Estimation of the free [Ca2+] gradient across endoplasmic reticulum membranes by a null-point method.
The rate of unidirectional efflux of 45Ca from rat liver microsomal vesicles loaded with 45Ca and then treated with thapsigargin is not inhibited by increased [Ca2+] in the external medium, although the net efflux rate is substantially inhibited. We have used this property to measure the electrochemical gradient of Ca2+ from the inside to the outside of the vesicles at a series of Ca2+ loadings, by measuring the external [Ca2+]free at which there is zero net efflux. At a loading of 7.9 +/- 0.6 nmol/mg of microsomal protein, the apparent internal [Ca2+]free is 21 +/- 1.6 microM. As the loading is increased, the internal [Ca2+]free increases linearly up to a value of 47 +/- 3.6 microM at a loading of 21.6 +/- 1.6 nmol/mg. Using a similar technique, the value for [Ca2+]free in the endoplasmic reticulum of permeabilized L1210 cells was found to be 12.5 microM.
Dawson
A P
AP
School of Biological Sciences, University of East Anglia, Norwich, U.K.
Rich
G T
GT
Loomis-Husselbee
J W
JW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002463:Cell Membrane Permeability; D004072:Digitonin; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D007939:Leukemia L1210 / Q000378:metabolism / Q000473:pathology; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D013729:Terpenes; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D004072:Digitonin; D002118:Calcium