30175-30184, August 26, 2005

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Sequential Phosphorylation Mediates Receptor- and Kinase-induced Inhibition of TREK-1 Background Potassium Channels^*

Janet Murbartián, Qiubo Lei, Julianne J. Sando, and Douglas A. Bayliss¶

From the Departments of Pharmacology and Anesthesiology, University of Virginia, Charlottesville, Virginia 22908

Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K⁺ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G_q-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R_p)-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.

Received for publication, April 11, 2005 , and in revised form, May 31, 2005.

^* This work was supported by United States Public Health Service Grant NS33583 (to D. A. B.). 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.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of Virginia Health System, P.O. Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735. Tel.: 434-982-4449; Fax: 434-982-3878; E-mail: dab3y{at}virginia.edu.

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