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 microM. A fast Ca2+ uptake mechanism was also found to play a dominant role in the clearance of Ca2+ after flashes at high intracellular free Ca2+ concentrations ([Ca2+]), 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 approximately 1 mM.
Xu
T
T
Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany.
Naraghi
M
M
Kang
H
H
Neher
E
E
D000085:Acetates; D000313:Adrenal Medulla / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002614:Chelating Agents; D019439:Chromaffin Cells / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D005029:Ethylenediamines; D016257:Fura-2; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010782:Photolysis; D014466:Ultraviolet Rays
D000085:Acetates; D002614:Chelating Agents; D005029:Ethylenediamines; C057014:DM-nitrophen; D002118:Calcium; D016257:Fura-2