Kidney Week

Abstract: TH-OR004

Potassium Directly Regulates WNK (With No Lysine Kinases)

Session Information

• Advances in Fluid and Electrolyte Handling: Basic Physiology
  November 07, 2019 | Location: 146 C, Walter E. Washington Convention Center
  Abstract Time: 05:06 PM - 05:18 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Rodan, Aylin R., University of Utah, Salt Lake City, Utah, United States

Aylin R. Rodan, MD, PhD, FASN



Dr. Rodan is a nephrologist and physician-scientist interested in ion transporters and channels and their regulation. Dr. Rodan’s lab uses biochemistry, molecular biology and genetics in the fly, Drosophila melanogaster, to understand the molecular basis of ion transport physiology relevant to human physiology and disease.

• Zhang, Feng, University of Utah, Salt Lake City, Utah, United States

Feng Zhang,

• Pleinis, John, University of Utah, Salt Lake City, Utah, United States

John Pleinis,

• Sosa, Jason O., University of Utah, Salt Lake City, Utah, United States

Jason O. Sosa,

• Humphreys, John M., University of Texas Southwestern Medical Center, Dallas, Texas, United States

John M. Humphreys,

• Akella, Radha, University of Texas Southwestern Medical Center, Dallas, Texas, United States

Radha Akella,

• He, Haixia, University of Texas Southwestern Medical Center, Dallas, Texas, United States

Haixia He,

• Goldsmith, Elizabeth J., University of Texas Southwestern Medical Center, Dallas, Texas, United States

Elizabeth J. Goldsmith,

Background

Plasma potassium concentration is maintained within a narrow range, implying the ability to sense deviations from normal. WNK mutations in mice and humans result in abnormal potassium concentrations, and WNKs have been proposed to indirectly sense plasma potassium via effects on intracellular chloride. Here, we investigate whether WNKs are directly regulated by potassium. Our lab has previously shown that the WNK signaling cascade is conserved in Drosophila Malpighian (renal) tubules.

Methods

We used differential scanning fluorimetry and mass spectrometry to measure WNK kinase domain thermal stability and autophosphorylation in vitro in the presence of varying concentrations of potassium. We also examined the activity of DmWNK (Drosophila WNK) and HsWNK3 (human WNK3) activity in the Drosophila Malpighian tubule, using phosphorylation of transgenically expressed kinase-dead rat SPAK as a readout. We developed baths with varying extracellular potassium and fixed intracellular chloride concentrations (16 mM or 30 mM, measured using the transgenic sensor ClopHensor). We measured intracellular potassium in the tubule using inductively coupled plasma mass spectrometry.

Results

Potassium directly binds to the kinase domain of DmWNK (Drosophila WNK) and human WNK1 in vitro, as assayed by differential scanning fluorimetry. Potassium also inhibits autophosphorylation, required for kinase activation, of DmWNK and HsWNK3 (human WNK3) kinase domains in vitro. We also examined the activity of DmWNK or HsWNK3 in Malpighian tubules. Compared to the normal potassium bath, there was no change in DmWNK or HsWNK3 activity in low potassium bath, but there was no change in intracellular potassium under these conditions. Intracellular potassium was increased in the high potassium bath, and high potassium inhibited both DmWNK and HsWNK3 in both the 16 mM and 30 mM intracellular chloride conditions. High potassium bath also inhibited chloride-insensitive HsWNK3^L295F activity in the tubule.

Conclusion

Our data suggest that potassium directly inhibits WNK kinases, in a manner that is additive to chloride inhibition, with implications for potassium sensing in the kidney and other organs.

Funding

• NIDDK Support