Subcellular Ca2+ 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 Ca2+ signals in adrenal chromaffin cells. Therefore, accurate measurements of Ca2+ inside cytoplasmic organella are essential for a comprehensive analysis of the Ca2+ redistribution that follows cell stimulation. We have engineered the Ca2+-sensitive photoprotein aequorin to monitor selectively Ca2+ 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 InsP3-sensitive Ca2+ pools and the presence of the Ca2+-induced Ca2+ release (CICR) mechanism in chromaffin cells. We find that ER Ca2+ pools responding to caffeine and to InsP3 mostly overlap and that CICR can be induced by Ca2+ entry elicited by high K+ depolarization. Moreover, the activation of Ca2+ channels, either the voltage-gated Ca2+ channels on the plasma membrane or the channels on the endoplasmic reticulum (ER), generates subplasmalemmal high [Ca2+]c domains that induce Ca2+ 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 Ca2+ efficiently, and the [Ca2+]M reaches values of 300-500 ?M.
subcellular aequorin chromaffin organelle amplicon calcium