Stimulation of human and mouse erythrocyte Na+-K+-2Cl- co-transport by osmotic shrinkage does not involve AMP-activated protein kinase, but is associated with STE20/SPS1-related proline/alanine-rich kinase activation.

Sid, Brice;Miranda, Lisa;Vertommen, Didier;Viollet, Benoit;Rider, Mark H.
(2010) The Journal of physiology — Vol. 588, n° Pt 13, p. 2315-2328 (2010)

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Authors
  • Sid, BriceUCLouvain
    Author
  • Miranda, Lisa
    Author
  • Vertommen, Didierorcid-logoUCLouvain
    Author
  • Viollet, Benoit
    Author
  • Rider, Mark H.UCLouvain
    Author
Abstract
This study was undertaken to investigate whether the mechanism of increased Na+-K+-2Cl- (NKCC1) co-transporter activation by osmotic shrinkage involved the AMP-activated protein kinase (AMPK). AMPK was found to phosphorylate a recombinant GST-dogfish (1-260) NKCC1 fragment at Ser38 and Ser214, corresponding to Ser77 and Ser242 in human NKCC1, respectively. Incubation of human erythrocytes with 20 muM A-769662 AMPK activator increased Ser242 NKCC1 phosphorylation but did not stimulate 86Rb+ uptake. Under hypertonic conditions in human red blood cells (RBCs) incubated with 0.3 M sucrose, NKCC1 activity increased as measured by bumetanide-sensitive 86Rb+ uptake and AMPK was activated. However, there was no effect of AMPKalpha1 deletion in mouse RBCs on the increased rate of 86Rb+ uptake induced by hyperosmolarity. AMPK activation by osmotic shrinkage of mouse RBCs was abrogated by 10 muM STO-609 CaMKKbeta inhibitor, but incubation with STO-609 did not affect the increase in 86Rb+ uptake induced by hyperosmolarity. Osmotic shrinkage of human and mouse RBCs led to activation loop phosphorylation of the STE20/SPS1-related proline/alanine-rich kinase (SPAK) at Thr233, which was accompanied by phosphorylation of NKCC1 at Thr203/207/212, one of which (Thr207) is responsible for co-transporter activation. Therefore, phosphorylation-induced activation of NKCC1 by osmotic shrinkage does not involve AMPK and is likely due to SPAK activation.
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Sid, B., Miranda, L., Vertommen, D., Viollet, B., & Rider, M. H. (2010). Stimulation of human and mouse erythrocyte Na+-K+-2Cl- co-transport by osmotic shrinkage does not involve AMP-activated protein kinase, but is associated with STE20/SPS1-related proline/alanine-rich kinase activation. The Journal of physiology, 588(Pt 13), 2315-2328. https://doi.org/10.1113/jphysiol.2009.185900 (Original work published 2010)