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.