ESTIMATION OF THE FREE [CA2+] GRADIENT ACROSS ENDOPLASMIC-RETICULUM MEMBRANES BY A NULL-POINT METHOD

The rate of unidirectional efflux of Ca-45 from rat liver microsomal vesicles loaded with Ca-45 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 mu M. As the loading is increased, the internal [Ca2+](free) increases linearly up to a value of 47+/-3.6 mu M 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 mu M.