Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium Oscillations*

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca²⁺ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca²⁺ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP₃R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP₃R. The FRET effect and ouabain-induced Ca²⁺ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH₂ terminus of Na,K-ATPase catalytic α1-subunit abolished Ca²⁺ oscillations and downstream activation of NF-κB. Ouabain-induced Ca²⁺ oscillations occurred in cells expressing an InsP₃ sponge and were hence independent of InsP₃ generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.