The Role of Janus Kinase 3 in the Regulation of Na+/K+ ATPase under Energy Depletion

Abstract

Background/Aims: Janus kinase-3 (JAK3) is activated during energy depletion. Energy-consuming pumps include the Na⁺/K⁺-ATPase. The present study explored whether JAK3 regulates Na⁺/K⁺-ATPase in dendritic cells (DCs). Methods: Ouabain (100 µM)-sensitive (I_ouabain) and K⁺-induced (I_pump) outward currents were determined by utilizing whole cell patch-clamp, Na⁺/K⁺-ATPase α1-subunit mRNA levels by RT-PCR, Na⁺/K⁺-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3^-/-) or wild-type mice (jak3^+/+). I_pump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active ^A568VJAK3 or inactive ^K851AJAK3. Results: Na⁺/K⁺-ATPase α1-subunit mRNA and protein levels, as well as I_pump and I_ouabain were significantly higher in jak3^-/-DCs than in jak3^+/+DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased I_pump in jak3^+/+ DCs but not in jak3^-/-DCs. Cellular ATP was significantly lower in jak3^-/-DCs than in jak3^+/+DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3^-/-DCs than in jak3^+/+DCs and strongly blunted by ouabain in both jak3^+/+ and jak3^-/-DCs. I_pump and I_ouabain in oocytes were decreased by expression of JAK3 and of ^A568VJAK3 but not of ^K851AJAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) enhanced I_pump and I_ouabain in JAK3 expressing oocytes. The difference between ^A568VJAK3 and ^K851AJAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM). Conclusions: JAK3 down-regulates Na⁺/K⁺-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.