Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP(3)). Destructive chemical approaches required to measure [InsP(3)] limit spatiotemporal understanding of subcellular InsP(3) signaling. We constructed novel fluorescence resonance energy transfer-based InsP(3) biosensors called FIRE (fluorescent InsP(3)-responsive element) by fusing plasmids encoding the InsP(3)- binding domain of InsP(3) receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an similar to 11% increase in the fluorescence ratio (F-530/F-480) at saturating [InsP(3)] (apparent K-d = 31.3 +/- 6.7 nM InsP(3)). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP(3)] using confocal microscopy (with free [InsP(3)] rising to similar to 30 nM). Local entry of intracellular InsP(3) via membrane rupture by a patch pipette (containing InsP(3)) in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP(3) diffusion. Both endothelin-1-induced and direct InsP(3) application ( via pipette rupture) revealed that InsP(3) diffusion into the nucleus occurs with a delay and blunted rise of [InsP(3)] versus cytosolic [InsP(3)]. These new biosensors allow studying InsP(3) dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP(3) signaling in intact cells.