title: Phosphorylation of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, by cyclic AMP-dependent protein kinase.
abstract: Activity of the recently cloned ATP-sensitive epithelial K+ channel, ROMK (Ho, K., Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. M., Kanazirska, M. V., and Hebert, S. C. (1993) Nature 362, 31-38), is regulated by phosphorylation-dephosphorylation processes with cAMP-dependent protein kinase (PKA)-dependent phosphorylation events being required for maintenance of channel activity in excised membrane patches (McNicholas, C. M., Wang, W., Ho, K., Hebert, S. C., and Giebisch, G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 8077-8081; Kubokawa, M., McNicholas, C. M., Higgins, M. A., Wang, W., and Giebisch, G. (1995) Am. J. Physiol. 269, F355-F362). To determine whether this channel is a substrate for PKA, ROMK tagged with the hemagglutinin epitope was transiently transfected into HEK293 cells. In vitro labeling of immunoprecipitated proteins from transfected cells showed that ROMK could be phosphorylated by PKA. Metabolic labeling of ROMK resulted in a significantly increased phosphorylation upon pretreatment of the cells with forskolin, consistent with an action of cAMP-dependent protein kinase. Phosphoamino acid analyses of the ROMK phosphoproteins revealed that phosphate was attached exclusively to serine residues. Three putative PKA phosphorylation sites containing serine residues in the predicted ROMK proteins are shown directly to be substrates for PKA. Site-directed mutagenesis of each of these sites or double mutation of any two sites showed that ROMK proteins retained the ability to be phosphorylated by PKA both in vivo and in vitro to a variable extent, while triple mutation of all three PKA sites abolished the phosphorylation induced by cAMP agonists in transfected cells. Two-electrode voltage clamp experiments showed that PKA-dependent phosphorylation was required for ROMK channel activity and that at least two of the three sites were required for channel function when expressed in X. laevis oocytes. Taken together, these results provide strong evidence that direct phosphorylation of the channel polypeptide by PKA is involved in channel regulation and PKA-dependent phosphorylation is essential for ROMK channel activity.
authors.affiliation: Laboratory of Molecular Physiology and Biophysics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: Z C
authors.initials: ZC
authors.lastname: Xu
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: S C
authors.initials: SC
authors.lastname: Hebert
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D005260:Female; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011188:Potassium / Q000494:pharmacology; D015221:Potassium Channels / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D017931:DNA Primers; C086989:KCNJ1 protein, human; D010750:Phosphoproteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D013757:Tetraethylammonium Compounds; D010768:Phosphoserine; D005576:Colforsin; D019789:Tetraethylammonium; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011188:Potassium