Synthesis and two-photon photolysis of 6-(ortho-nitroveratryl)-caged IP3 in living cells.
The synthesis of a photolabile derivative of inositol-1,4,5-trisphosphate (IP3) is described. This new caged second messenger (6-ortho-nitroveratryl)-IP3 (6-NV-IP3) has an extinction coefficient of 5000 M(-1) cm(-1) at 350 nm, and a quantum yield of photolysis of 0.12. Therefore, 6-NV-IP3 is photolyzed with UV light about three times more efficiently than the widely used P(4(5))-1-(2-nitrophenyl)ethyl-caged IP3 (NPE-IP3). 6-NV-IP3 has a two-photon cross-section of about 0.035 GM at 730 nm. This absorbance is sufficiently large for effective two-photon excitation in living cells at modest power levels. Using near-IR light (5 mW, 710 nm, 80 MHz, pulse-width 70 fs), we produced focal bursts of IP3 in HeLa cells, as revealed by laser-scanning confocal imaging of intracellular Ca2+ concentrations. Therefore, 6-NV-IP3 can be used for efficient, subcellular photorelease of IP3, not only in cultured cells but also, potentially, in vivo. It is in the latter situation that two-photon photolysis should reveal its true forte.
Kantevari
Srinivas
S
Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.
Hoang
Caroline J
CJ
Ogrodnik
Jakob
J
Egger
Marcel
M
Niggli
Ernst
E
Ellis-Davies
Graham C R
GC
D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D008968:Molecular Conformation; D010777:Photochemistry; D010782:Photolysis; D017785:Photons; D013997:Time Factors