DYNAMICS OF [CA2-RETICULUM AND CYTOPLASM OF INTACT HELA-CELLS - A COMPARATIVE-STUDY(] IN THE ENDOPLASMIC)

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 endop lasmic reticulum-targeted, low Ca2+ affinity aequorin reconstituted wi th coelenterazine n. Aequorin consumption was much slower at 22 degree s C, and this allowed performing a much longer study of the dynamics o f [Ca2+](er). The steady-state [Ca2+](er) (500-600 mu M) was not modif ied by the temperature, although both the rates of pumping and leak we re decreased at 22 degrees C, The behavior of both [Ca2+](er) and cyto plasmic [Ca2+] ([Ca2+](c)) after the addition of increasing concentrat ions of agonists and/or Ca2+-ATPase inhibitors, or following incubatio n in Ca2+-free medium were compared. We show that agonists induce a fa st but relatively small decrease in [Ca2+](er), which is enough to pro duce a sharp increase in [Ca2+](c). Termination of Ca2+ release is con trolled by feedback inhibition of the inositol 1,4,5-trisphosphate rec eptors by [Ca2+](c), a mechanism that appears to be designed to releas e 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 mu M, even in the absence of Ca2+ pumping or [Ca2+](c) increase. This effect is con sistent with a regulation of the inositol 1,4,5-trisphosphate-gated ch annels by [Ca2+](er).