Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells

Real-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.