Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis

Abstract

 We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca²⁺ indicators in response to large (50–200 μM), short duration (<100 ns), free [Ca²⁺] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca²⁺). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca²⁺ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca²⁺] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca²⁺ signaling events and the use of flash photolysis in cells and model systems.