Dense core secretory vesicles revealed as a dynamic Ca²⁺ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

The role of dense core secretory vesicles in the control of cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_c) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2–synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet β-cells: (a) increases in [Ca²⁺]_c cause a prompt increase in intravesicular-free Ca²⁺ concentration ([Ca²⁺]_SV), which is mediated by a P-type Ca²⁺-ATPase distinct from the sarco(endo) plasmic reticulum Ca²⁺-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca²⁺ pumps; (b) steady state Ca²⁺ concentrations are 3–5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca²⁺; (c) inositol (1,4,5) trisphosphate has no impact on [Ca²⁺]_SV in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca²⁺]_SV. Thus, secretory vesicles represent a dynamic Ca²⁺ store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca²⁺-induced Ca²⁺ release from vesicles docked at the plasma membrane could participate in triggering exocytosis.