Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels.
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 alpha(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.
Murbartián
Janet
J
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
Lei
Qiubo
Q
Sando
Julianne J
JJ
Bayliss
Douglas A
DA
D000409:Alanine / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D064446:Orexin Receptors; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000737:chemistry*; D015221:Potassium Channels / Q000737:chemistry; D024683:Potassium Channels, Tandem Pore Domain / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000737:chemistry; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D007211:Indoles; D008301:Maleimides; D064446:Orexin Receptors; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; C103742:potassium channel protein TREK-1; C074155:8-chloroadenosine-3',5'-cyclic monophosphorothioate; D005576:Colforsin; D001224:Aspartic Acid; D015240:Phorbol 12,13-Dibutyrate; D012694:Serine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C088060:bisindolylmaleimide; D000409:Alanine; D011188:Potassium