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 (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM 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 IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.
Sugimoto
Kenji
K
Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan.
Nishida
Motohiro
M
Otsuka
Masami
M
Makino
Keisuke
K
Ohkubo
Katsutoshi
K
Mori
Yasuo
Y
Morii
Takashi
T
D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054803:Phospholipase C delta; D051381:Rats; D013997:Time Factors; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D015335:Molecular Probes; D010446:Peptide Fragments; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D002118:Calcium