Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the "closed" conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/ E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.

Although CAPS1 was originally identified as a soluble factor that reconstitutes Ca 2؉ -dependent secretion from permeabilized neuroendocrine cells, its exact function in intact mammalian cells remains controversial. Here we investigate the role for CAPS1 by generating stable cell lines in which CAPS1 is strongly down-regulated. In these cells, Ca 2؉ -dependent secretion was strongly reduced not only of catecholamine but also of a transfected neuropeptide. These secretion defects were rescued by infusion of CAPS1-containing brain cytosol or by transfectionmediated expression of CAPS1. Whole cell patch clamp recording revealed significant reductions in slow burst and sustained release components of exocytosis in the knockdown cells. Unexpectedly, they also accumulated higher amounts of endogenous and exogenous transmitters, which were attributable to reductions in constitutive secretion. Electron microscopy did not reveal abnormalities in the number or docking of dense core vesicles. Our results indicate that CAPS1 plays critical roles not only in Ca 2؉ -dependent, regulated exocytosis but also in constitutive exocytosis downstream of vesicle docking. However, they do not support the role for CAPS1 in loading transmitters into dense core vesicles.Catecholamines such as dopamine and noradrenaline are released from secretory vesicles through regulated and constitutive secretory processes. Distinct types of secretory vesicles account for the storage and secretion of different neurotransmitters and peptides/neuromodulators. Synaptic vesicles are responsible for the storage and secretion of classic neurotransmitters such as glutamate, glycine, and ␥-aminobutyric acid, whereas dense core vesicles are responsible for the storage and secretion of peptides and catecholamines. Ca

Voa1 and Voa2 cooperatively regulate the acidification and transmitter uptake/storage of dense-core vesicles, although they might not be as critical for exocytosis as recently proposed.