Molecular characterization of two distinct secretory vesicle priming steps in adrenal chromaffin cells

Priming of large dense-core vesicles (LDCVs) is a Ca-dependent step by which LDCVs enter a release-ready pool. The underlying molecular step involves the formation of the SNAREcomplex consisting of syntaxin, SNAP-25 and synaptobrevin, which is facilitated by priming proteins such as Munc13s. By using mutant mice lacking both isoforms of the calcium dependent activator protein for secretion (CAPS), we show that the LDCV priming process in adrenal chromaffin cells entails two distinct steps. Deletion of both CAPS isoforms reduces total secretion, the size of the readily releasable LDCV pool, and the amount of sustained secretion in the continued presence of high Ca concentrations. The effects of CAPS loss on sustained release are much greater than those on the readily releasable LDCV pool. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool upon CAPS loss is rescued by a constitutively open form of syntaxin, but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Though CAPS appears to function downstream of Munc13s, a functional interaction between these two proteins seems to be required throughout the entire LDCV priming process, including the opening of syntaxin.