The E3 ubiquitin-protein ligase Nedd4-2 regulates the sodium chloride cotransporter NCC but is not required for a potassium-induced reduction of NCC expression.

Na and K balance is influenced by the activity of the sodium chloride cotransporter NCC in the distal convoluted tubule. NCC activity and abundance are reduced by high extracellular K. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) has been proposed as a modulator of NCC abundance. Here, we examined the functional role of Nedd4-2 on NCC regulation and whether Nedd4-2 is important for the effects of high extracellular K on NCC. Total and plasma membrane levels of ubiquitylated NCC were lower in NCC-expressing MDCKI cells after Nedd4-2 deletion. NCC and phosphorylated NCC (pT58-NCC) levels were higher after Nedd4-2 deletion, and NCC levels on the plasma membrane were elevated. No significant changes were seen after Nedd4-2 knockdown in the levels of SPAK and phosphorylated SPAK (pS373-SPAK), the major NCC regulatory kinase. Nedd4-2 deficiency had no effect on the internalization rate of NCC from the plasma membrane, but NCC protein half-life was increased. In experiments with kidney tubule suspensions from Nedd4-2 knockout (KO) mice, high K reduced total and pT58-NCC regardless of genotype. We conclude that Nedd4-2 is involved in ubiquitylation of NCC and modulating its plasma membrane levels and degradation. However, Nedd4-2 does not appear to be important for K induced reductions in NCC abundance.