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 Ca²⁺-specific chelator. This compound exhibits good two-photon absorption at 705 nm, has a low Mg²⁺ affinity (≃ 7 mM), a K(d) 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 (K(d) for Ca²⁺ 5 nM, K(d) for Mg²⁺ 2.5 μM, 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 Ca²⁺-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⁺.