Negative charge at the consensus sequence for the serum- and glucocorticoid-inducible kinase, SGK1, determines pH sensitivity of the renal outer medullary K+ channel, ROMK1.
The renal outer medullary K(+)-channel ROMK1 is upregulated by the serum- and glucocorticoid-inducible kinase SGK1, an effect potentiated by Na(+)/H(+)-exchanger-regulating-factor NHERF2. SGK1 phosphorylates ROMK1 at serine44. To explore the role of SGK1 phosphorylation, serine44 was replaced by an alanine ([S44A]ROMK1) or an aspartate ([S44D]ROMK1). Wild type ROMK1, [S44A]ROMK1, and [S44D]ROMK1 were expressed in Xenopus oocytes with or without constitutively active [S422D]SGK1 and NHERF2, and K(+) current (I(KR)) determined. Cytosolic pH required for halfmaximal I(KR) (pK(a)) amounted to 7.05+/-0.01 for ROMK1, 7.07+/-0.02 for [S44A]ROMK1, and 6.83+/-0.05 for [S44D]ROMK1. Maximal I(KR) was [S44D]ROMK1>wild type ROMK1>[S44A]ROMK1. Coexpression of [S422D]SGK1 and NHERF2 enhanced the activity of ROMK1, [S44A]ROMK1 and [S44D]ROMK1, but led to a significant shift of pK(a) only in wild type ROMK1 (6.95+/-0.03). In conclusion, phosphorylation by SGK1 or introduction of a negative charge at serine44 shifts the pH sensitivity of the channel and contributes to the stimulation of maximal channel activity by the kinase.
Palmada
Monica
M
Department of Physiology I, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.
Embark
Hamdy M
HM
Wyatt
Amanda W
AW
Böhmer
Christoph
C
Lang
Florian
F
D000409:Alanine / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002478:Cells, Cultured; D016384:Consensus Sequence; D004553:Electric Conductivity; D006863:Hydrogen-Ion Concentration; D017874:Immediate-Early Proteins; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D014981:Xenopus; D000099184:Serum-Glucocorticoid Regulated Kinases
D000409:Alanine; D001224:Aspartic Acid; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D000099184:Serum-Glucocorticoid Regulated Kinases