Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3–Ubiquitin Ligases

Abstract

The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of cullins (Cul1, 3, 4, and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

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

“…After determining that Nedd4-2 activity is important for modulating NCC abundance and degradation in MDCKI cells, we next tested if Nedd4-2 was important for lowering NCC abundance in response to high extracellular K + . As long-term effects on NCC are technically difficult to study in the inducible MDCKI-hNCC cell system, we switched to using ex vivo kidney tubule preparations ( Cheng et al, 2019 ; Kortenoeven et al, 2021 ; Murali et al, 2021 ; Poulsen et al, 2021 ) from mice lacking Nedd4-2 in the kidney tubule (referred to as Nedd4-2 KO) and relevant control mice. Tubules were incubated with different concentrations of K + (0.5, 3.5, and 8 mM) for 24 h and protein abundances were assessed using western blotting.…”

“…The lower NCC phosphorylation following high dietary K + intake is due to reduced activity of the inwardly-rectifying potassium channels Kir4.1/Kir5.1 (a heterotetramer), changes in the basolateral plasma membrane potential, and reduced activity of the WNK-SPAK kinase signaling pathway ( Piala et al, 2014 ; Bazua-Valenti et al, 2015 ; Cuevas et al, 2017 ; Wu et al, 2019 ). Altered dietary K + also results in changes in activity of the Kelch-like protein 3 (KLHL3) and Cullin 3 (Cul3) E3 ubiquitin ligase complex that targets WNK kinases for degradation, altering NCC activity ( Ishizawa et al, 2016 ; Cheng et al, 2019 ; Kortenoeven et al, 2021 ; Murali et al, 2021 ; Poulsen et al, 2021 ).…”

Ubiquitination of NKCC2 by the cullin-RING E3 ubiquitin ligase family in the thick ascending limb of the loop of Henle

“…To the best of our knowledge, this is the first report showing an increase in cullin-1 expression in TALs from Sprague–Dawley rats when cullin neddylation is inhibited. However, this was not observed in homogenized whole kidney samples ( 80 ). Although the data suggest that cullin-1 is autoregulated, the data do not allow us to conclude that cullin-1 is responsible for mediating the cGMP-dependent increase in NKCC2 ubiquitination.…”

“…Moreover, Ishizawa et al ( 88 ) showed that a reduction in K + intake stimulates NCC phosphorylation while decreasing ubiquitination. On the other hand, Murali et al ( 80 ) showed that the increase in NCC ubiquitination due to high K + intake was attenuated when cullin activity was inhibited. Taken together, these studies suggest that an increase in cullin activation is indirectly related to NCC phosphorylation.…”

“…Taken together, these studies suggest that an increase in cullin activation is indirectly related to NCC phosphorylation. Furthermore, Murali et al ( 80 ) showed that in the absence of cullin-3, an increase in K + can still reduce phosphorylation of NCC, suggesting that another member of the cullin family may regulate the K + -dependent regulation of NCC phosphorylation. Based on the proximity and similarities between NCC and NKCC2, the phosphorylation and ubiquitination of NKCC2 may be regulated in a manner similar to those of NCC.…”