Abstract
Abnormal activity of red cell KCl cotransport (KCC) is involved in pathogenesis of sickle cell anaemia (SCA). KCC-mediated solute loss causes shrinkage, concentrates HbS, and promotes HbS polymerisation. Red cell KCC also responds to various stimuli including pH, volume, urea, and oxygen tension, and regulation involves protein phosphorylation. The main aim of this study was to investigate the role of the WNK/SPAK/OSR1 pathway in sickle cells. The pan WNK inhibitor WNK463 stimulated KCC with an EC50 of 10.9 ± 1.1 nM and 7.9 ± 1.2 nM in sickle and normal red cells, respectively. SPAK/OSR1 inhibitors had little effect. The action of WNK463 was not additive with other kinase inhibitors (staurosporine and N-ethylmaleimide). Its effects were largely abrogated by pre-treatment with the phosphatase inhibitor calyculin A. WNK463 also reduced the effects of physiological KCC stimuli (pH, volume, urea) and abolished any response of KCC to changes in oxygen tension. Finally, although protein kinases have been implicated in regulation of phosphatidylserine exposure, WNK463 had no effect. Findings indicate a predominant role for WNKs in control of KCC in sickle cells but an apparent absence of downstream involvement of SPAK/OSR1. A more complete understanding of the mechanisms will inform pathogenesis whilst manipulation of WNK activity represents a potential therapeutic approach.
Highlights
The family of cation-chloride cotransporters (CCCs) comprise the Na+-Cl, the Na+-K+-Cl, and the K+-Cl- cotransporters (NCC, Na+-K+-2Cl- cotransporter (NKCC), and KCl cotransport (KCC))
Red cell KCC is sensitive to a number of stimuli including volume, pH, urea, and oxygen tension [25]
These modalities appear to affect the transporter by proteinphosphorylation [33, 34], with pharmacological evidence for the presence of both serine/threonine and tyrosine phosphoresidues [2, 10]
Summary
The family of cation-chloride cotransporters (CCCs) comprise the Na+-Cl-, the Na+-K+-Cl-, and the K+-Cl- cotransporters (NCC, NKCCs, and KCCs) They have been identified in many tissues – notably red cells, epithelia, and neurons – in Pflugers Arch - Eur J Physiol (2019) 471:1539–1549 which they contribute extensively to ion and water homeostasis, both cellular and transepithelial [23]. Many of these transporters were functionally identified in the late 1970s/early 1980s as Cl--dependent cation fluxes, with red cells and Ehrlich ascites tumour cells constituting pivotal model tissues [18, 28, 32, 38]. The sole NCC isoform, SLC12A3, is found in the kidney [24]
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