Intracellular Chloride and Scaffold Protein Mo25 Cooperatively Regulate Transepithelial Ion Transport through WNK Signaling in the Malpighian Tubule

Abstract

<b>Background</b> With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the <i>Drosophila</i> Malpighian tubule.<b>Methods</b> Using <i>in vitro</i> assays and transgenic <i>Drosophila</i> lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux.<b>Results</b><i>In vitro</i>, autophosphorylation of purified <i>Drosophila</i> WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, <i>Drosophila</i> WNK activity acutely increased. <i>Drosophila</i> WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of <i>Drosophila</i> WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In <i>in vitro</i> kinase assays, <i>Drosophila</i> Mo25 enhanced the activity of the <i>Drosophila</i> WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of <i>Drosophila Mo25</i> in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type <i>Drosophila WNK</i>, with or without <i>Drosophila Mo25</i>, did not affect transepithelial ion transport, <i>Drosophila Mo25</i> overexpressed with chloride-insensitive <i>Drosophila WNK</i> increased ion flux.<b>Conclusions</b> Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.