Burmese Cat Mutation, the C-Terminus-Truncated WNK4, Diminishes NCC Activation by Potassium Depletion
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders
- 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic
- Su, Xiao-Tong, Oregon Health & Science University, Portland, Oregon, United States
- Carbajal-Contreras, Hector, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico
- Castañeda-Bueno, Maria, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico
- Sharma, Avika, Oregon Health & Science University, Portland, Oregon, United States
- Gamba, Gerardo, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico
- Delpire, Eric J., Vanderbilt University, Nashville, Tennessee, United States
- Yang, Chao-Ling, Oregon Health & Science University, Portland, Oregon, United States
- Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States
The Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT) plays an important role in regulating renal potassium (K) excretion by controlling sodium delivery to the distal nephron. NCC is inhibited when K intake is high and activated when K intake is low to modulate K excretion. WNKs (with-no-lysine kinases, mainly WNK1 and WNK4 in DCT) phosphorylate and activate SPAK (SPS1-related proline/alanine-rich kinase), which stimulates NCC phosphorylation and activation. An autosomal recessive mutation in Burmese cats, a popular pet breed, leads to hypokalemia. This mutation is responsible for a premature WNK4 protein truncation lacking the C-terminus, which contains a WNK/WNK interaction domain, SPAK binding motif and several phosphorylation sites. Here, we tested whether this mutation inhibits NCC activity by abrogating WNK activity in vivo and in vitro.
The Burmese cat mutation was introduced to create Burmese cat mouse line using CRISPR. Control and Burmese cat mice were treated with either control (NK) or low K (LK) diet for 7 days and blood and kidney were harvested.
Burmese cat mice do not have any phenotype in blood chemistry at baseline. Yet, when challenged with LK diet for 7 days, they showed obvious hypokalemia (2.8 mM). Total and phospho-NCC were reduced in Burmese cat mice at baseline, and LK induced an increase in pNCC. Yet, the expression level was still lower than that in the control mice. WNK4 can be observed within WNK bodies in control mice on LK diet; whereas it is present within WNK bodies in Burmese cat mice on both NK and LK diets. Immunofluorescence showed less apical pS383- and pT243-SPAK, but more pS383-SPAK within WNK bodies in Burmese cat mice. WNK1/KS-WNK1 is not visible in control mice on both diets but is more evident within WNK bodies in Burmese cat mice. When the truncated WNK4 was transfected into HEK293 cells, direct WNK4-SPAK interaction was abolished. Thus, preserved WNK-WNK interaction may be responsible for the recruitment and phosphorylation of SPAK in WNK bodies.
Hypokalemia in Burmese cats results from defective NCC function, as the WNK4 C-terminus is crucial for SPAK-mediated effects. Loss of function in WNK4 leads to a compensatory increase in WNK1/KS-WNK1 WNK body formation.
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