Phosphoregulation of K⁺‐Cl⁻ cotransporters during cell swelling: Novel insights

Abstract

The K + -Cl − cotransporters (KCCs) belong to the cation-Cl − cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K + and Cl − ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl − ] (Cl i ), but the mechanisms at play are still ill-defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine-deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1-related proline/alanine-rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C-terminus of full-length KCC4 led to higher levels of heterologous K + -Cl − cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric-sensitive kinase-dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation. K E Y W O R D Scation-Cl − cotransporters, K + -Cl − cotransporters, lysine-deficient protein kinases, membrane transport, protein phosphatase 1

Endocytic recycling of Na+–K+–Cl− cotransporter type 2: importance of exon 4

“…; Frenette‐Cotton et al . ; Marcoux et al . ) to study the effects of cell swelling or cell shrinkage while [Cl − ] i is known to be low and similar regardless of external osmolality (Bergeron et al .…”

“…The measurements were obtained by atomic absorption spectrophotometry as detailed in several of our previous publications (Frenette‐Cotton et al . ; Marcoux et al . ).…”

“…All were subcloned in a pGEM-derived oocyte expression vector called Pol1. This vector is composed of the following sequences (from 5 to 3 ): the T7 bacterial promoter, the untranslated regions of the X. laevis β-globin cDNA, a polyA tail and linearizing sites (Caron et al 2000;Frenette-Cotton et al 2018). Four of the cDNA constructs used, rabbit (rb) NKCC2A (U07547.1), rbNKCC2B (U07548.1), rbNKCC2F (U07549.1) and mouse WNK3 (NM 001271679.1), were available from previous work (Gagnon et al 2003;Marcoux et al 2019).…”

Physiological roles and molecular mechanisms of K+‐Cl− cotransport in the mammalian kidney and cardiovascular system: where are we?

“…; Frenette‐Cotton et al . ). Two of them drew much attention a few years ago when they were linked to pseudohypoaldosteronism type II (PHAII), a rare hereditary disorder of renal electrolyte handling characterized by high blood pressure, metabolic acidosis and hyperkalaemia (Wilson et al .…”

“…; Frenette‐Cotton et al . ) and that loss‐of‐function mutations in WNK4 should therefore increase K + ‐Cl − cotransport in the renal epithelium. Curiously, however, the KCCs are also inhibited by WNK1 while gain‐of‐function mutations in this enzyme lead to increased Na + ‐Cl − cotransport by relieving the inhibitory effect of WNK4 on NCC.…”