Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.
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 microM), 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.
Escobar
A L
AL
Department of Physiology, UCLA School of Medicine 90024, USA.
Velez
P
P
Kim
A M
AM
Cifuentes
F
F
Fill
M
M
Vergara
J L
JL
D000085:Acetates / Q000493:pharmacokinetics*; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D005029:Ethylenediamines / Q000493:pharmacokinetics*; D005453:Fluorescence; D005456:Fluorescent Dyes; D005544:Forecasting; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000493:pharmacokinetics*; D007700:Kinetics; D008956:Models, Chemical; D009930:Organic Chemicals; D009994:Osmolar Concentration; D010775:Photic Stimulation; D010782:Photolysis; D013997:Time Factors; D014466:Ultraviolet Rays
D000085:Acetates; D002021:Buffers; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009930:Organic Chemicals; C071335:calcium orange; C057014:DM-nitrophen; D002118:Calcium