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?-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(+) 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.

SUBMITTER: Dimke H

PROVIDER: S-EPMC3060444 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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γ-Adducin stimulates the thiazide-sensitive NaCl cotransporter.

Dimke Henrik H San-Cristobal Pedro P de Graaf Mark M Lenders Jacques W JW Deinum Jaap J Hoenderop Joost G J JG Bindels René J M RJ

Journal of the American Society of Nephrology : JASN 20101216 3

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 γ-adducin interacts with the NCC transporter. γ-Adducin colocalized with NCC to the distal convoluted tubule. (22)Na(+) uptake experiments in the Xenopus laevis oocyte showed that γ-adducin stimulat ...[more]

PMID: 21164023

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Project description:BackgroundThe potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1 in vitro. Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown.MethodsWe used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both.ResultsDeletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel α-subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice.ConclusionsNedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.

Dataset Information

?-Adducin stimulates the thiazide-sensitive NaCl cotransporter.

Publications

γ-Adducin stimulates the thiazide-sensitive NaCl cotransporter.

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