Dissection of three Ca2+-dependent steps leading to secretion in chromaffin cells from mouse adrenal slices

In neurosecretory cells, intracellular Ca2+ ([Ca2+](i)) not only acts as the trigger for secretion but also regulates earlier steps in the secretory pathway. Here, a novel approach was developed to control [Ca2+](i) over a broad concentration range, which allowed the quantification of three distinct actions of [Ca2+](i) on large dense-core vesicle (LDCV) fusion in chromaffin cells from mouse adrenal slices. Basal [Ca2+](i) regulated the transfer of vesicles toward a slowly releasable state, whereas further maturation to the readily releasable state was Ca2+ independent. [Ca2+](i) levels above 3 muM triggered exocytosis of all readily and slowly releasable vesicles in two parallel, kinetically distinct fusion reactions. In a molecular context, these results suggest that Ca2+ acts both before and after trans-SNARE complex formation to regulate fusion competence and fusion kinetics of LDCVs.