title: A phogrin-aequorin chimaera to image free Ca2+ in the vicinity of secretory granules.
abstract: Microdomains of high Ca2+ concentration ([Ca2+]) may be critical to the control of intracellular processes such as secretion and metabolism without compromising other cell functions. To explore changes in [Ca2+] in the outer mantle (< 30 nm deep) that surrounds the surface of dense-core secretory granules, we have designed a recombinant chimaera between the granule protein phogrin and aequorin. When expressed in populations of insulin-secreting MIN6 or phaeochromocytoma PC12 cells, the chimaera was targeted to secretory granules as expected. The recombinant protein reported a similar [Ca2+] at the granule surface to that in the bulk cytosol, measured with untargeted aequorin. This was the case both at rest (-Ca2+- = 80-120 nM) and after stimulation with agents that provoke Ca2+ entry or Ca2+ mobilization from intracellular pools, and during activated secretion. Thus depolarization of MIN6 cell populations with high K+ increased [Ca2+] both in the bulk cytosol and close to the granules to approx. 4 microM, with near-identical kinetics of increase and recovery. Similarly, stimulation of PC12 cells with ATP provoked an increase in -Ca2+- in either domain to 1.3 microM. These data argue that, in MIN6 and PC12 neuroendocrine cells (i) significant mobilization of Ca2+ from most secretory granules probably does not occur during activated Ca2+ influx or mobilization of internal Ca2+ stores, and (ii) agonist-stimulated Ca2+-dependent secretion can occur without development of a large gradient of [Ca2+] between the surface of most secretory vesicles and the rest of the cytosol.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
authors.forename: A E
authors.initials: AE
authors.lastname: Pouli
authors.forename: N
authors.initials: N
authors.lastname: Karagenc
authors.forename: C
authors.initials: C
authors.lastname: Wasmeier
authors.forename: J C
authors.initials: JC
authors.lastname: Hutton
authors.forename: N
authors.initials: N
authors.lastname: Bright
authors.forename: S
authors.initials: S
authors.lastname: Arden
authors.forename: J G
authors.initials: JG
authors.lastname: Schofield
authors.forename: G A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000331:Aequorin / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans; D007700:Kinetics; D008163:Luminescent Measurements; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000378:metabolism*; D008565:Membrane Proteins; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000378:metabolism*; D016716:PC12 Cells; D011188:Potassium / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases; D051381:Rats; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
chemical_list: D007328:Insulin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D000255:Adenosine Triphosphate; C102889:PTPRN2 protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011188:Potassium; D002118:Calcium