Two-photon and UV-laser flash photolysis of the Ca2+ cage, dimethoxynitrophenyl-EGTA-4.
We report efficient two-photon and UV-laser flash photolysis of dimethoxynitrophenyl-EGTA-4 (DMNPE-4), a newly-developed photolabile Ca(2+)-specific chelator. This compound exhibits good two-photon absorption at 705 nm, has a low Mg2+ affinity (approximately 7 mM), a Kd for Ca2+ of 19 nM, a quantum yield of 0.20 and changes its Ca2+ affinity by 21,000-fold upon photolysis. Two-photon excitation photolysis (TPP) experiments were performed with a Ti:Sapphire laser in solutions containing DMNPE-4 with either 0 or 10 mM Mg2+ and compared to that of the widely used Ca2+ cage, DM-nitrophen (Kd for Ca2+ 5 nM, Kd for Mg2+ 2.5 microM, quantum yield 0.18, affinity change 600,000-fold). The resulting Ca2+ signals were recorded with the fluorescent Ca2+ indicator fluo-3 and a laser-scanning confocal microscope in the line-scan mode. In vitro, photolysis of DMNPE-4:Ca2+ produced Ca(2+)-release signals that had comparable amplitudes and time courses in the presence and absence of Mg2+. However, photorelease of Ca2+ from DM-nitrophen was obviated by the presence of Mg2+. In patch-clamped isolated cardiac myocytes, equivalent TPP results were obtained in analogous experiments. Single-photon excitation of DMNPE-4 by Nd:YAG laser flashes produced Na-Ca exchange currents of comparable amplitude in the absence and presence of Mg2+. However, only very small currents were observed in DM-nitrophen solution containing 10 mM Mg2+. In conclusion, both DMNPE-4 and DM-nitrophen undergo TPP, however, only DMNPE-4 exhibits efficient release of Ca2+ in the presence of Mg2+.
DelPrincipe
F
F
Department of Physiology, University of Bern, Switzerland.
Egger
M
M
Ellis-Davies
G C
GC
Niggli
E
E
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006168:Guinea Pigs; D015394:Molecular Structure; D010782:Photolysis; D017785:Photons; D014466:Ultraviolet Rays
C117677:dimethoxynitrophenyl-EGTA-4; D004533:Egtazic Acid; D002118:Calcium