Flash Photolytic Control of the Ca-concentration

Here we describe the subcellularly uniform control of the intracellular Ca2+ concentration ([Ca2+]i) by flash photolysis of caged Ca2+ or a caged Ca2+ buffer. A mixture of the two Ca2+ indicators fluo-3 and Fura-Red was used together with a laser-scanning confocal microscope to reveal spatial aspects of intracellular Ca2+ signals. The patch clamp technique in the whole-cell variant was applied to load the cells with the indicator mixture together with either DM-Nitrophen or diazo-2 and to measure changes in the membrane current. An in-vivo calibration was performed to convert the fluo-3/Fura-Red-fluorescence ratios into [Ca2+] values. The resulting calibration curve suggested an apparent kD of 1.6 μM, Rmax of 2.15, Rmin of 0.08 and a Hill-coefficient of 0.75 for the mixture. Controlled rupture of the cell membrane revealed a large fraction of immobile intracellular Fura-Red fluorescence that may account for the reduced in-vivo Rmax value when compared to the invitro value of 3.1. In cardiac myocytes flash photolytic release of Ca2+ from DM-Nitrophen generated inwardly directed Na+/Ca2+ exchange currents and Ca2+ signals that were graded with the discharged flash-energy. Rapid line-scans revealed subcellularly homogeneous [Ca2+] jumps regardless of the discharged flash energy. Ca2+ signals evoked by L-type Ca2+ currents (ICa) could be terminated rapidly in a spatially homogeneous manner by UVflash photolysis of diazo-2. No side-effects of the photolytic products of DM-Nitrophen or diazo-2 with the mixture of fluo-3/Fura-Red were detectable in our experiments. The combination of UV flash photolysis and laser scanning confocal microscopy enabled us to control [Ca2+]i homogeneously on the subcellular level. This approach may improve our understanding of the subcellular properties of cardiac Ca2+ signalling. The technique can also be applied in other cell types and with other signalling systems for which caged compounds are available. CELL CALCIUM (1996) 255-266.