Abstract: SA-PO1046

The Sensibility of WNK3 and WNK4 for Intracellular Chloride Concentration and Cell Volume Is Opposite

Session Information

  • Na+, K+, Cl-
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 703 Na+, K+, Cl- Basic

Authors

  • Carrillo Perez, Diego Luis, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, Mexico
  • Leyva-Rios, Karla, Universidad Panamericana, Mexico City, Mexico
  • Mercado, Adriana P., Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
  • Hernandez Mercado, Elisa, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
  • Moreno, Erika, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, Mexico
  • Vázquez, Norma Hilda, Instituto de Investigaciones Biomédicas, UNAM, Mexico City, Mexico
  • Pacheco-Alvarez, Diana, Universidad Panamericana, Mexico City, Mexico
  • Gamba, Gerardo, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan, Mexico City, Mexico
Background

The activity of the electroneutral chloride cotransporters (CCCs) such as the Na-K-2Cl and the K-Cl cotransporters is modulated by intracellular chloride concentration [Cl-]i and cell volume. Depletion of [Cl-]i and cell shrinkage induce phosphorylation of CCCs, while increase of [Cl-]i and cell swelling induces dephosphorylation. However, cell shrinkage increases [Cl-]i and cell swelling decreases [Cl-]i. It is known that the effect of [Cl-]i towards CCCs is traduced by the WNK1 or WNK4 kinases. Thus, the effect of cell volume towards CCCs must be traduced by a different kinase. Because WNK3 bypasses the tonicity requirements for regulation of the CCCs (PNAS 2005 and 2006), we tested the hypothesis that WNK3 is sensitive to cell volume, rather than to the [Cl-]i.

Methods

Xenopus oocytes were microinjected with N(K)CCs or KCCs cRNA alone or together with WNK3 wild type or mutants in which catalytic activity and/or the chloride binding site has been eliminated (WNK3-DA; WNK3 L295/297F; WNK3-DA-LLFF). The effect of [Cl-]i or extracellular tonicity on WNKs was assessed. WNKs phosphorylation at the activating serine of the T-loop with specific antibodies was analyzed as a surrogate of WNKs activity.

Results

Depletion of [Cl-]i increases the activity of WNK1 and WNK4 and thus increases the NCC activity, while it had not effect on the WNK3 and its effect towards NCC. In contrast to what we previously observed for WNK1 and WNK4, elimination of the chloride-binding site in WNK3 had no effect on the activity of the kinase. WNK3, but not WNK4, phosphorylation was sensitive to changes in cell volume. Compared to isotonic condition, WNK3 phosphorylation significantly diminished or increased by 50% in hypotonic or hypertonic conditions, respectively. The phosphorylation of WNK4 was not affected by similar changes in tonicity.

Conclusion

Our data show that WNK3 is not sensitive to changes of the [Cl-]i, but is modulated by changes in cell volume in the expected direction according to the effect of WNK3 wild type and the catalytically inactive WNK3 on the CCCs. We propose that WNK3 is a volume sensitive kinase modulating the effect of cell volume changes in CCCs.

Funding

  • Government Support - Non-U.S.