Signaling transduction mediated by protein aggregates within specific microdomains has been receiving increased attention. We previously showed that Na,K-ATPase, partially inhibited by ouabain, induces intracellular calcium (Ca(2+)) oscillations which involve Ca(2+) release from the endoplasmic reticulum (ER). Plasma membrane bound Na,K-ATPase and proteins in the ER are in close proximity to each other, and signal transduction may occur via a physical interaction or a microdomain. To study these signaling pathways and intricate microenvironments, sophisticated methods are required. One way to detect molecular interactions in the nanometer scale (1-10 nm) is fluorescence resonance energy transfer (FRET). Thus, FRET provides vital insight into the action of Na,K-ATPase to trigger intracellular signaling events.