Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin.

We use here a new very low-Ca(2+)-affinity targeted aequorin to measure the [Ca(2+)] in the endoplasmic reticulum ([Ca(2+)]ER). The new aequorin chimera has the right Ca(2+)-affinity to make long-lasting measurements of [Ca(2+)]ER in the millimolar range. Moreover, previous Ca(2+)-depletion of the ER is no longer required. The steady-state [Ca(2+)]ER obtained is 1-2 mM, higher than previously reported. In addition, we find evidence that there is significant heterogeneity in [Ca(2+)]ER among different regions of the ER. About half of the ER had a [Ca(2+)]ER of 1 mM or below, and the rest had [Ca(2+)]ER values above 1mM and in some parts even above 2 mM. About 5% of the ER was also found to have high [Ca(2+)]ER levels but to be thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca(2+) of the ER was almost linearly dependent on the extracellular [Ca(2+)] between 0.1 and 3 mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca(2+)] microdomains connecting the store operated Ca(2+) channels with the ER Ca(2+) pumps may be important during refilling.