γ-Adducin Stimulates the Thiazide-sensitive NaCl Cotransporter

Abstract

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 ␥-adducin interacts with the NCC transporter. ␥-Adducin colocalized with NCC to the distal convoluted tubule.22 Na ϩ uptake experiments in the Xenopus laevis oocyte showed that ␥-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 ␥-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 ␥-adducin on the transporter. ␥-Adducin failed to increase NCC activity when these phosphorylation sites were constitutively inactive or active. In addition, ␥-adducin bound only to the dephosphorylated N terminus of NCC. Taken together, our observations suggest that ␥-adducin dynamically regulates NCC, likely by amending the phosphorylation state, and consequently the activity, of the transporter. These data suggest that ␥-adducin may influence BP homeostasis by modulating renal NaCl transport.

Effects of ACE inhibition and ANG II stimulation on renal Na-Cl cotransporter distribution, phosphorylation, and membrane complex properties

“…The model predicts that ␥-adducin would be released from NCC after phosphorylation. Our results indicate colocalization of NCC and ␥-adducin in the same 700 kDa complex, as well as complex formation with unphosphorylated NCC but not NCCpT58, providing in vivo support for the previous in vitro findings (7). Clearly, it will be interesting to apply this technique to determine whether SPAK, OSR1, WNKs, or phosphatases are also 700 kDa complex members and whether proteins involved in targeting NCC to degradation pathways are present in the 400 kDa complex (49).…”

“…SPAK and OSR1 both phosphorylate rodent NCC at multiple conserved Ser and Thr residues in the cytoplasmic NH 2 -terminal domain. Phosphorylation at Thr58 appears to play a very critical role in the phosphorylation and activation of NCC as 1) experimental mutation of this site prevents the phosphorylation of the other NH 2 -terminal phos- [6][7][8], pelleted, subjected to nondenaturing BN-PAGE, transferred to PVDF membrane, and immunoblotted for NCC total, NCCpT53, and NCCpS71 as described in METHODS (note: control, captopril-, and ANG II-treated samples were assayed on the same gel). Apparent oligomeric size was determined by comparison with NativeMark molecular weight markers (Invitrogen).…”

“…The only candidate we tested for coexpression in the complex was ␥-adducin based on the recent study of Dimke et al (7) in heterologous expression systems that suggested ␥-adducin binds to unphosphorylated NCC, where it can attract SPAK and OSR1 binding to the complex and phosphorylate NCC (6). The model predicts that ␥-adducin would be released from NCC after phosphorylation.…”

The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase

“…Fluorescently labeled DCT/CNT tubules were isolated from transgenic animals, using a Complex Object Parametric Analyzer and Sorter (COPAS) sorter (Union Biometrica, Somerville, MA) as previously described (25,26). The animal ethics board of the Radboud University Nijmegen approved all of the experimental procedures.…”

Genetic basis of the impaired renal myogenic response in FHH rats

“…Add3 has been reported to play a role in NaCl cotransporter (NCC) activity in luminal cells of the distal convoluted tubule by binding to phosphorylation sites on the NCC channel to stimulate NCC activity (10). Knockout of Add3 in mice (34) had no effect on blood pressure or red blood cell (RBC) and platelet function.…”