title: Cell surface expression of the ROMK (Kir 1.1) channel is regulated by the aldosterone-induced kinase, SGK-1, and protein kinase A.
abstract: The Kir1.1 (ROMK) subtypes of inward rectifier K+ channels mediate potassium secretion and regulate sodium chloride reabsorption in the kidney. The density of ROMK channels on the cortical collecting duct apical membrane is exquisitely regulated in concert with physiological demands. Although protein kinase A-dependent phosphorylation of one of the three phospho-acceptors in Kir1.1, Ser-44, also a canonical serum-glucocorticoid-regulated kinase (SGK-1) phosphorylation site, controls the number of active channels, it is unknown whether this involves activating dormant channels already residing on the plasma membrane or recruiting new channels to the cell surface. Here we explore the mechanism and test whether SGK-1 phosphorylation of ROMK regulates cell surface expression. Removal of the phosphorylation site by point mutation (Kir1.1, S44A) dramatically attenuated the macroscopic current density in Xenopus oocytes. As measured by antibody binding of external epitope-tagged forms of Kir1.1, surface expression of Kir1.1 S44A was inhibited, paralleling the reduction in macroscopic current. In contrast, surface expression and macroscopic current density was augmented by a phosphorylation mimic mutation, Kir1.1 S44D. In vitro phosphorylation assays revealed that Ser-44 is a substrate of SGK-1 phosphorylation, and expression of SGK-1 with the wild type channel increased channel density to the same level as the phosphorylation mimic mutation. Moreover, the stimulatory effect of SGK-1 was completely abrogated by mutation of the phosphorylation site. In conclusion, SGK-1 phosphorylation of Kir1.1 drives expression on the plasmalemma. Because SGK-1 is an early aldosterone-induced gene, our results suggest a possible molecular mechanism for aldosterone-dependent regulation of the secretory potassium channel in the kidney.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Dana
authors.initials: D
authors.lastname: Yoo
authors.forename: Bo Young
authors.initials: BY
authors.lastname: Kim
authors.forename: Cristina
authors.initials: C
authors.lastname: Campo
authors.forename: Latreece
authors.initials: L
authors.lastname: Nance
authors.forename: Amanda
authors.initials: A
authors.lastname: King
authors.forename: Djikolngar
authors.initials: D
authors.lastname: Maouyo
authors.forename: Paul A
authors.initials: PA
authors.lastname: Welling
mesh_terms: D000450:Aldosterone / Q000494:pharmacology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002462:Cell Membrane / Q000502:physiology*; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D017874:Immediate-Early Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018075:RNA, Complementary / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D000450:Aldosterone; D017868:Cyclic AMP-Dependent Protein Kinases; D017931:DNA Primers; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D017346:Protein Serine-Threonine Kinases; D018075:RNA, Complementary; D000099184:Serum-Glucocorticoid Regulated Kinases; C510693:Kcnj1 protein, mouse