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Published online 24 September 2001. doi:10.1083/jcb.200103145
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© The Rockefeller University Press, 0021-9525/2001/10/41 $5.00
The Journal of Cell Biology, Volume 155, Number 1, October 1, 2001 41-52

Article

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



Kathryn J. Mitchell1, Paolo Pinton2,3, Aniko Varadi1, Carlo Tacchetti4, Edward K. Ainscow1, Tullio Pozzan3, Rosario Rizzuto2 and Guy A. Rutter1

1 Departments of Biochemistry, University of Bristol, BS8 1TD Bristol, United Kingdom
2 Experimental and Diagnostic Medicine Section of General Pathology, University of Ferrara, 44100 Ferrara, Italy
3 Biomedical Sciences and CNR Center for Study of Biological Membranes, University of Padova, 35121 Padova 17, Italy
4 Experimental Medicine, University of Genova Medical School, 16132 Genova, Italy

Address correspondence to Guy Rutter, Department of Biochemistry, University of Bristol, BS8 1TD Bristol, UK. Tel.: (44) 117-954-6401. Fax: (44) 117-928-8274. E-mail: g.a.rutter{at}bris.ac.uk

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 ß-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.

Key Words: calcium; secretory vesicle; insulin; ryanodine receptor; aequorin


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