Measurement of cytoplasmic free Ca changes during activation of outward Na/Ca exchange currents in BHK cells. The pipette solution contains 0.5 mM EGTA and 0.25 mM Ca (i.e., 0.4 μM free Ca) with 3 μM Fluo 5N (Kd, 90 μM). Here, and in all figures, Ca concentrations given indicate the free Ca concentration. (A) Typical fluorescence and current records. When 2 mM Ca is applied for 4 s, current rises rapidly to a peak of 175 pA and decays to baseline within <1 s when Ca is removed. Peak fluorescence occurs at 3 s and begins to decay rapidly before current is deactivated. Upon perfusion of the pipette with 1 mM additional Ca, fluorescence rises to a steady “maximal” level with a time constant of ∼20 s. (B) Free and total Ca are calculated from the fluorescence and current records as described in the text. Peak free Ca is estimated to be 0.20 mM at a time point when total Ca influx amounts to 3.0 mmol/liter cell volume. (C) Peak free and total Ca influx values obtained 14 control cells and 5 cells perfused with 2 μM thapsigargin and 2 mM AMP-PNP to block Ca pumping. Results are not significantly different.
