SPAK and OSR1 Dependent Down-Regulation of Murine Renal Outer Medullary K<sup>+</sup> Channel ROMK1

Abstract

<b><i>Background/Aims: </i></b>The kinases SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) participate in the regulation of the NaCl cotransporter NCC and the Na<sup>+</sup>,K<sup>+</sup>,2Cl<sup>-</sup> cotransporter NKCC2. The kinases are regulated by WNK (with-no-K[Lys]) kinases. Mutations of genes encoding WNK kinases underly Gordon's syndrome, a monogenic disease leading to hypertension and hyperkalemia. WNK kinases further regulate the renal outer medullary K<sup>+</sup> channel ROMK1. The present study explored, whether SPAK and/or OSR1 have similarly the potential to modify the activity of ROMK1. <b><i>Methods: </i></b>ROMK1 was expressed in <i>Xenopus</i> oocytes with or without additional expression of wild-type SPAK, constitutively active <sup>T233E</sup>SPAK, catalytically inactive <sup>D212A</sup>SPAK, wild-type OSR1, constitutively active <sup>T185E</sup>OSR1 and catalytically inactive <sup>D164A</sup>OSR1. Channel activity was determined utilizing dual electrode voltage clamp and ROMK1 protein abundance in the cell membrane utilizing chemiluminescence of ROMK1 containing an extracellular hemagglutinin epitope (ROMK1-HA). <b><i>Results: </i></b>ROMK1 activity and ROMK1-HA protein abundance were significantly down-regulated by wild-type SPAK and <sup>T233E</sup>SPAK, but not by <sup>D212A</sup>SPAK. Similarly, ROMK1 activity and ROMK1-HA protein abundance were significantly down-regulated by wild-type OSR1 and <sup>T185E</sup>OSR1, but not by <sup>D164A</sup>OSR1. <b><i>Conclusion: </i></b>ROMK1 protein abundance and activity are down-regulated by SPAK and OSR1.