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. (C) 2003 Elsevier Inc. All rights reserved.