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.
Estimation of the free [Ca2+] gradient across endoplasmic reticulum membranes by a null-point method
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A P Dawson, G T Rich, J W Loomis-Husselbee; Estimation of the free [Ca2+] gradient across endoplasmic reticulum membranes by a null-point method. Biochem J 1 September 1995; 310 (2): 371–374. doi: https://doi.org/10.1042/bj3100371
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