Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their p recise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca2+-dependent activator protein for secretion) , a protein required for Ca2+-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clam p membrane capacitance measurements. Flash photolysis of caged Ca2+ elicite d biphasic capacitance increases consisting of rapid and slow components wi th distinct Ca2+ dependencies. A threshold of approximate to 10 mu M Ca2+ w as required to trigger the slow component, while the rapid capacitance incr ease was recorded already at a intracellular Ca2+ activity < 10 mu M. Both kinetic membrane capacitance components were abolished by botulinum neuroto xin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmalei mide-sensitive factor attachment protein receptor)-dependent vesicle fusion . The rapid but not the slow component was inhibited by CAPS antibody. Thes e results were further clarified by immunocytochemical studies that reveale d that CAPS was present on only a subset of dense-core vesicles. Overall, t he results indicate that dense-core vesicle exocytosis in melanotrophs occu rs by two parallel pathways. The faster pathway exhibits high sensitivity t o Ca2+ and requires the presence of CAPS, which appears to act at a late st age in the secretory pathway.