Protein Kinase A Is Central for Forward Transport of Two-pore Domain Potassium Channels K2P3.1 and K2P9.1

Acid-sensitive two-pore domain potassium channels (K(2P)3.1 and K(2P)9.1) play key roles in both physiological and pathophysiological mechanisms, the most fundamental of which is control of resting membrane potential of cells in which they are expressed. These background leak channels are constitutively active once expressed at the plasma membrane, and hence tight control of their targeting and surface expression is fundamental to the regulation of K+ flux and cell excitability. The chaperone protein, 14-3-3, binds to a critical phosphorylated serine in the channel C termini of K(2P)3.1 and K(2P)9.1 (Ser(393) and Ser(373), respectively) and overcomes retention in the endoplasmic reticulum by beta COP. We sought to identify the kinase responsible for phosphorylation of the terminal serine in human and rat variants of K(2P)3.1 and K(2P)9.1. Adopting a bioinformatic approach, three candidate protein kinases were identified: cAMP-dependent protein kinase, ribosomal S6 kinase, and protein kinase C. In vitro phosphorylation assays were utilized to determine the ability of the candidate kinases to phosphorylate the channel C termini. Electrophysiological measurements of human K(2P)3.1 transiently expressed in HEK293 cells and cell surface assays of GFP-tagged K(2P)3.1 and K(2P)9.1 enabled the determination of the functional implications of phosphorylation by specific kinases. All of our findings support the conclusion that cAMP-dependent protein kinase is responsible for the phosphorylation of the terminal serine in both K(2P)3.1 and K(2P)9.1.