Dynamics of [Ca2+] in the endoplasmic reticulum and cytoplasm of intact HeLa cells. A comparative study

Abstract

We have measured the [Ca2+] in the endoplasmic reticulum ([Ca2+]er) of intact HeLa cells at both 22 degrees C and 37 degrees C using endoplamsic reticulum-targeted, low Ca2+ affinity aequorin reconstituted with coelenterazine n. Aequorin consumption was much slower at 22 degrees C, and this allowed performing a much longer study of the dynamics of [Ca2+]er. The steady-state [Ca2+]er (500-600 microM) was not modified by the temperature, although both the rates of pumping and leak were decreased at 22 degrees C. The behavior of both [Ca2+]er and cytoplasmic [Ca2+] ([Ca2+]c) after the addition of increasing concentrations of agonists and/or Ca2+-ATPase inhibitors, or following incubation in Ca2+-free medium were compared. We show that agonists induce a fast but relatively small decrease in [Ca2+]er, which is enough to produce a sharp increase in [Ca2+]c. Termination of Ca2+ release is controlled by feedback inhibition of the inositol 1,4,5-trisphosphate receptors by [Ca2+]c, a mechanism that appears to be designed to release the minimum amount of Ca2+ necessary to produced the required [Ca2+]c signal. We also show that Ca2+ release is inhibited progressively when [Ca2+]er decreases below a threshold of about 150 microM, even in the absence of Ca2+ pumping or -Ca2+-c increase. This effect is consistent with a regulation of the inositol 1,4,5-trisphosphate-gated channels by [Ca2+]er.