title: A dynamic pool of calcium in catecholamine storage vesicles. Exploration in living cells by a novel vesicle-targeted chromogranin A-aequorin chimeric photoprotein.
abstract: Chromaffin vesicles contain very high concentration of Ca2+ (approximately 20-40 mM total), compared with approximately 100 nM in the cytosol. Aequorin, a jellyfish photoprotein with Ca(2+)-dependent luminescence, measures [Ca2+] in specific subcellular compartments wherein proteins with organelle-specific trafficking domains are fused in-frame to aequorin. Because of the presence of vesicular trafficking domain within CgA we engineered sorting of an expressed human CgA-Aequorin fusion protein (hCgA-Aeq) into the vesicle compartment as confirmed by sucrose density gradients and confocal immunofluorescent co-localization studies. hCgA-Aeq and cytoplasmic aequorin (Cyto-Aeq) luminescence displayed linear functions of [Ca2+] in vitro, over >5 log10 orders of magnitude (r > 0.99), and down to at least 10(-7) M sensitivity. Calibrating the pH dependence of hCgA-Aeq luminescence allowed estimation of [Ca2+]ves at granule interior pH (approximately 5.5). In the cytoplasm, Cyto-Aeq accurately determined [Ca2+]cyto under both basal ([Ca2+]cyto = 130 +/- 35 nM) and exocytosis-stimulated conditions, confirmed by an independent reference technique (Indo-1 fluorescence). The hCgA-Aeq chimera determined vesicular free [Ca2+]ves = 1.4 +/- 0.3 microM under basal conditions indicating that >99% of granule total Ca2+ is in a "bound" state. The basal free [Ca2+]ves/[Ca2+]cyto ratio was thus approximately 10.8-fold, indicating active, dynamic Ca2+ uptake from cytosol into the granules. Stimulation of exocytotic secretion revealed prompt, dynamic increases in both [Ca2+](ves) and [Ca2+]cyto, and an exponential relation between the two (y = 0.99 x e(1.53x), r = 0.99), reflecting a persistent [Ca2+]ves/[Ca2+]cyto gradient, even during sharp increments of both values. Studies with inhibitors of Ca2+ translocation (Ca(2+)-ATPase), Na+/Ca(+)-exchange, Na+/H(+)-exchange, and vesicle acidification (H(+)-translocating ATPase), documented a role for these four ion transporter classes in accumulation of Ca2+ inside the vesicles.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla 92093-0838, USA.
authors.forename: Nitish R
authors.initials: NR
authors.lastname: Mahapatra
authors.forename: Manjula
authors.initials: M
authors.lastname: Mahata
authors.forename: Partha P
authors.initials: PP
authors.lastname: Hazra
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McDonough
authors.forename: Daniel T
authors.initials: DT
authors.lastname: O'Connor
authors.forename: Sushil K
authors.initials: SK
authors.lastname: Mahata
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000331:Aequorin / Q000737:chemistry*; D000818:Animals; D001669:Biochemical Phenomena; D001671:Biochemistry; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002138:Calibration; D002395:Catecholamines / Q000737:chemistry* / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D019439:Chromaffin Cells / Q000378:metabolism; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry*; D004396:Coloring Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005089:Exocytosis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D007477:Ions; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009638:Norepinephrine / Q000494:pharmacology; D016716:PC12 Cells; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011522:Protons; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013347:Subcellular Fractions; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection
chemical_list: C506150:CHGA protein, human; D002395:Catecholamines; D053379:Chromogranin A; D002864:Chromogranins; D004396:Coloring Agents; D018076:DNA, Complementary; D007211:Indoles; D007477:Ions; D011522:Protons; D011993:Recombinant Fusion Proteins; C506153:chromogranin A, rat; D000331:Aequorin; D013395:Sucrose; D000251:Adenosine Triphosphatases; C048960:indo-1; D002118:Calcium; D009638:Norepinephrine