title: Protein kinase A is central for forward transport of two-pore domain potassium channels K2P3.1 and K2P9.1.
abstract: Acid-sensitive two-pore domain potassium channels (K2P3.1 and K2P9.1) 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 K2P3.1 and K2P9.1 (Ser(393) and Ser(373), respectively) and overcomes retention in the endoplasmic reticulum by βCOP. We sought to identify the kinase responsible for phosphorylation of the terminal serine in human and rat variants of K2P3.1 and K2P9.1. 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 K2P3.1 transiently expressed in HEK293 cells and cell surface assays of GFP-tagged K2P3.1 and K2P9.1 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 K2P3.1 and K2P9.1.
authors.affiliation: Division of Human Genetics, Centre for Human Development, Stem Cells and Regeneration, University of Southampton, Duthie Building, Mail Point 808, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Mant
authors.forename: David
authors.initials: D
authors.lastname: Elliott
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Eyers
authors.forename: Ita M
authors.initials: IM
authors.lastname: O'Kelly
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D018833:Chaperonins / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004594:Electrophysiology; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010766:Phosphorylation; D024683:Potassium Channels, Tandem Pore Domain / Q000737:chemistry*; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D014982:Xenopus laevis
chemical_list: C498655:KCNK9 protein, human; C407082:Kcnk9 protein, rat; D009419:Nerve Tissue Proteins; D024683:Potassium Channels, Tandem Pore Domain; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C420132:potassium channel subfamily K member 3; D012694:Serine; D019893:Ribosomal Protein S6 Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D018833:Chaperonins