Subcellular Ca(2+) dynamics measured with targeted aequorin in chromaffin cells

In the last years, intracellular organella have emerged as key components in the generation and transduction of Ca(2+) signals in adrenal chromaffin cells. Therefore, accurate measurements of Ca(2+) inside cytoplasmic organella are essential for a comprehensive analysis of the Ca(2+) redistribution that follows cell stimulation. We have engineered the Ca(2+)-sensitive photoprotein aequorin to monitor selectively Ca(2+) within the endoplasmic reticulum and the mitochondria. The targeted aequorins were delivered to the appropriate organelles of bovine chromaffin cells by using a herpes simplex virus-based amplicon vector, which permits efficient gene transfer and high levels of expression in infected cells. We have investigated the relationship between the caffeine and InsP(3)-sensitive Ca(2+) pools and the presence of the Ca(2+)-induced Ca(2+) release (CICR) mechanism in chromaffin cells. We find that ER Ca(2+) pools responding to caffeine and to InsP(3) mostly overlap and that CICR can be induced by Ca(2+) entry elicited by high K(+) depolarization. Moreover, the activation of Ca(2+) channels, either the voltage-gated Ca(2+) channels on the plasma membrane or the channels on the endoplasmic reticulum (ER), generates subplasmalemmal high [Ca(2+)](c) domains that induce Ca(2+) uptake by mitochondria. Interestingly, only a subpopulation of mitochondria, the one contained in the pool located close to the plasma membrane and the ryanodine receptors, take up Ca(2+) efficiently, and the [Ca(2+)](M) reaches values of 300-500 micro M.