γ-Adducin Stimulates the Thiazide-sensitive NaCl Cotransporter

The thiazide-sensitive NaCl cotransporter (NCC) plays a key role in renal salt reabsorption and the determination of systemic BP, but the molecular mechanisms governing the regulation of NCC are not completely understood. Here, through pull-down experiments coupled to mass spectrometry, we found that gamma-adducin interacts with the NCC transporter. gamma-Adducin colocalized with NCC to the distal convoluted tubule. Na-22(+) uptake experiments in the Xenopus laevis oocyte showed that gamma-adducin stimulated NCC activity in a dose-dependent manner, an effect that occurred upstream from With No Lysine (WNK) 4 kinase. The binding site of gamma-adducin mapped to the N terminus of NCC and encompassed three previously reported phosphorylation sites. Supporting this site of interaction, competition with the N-terminal domain of NCC abolished the stimulatory effect of gamma-adducin on the transporter. gamma-Adducin failed to increase NCC activity when these phosphorylation sites were constitutively inactive or active. In addition, gamma-adducin bound only to the dephosphorylated N terminus of NCC. Taken together, our observations suggest that gamma-adducin dynamically regulates NCC, likely by amending the phosphorylation state, and consequently the activity, of the transporter. These data suggest that gamma-adducin may influence BP homeostasis by modulating renal NaCl transport.