Intracellular chloride depletion increases activity and amino terminal phosphorylation of the renal Na‐Cl cotransporter (NCC)

Abstract

NCC activity has been associated with changes in arterial blood pressure levels (Gitelman's and Gordon's disease) and thiazides are the first line therapy of hypertension. Little is known, however, about mechanisms that modulate NCC activity. Here, we investigated the effect of lowering the intracellular chloride concentration [Cl−i upon NCC activity and amino terminal domain phosphorylation using Xenopus laevis oocytes. To promote a decrease in [Cl−]i, water or NCC cRNA-injected oocytes, were exposed to overnight incubation in a Cl−-free hypotonic solution and/or co-injection of NCC cRNA, with the K+:Cl− cotransporter KCC2 cRNA. Then, thiazide-sensitive 22Na+ uptake was assessed. Phosphorylation status of the threonine residues T53 and T58 was assessed using the R5 phospho-antibodies that in the Na+:K+:2Cl−cotransporter recognizes these conserved residues when are phosphorylated. Observed uptakes in studied groups were (in nmol/oocytes/h): water-injected control 0.17 ± 0.03; NCC 2.84 ± 0.1; NCC-Cl−-free 12.4 ± 0.9; NCC-KCC2 11.7 ± 0.5, and NCC-KCC2-Cl−-free 16.4 ± 0.6 (p<0.01). These maneuvers were associated with increased phosphorylation of T53 and T58. Surface expression analysis of EGFP-tagged-NCC revealed that increased activity of NCC is not dependent on the amount of cell surface EGFP-NCC signal. Substitution of T53 with alanine (T53A) exhibited a similar behavior than wild type NCC. In contrast, functional activity of T58A mutant was completely reduced. We conclude that decreasing [Cl−i is associated with activation and phosphorylation of NCC proteins that are already inserted in plasma membrane.