Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells

The Ca2+ binding kinetics of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer, which determine the time course of Ca2+ changes after photolysis of DM-nitrophen, Were studied in bovine chromaffin cells. The in vivo Ca2+ association rate constants of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer were measured to be 5.17 X 10(8) M-1 s(-1), 3.5 X 10(7) M-1 s(-1), and 1.07 X 10(8) M-1 s(-1), respectively. The endogenous Ca2+ buffer appeared to have a low affinity for Ca2+ With a dissociation constant around 100 mu M. A fast Ca2+ uptake mechanism was also found to play a dominant role in the clearance of Ca2+ after flashes at high in intracellular free Ca2+ concentrations ([Ca2+](i)), causing a fast [Ca2+](i) decay within seconds. This Ca2+ clearance was identified as mitochondrial Ca2+ uptake. Its uptake kinetics were studied by analyzing the Ca2+ decay at high [Ca2+](i) after flash photolysis of DM-nitrophen. The capacity of the mitochondrial uptake corresponds to a total cytosolic Ca2+ load of similar to 1 mM.