The role of WNK in modulation of KCl cotransport activity in red cells from normal individuals and patients with sickle cell anaemia

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

Chapter 1 Ion Permeability of Membranes. From Lipid Bilayers to Biological Membranes

Although cells were first observed as early as in the 17th century, it is only over the last 100 years or so that our understanding of the permeability barrier imposed by the plasma membrane of cells started to emerge. The last 50 years, in particular, has seen a marked development in our knowledge of the characteristics of this barrier. The membrane itself comprises in the most part a bilayer of phospholipids, asymmetrically distributed, together with cholesterol and intrinsic and extrinsic proteins. Simple diffusion through the lipid phase can occur for lipid soluble moieties. In other cases, intrinsic membrane proteins provide pathways for solutes and water. Three types of pathways are described: channels, carriers, and pumps. What is known about flux through these proteins, together with their structure and function, is discussed-illustrated with reference to particularly significant pathways. The important part played by the red blood cell as a paradigm for membrane transport is explained. Existing gaps in our understanding of transport across membranes are emphasized. For example, how pumps like the quintessential membrane, the Na +-K + ATPase, operate remains unknown. In addition, the nature of the residual (or "leak") permeability, which remains when all such pathways are inhibited, is unclear. The residual permeability of biological membranes appears to be about 2 orders of magnitude greater than that of artificial lipid bilayers. An important caveat here is the existence of unknown pathways yet to be described. In this context, a novel permeability with characteristics of K +(Na +)/H + exchange is described which becomes manifest across the red blood cell membrane when cells are suspended in low ionic strength solution. Future discoveries will add to our understanding of membrane permeability. It is likely that the red blood cell will play an important part in this new chapter. © 2009 Elsevier Inc. All rights reserved

Identification of a 25 kDa polypeptide associated with the L antigen in low potassium-type sheep red cells.

Antisera to the L blood group antigen have been used, following radioiodination of low potassium-type sheep red cells and subsequent immunoprecipitation, to identify a polypeptide of the L antigen. Only LK, and not HK, cells express this 25 kDa component which is present in very low copy number

The conductance of red blood cells from sickle cell patients: ion selectivity and inhibitors

The abnormally high cation permeability in red blood cells (RBCs) from patients with sickle cell disease (SCD) occupies a central role in pathogenesis. Sickle RBC properties are notably heterogeneous, however, thus limiting conventional flux techniques that necessarily average out the behaviour of millions of cells. Here we use the whole-cell patch configuration to characterise the permeability of single RBCs from patients with SCD in more detail. A non-specific cation conductance was reversibly induced upon deoxygenation and was permeable to both univalent (Na+, K+, Rb+) and also divalent (Ca2+, Mg2+) cations. It was sensitive to the tarantula spider toxin GsMTx-4. Mn2+ caused partial, reversible inhibition. The aromatic aldehyde o-vanillin also irreversibly inhibited the deoxygenation-induced conductance, partially at 1mM and almost completely at 5mM. Nifedipine, amiloride and ethylisopropylamiloride were ineffective. In oxygenated RBCs, the current was pH sensitive showing a marked increase as pH fell from 7.4 to 6, with no change apparent when pH was raised from 7.4 to 8. The effects of acidification and deoxygenation together were not additive. Many features of this deoxygenation-induced conductance (non-specificity for cations, permeability toCa2+ andMg2+, pH sensitivity, reversibility, partial inhibition by DIDS and Mn2+) are shared with the flux pathway sometimes referred to as Psickle. Sensitivity to GsMTx-4 indicates its possible identity as a stretch-activated channel. Sensitivity to o-vanillin implies that activation requires HbS polymerisation but since the conductance was observed in whole-cell patches, results suggest that bulk intracellular Hb is not involved; rather a membrane-bound subfraction is responsible for channel activation. The ability to record P(sickle)-like activity in single RBCs will facilitate further studies and eventual molecular identification of the pathway involved