Role of Protein Phosphatase 1 (PP1) Upon KCCs Regulation by WNK3 Kinase During Cell Volume Regulation and Ion Transport

Abstract

Background: The K-Cl cotransporters (KCCs) are SLC12A cation/chloride cotransporters (CCCs) that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. These mechanisms are complex, and contain multiple sensors, transducers, and effectors, often operating in parallel pathways. It has been shown that KCCs activity is affected by direct phosphorylation and dephosphorylation. In particular dephosphorylation of important regulatory sites on KCCs is necessary to become fully activated, whereas phosphorylation by SPAK/OSR1 kinases will result in KCCs inhibition. Protein phosphatases (PP) inhibitors reduce KCCs function and so, it is generally accepted the presence of phosphatases on the KCCs signaling pathway. Several studies have proposed protein phosphatase 1 (PP1) as the major PP responsible for KCCs dephosphorylation. On the other hand, WNK3 kinases have been shown to directly regulate KCCs activity bypassing their osmotic regulation. That is wild type (WT) WNK3 phosphorylates KCCs under hypotonic conditions where they are normally active, whereas kinase dead WNK3 (DA) activates KCCs function under isotonic conditions where they are normally inhibited. On the present study we analyze the role of protein phosphatase 1 (PP1) upon KCCs regulation by WNK3 kinase during cell volume regulation and ion transport. Methods: HEK 293 cells and Xenopus laevis oocytes were either transfected or microinjected with KCC3 or KCC4 and one of the following PP1 plasmids: WT, H248K (inactive) or T320A (constitutive active), and/or WT WNK3 /DA WNK3. KCCs dephosphorylation in response to cell swelling, hypotonic conditions, was evaluated by Western blot analysis of KCCs regulatory phosphorylation site (pThr1048 in KCC3a). Phosphatase/kinase interaction and the possible formation of a complex was analyzed by transfecting cells or microinjecting oocytes with WNK3 PP1 putative binding sites mutants (WNK3 PP1a y PP1b). Phosphorylation of SPAK/OSR1 activating regulatory site Ser373 was analyzed in parallel under the same conditions. Results: WT or constitutive active mutant PP1 T320A induced KCCs Thr1048 dephosphorylation in HEK cells and X. laevis oocytes even under isotonic conditions, where KCCs are normally phosphorylated. WT WNK3 prevented KCCs T1048 dephosphorylation under hypotonic conditions, ablating their response to cell swelling. Nevertheless, when co-expressed with PP1 T320A, KCCs became dephosphorylated in both osmotic conditions eliminating WT WNK3 inhibitory effect. PP1 inactivating mutation H248K prevented KCCs Thr1048 dephosphorylation in response to hypotonic conditions. However, when co-expressed with DA WNK3, this KCCs regulatory site, became dephosphorylated under hypotonic conditions. SPAK Ser373 regulation was unaffected in any off the studied conditions. Conclusions: Our results show a direct role for PP1 on KCCs function. PP1WT and PP1T320A regulate KCCs activity by its dephosphorylation. In presence of WT WNK3, PP1 activity predominates over WNK3 surpassing the inhibitory phosphorylation of the regulatory Thr residue. KCCs are not dephosphorylated by PP1H248K under hypotonic conditions. Coexpression of kinase dead WNK3 overcomes this effect, supporting the hypothesis of a phosphatase/kinase complex regulating KCCs cell volume regulatory response. AM CF-2023-I-532, INCar 22-1345 PdlH Papiit IN228123. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.