The calcium concentration clamp: spikes and reversible pulses using the photolabile chelator DM-nitrophen.
New procedures are described for producing brief transients and reversible elevations in [Ca] that can be used to quantitatively control the concentration of cytoplasmic calcium. If the photolabile calcium chelator DM-nitrophen, partially bound to calcium, is exposed to steady illumination, [Ca] can be raised from a few nM to up to 10 microM for durations of 100 ms or longer, depending on light intensity and duration. An association rate of calcium with nitrophen of 1.5 x 10(6) M-1s-1 was estimated from measurements of [Ca] using the fluorescent indicator Fluo-3, and calcium was found to speed the photolysis of nitrophen 2.5-times. Partial photolysis of DM-nitrophen partly loaded with calcium elicits a [Ca] spike of over 100 microM lasting about 1 ms, depending on intensity and duration of the light flash. Simulations of the reactions involved predict changes in Fluo-3 fluorescence measured at high time resolution with a laser scanning confocal microscope. These procedures have been applied in physiological experiments to generate cytoplasmic [Ca] spikes and pulses and study the cellular responses to them.
Zucker
R S
RS
Department of Molecular and Cell Biology, University of California, Berkeley.
D000085:Acetates / Q000528:radiation effects; D000814:Aniline Compounds / Q000378:metabolism; D001667:Binding, Competitive; D002118:Calcium / Q000008:administration & dosage / Q000494:pharmacology*; D002138:Calibration; D002614:Chelating Agents / Q000528:radiation effects; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000528:radiation effects; D005029:Ethylenediamines / Q000528:radiation effects; D006859:Hydrogen / Q000378:metabolism / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D010782:Photolysis; D014966:Xanthenes / Q000378:metabolism
D000085:Acetates; D000814:Aniline Compounds; D002614:Chelating Agents; D005029:Ethylenediamines; D014966:Xanthenes; C052455:nitr 5; C057014:DM-nitrophen; C059715:Fluo-3; D004533:Egtazic Acid; D006859:Hydrogen; D008274:Magnesium; D002118:Calcium