Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

Abstract

Summary Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function.

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor and NaCl transport mechanisms in the renal distal nephron

“…However, the significance of phosphorylation in the regulation of MR function was unclear. Using a phospho-proteomics approach (Rinehart et al 2009), we identified 16 phosphorylation sites in full-length human MR (Shibata et al 2013a). Phosphorylation at S843 (the only site in the ligand-binding domain) had the greatest functional effect in a luciferase reporter assay, and previous studies have reported that this site is responsible for the difference in ligand selectivity between glucocorticoid receptor and MR (Ortlund et al 2007).…”

Differences in the Large Extracellular Loop between the K+-Cl− Cotransporters KCC2 and KCC4

“…Substitution of Tyr 1087 to aspartate in KCC2 or its congener in KCC1 reduced transport activity (21). Furthermore, replacement of threonines by alanine at positions 991 and 1048 in KCC3 or their congeners in KCC2 increased transport activity (22).…”

Protein Kinase WNK1 Promotes Cell Surface Expression of Glucose Transporter GLUT1 by Regulating a Tre-2/USP6-BUB2-Cdc16 Domain Family Member 4 (TBC1D4)-Rab8A Complex

“…It directly phosphorylates and activates SPAK and OSR1 protein kinases, which interact with and stimulate the activity of the cation chloride co-transporters NKCC1 (13)(14)(15)(16) or NCC (14,17) in HEK293 or HeLa cells. The expression of WNK1 appears also to be part of a pathway involved in inhibitory phosphorylation events of co-transporter KCC (18).…”