Novel Real-Time Sensors to Quantitatively Assess In Vivo Inositol 1,4,5-Trisphosphate Production in Intact Cells

AbstractReal-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP3) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) δ. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP3 binding pocket for enhanced IP3 selectivity and for rapid and direct visualization of intracellular IP3 ≥ 0.5 μM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP3 production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP3 probe was extremely effective to quantitatively assess real-time physiological IP3 production via those pathways formed only in the intact cellular configuration