TY - JOUR
T1 - Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells
AU - Sugimoto, Kenji
AU - Nishida, Motohiro
AU - Otsuka, Masami
AU - Makino, Keisuke
AU - Ohkubo, Katsutoshi
AU - Mori, Yasuo
AU - Morii, Takashi
PY - 2004/4
Y1 - 2004/4
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=1942541370&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=1942541370&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2004.03.019
DO - 10.1016/j.chembiol.2004.03.019
M3 - Article
C2 - 15123242
AN - SCOPUS:1942541370
SN - 1074-5521
VL - 11
SP - 475
EP - 485
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 4
ER -