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.
Highlights
From the ‡Department of Medicine, the §Division of Biology, and **Molecular Genetics, University of California, and the ‡‡Veterans Affairs San Diego Healthcare System, San Diego, California 92093-0838
We reasoned that a Chromogranin A (CgA)-Aequorin chimera would be trafficked to the chromaffin granules by using the N-terminal targeting information of CgA, and could monitor free vesicular [Ca2ϩ] under basal or secretion-stimulated conditions. Such a CgA-Aequorin chimera should be useful to probe questions about chromaffin cell Ca2ϩ that have remained elusive for decades, such as: (i) What is the free [Ca2ϩ] in the core of chromaffin granules? (ii) Are chromaffin granule Ca2ϩ stores predominantly free or bound? (iii) How do chromaffin granule Ca2ϩ stores respond to secretory stimuli? (iv) What transporters underlie the accumulation of granular Ca2ϩ? Our studies suggest that chromaffin granules actively accumulate Ca2ϩ against a substantial concentration gradient, that Ͼ99% of Ca2ϩ inside the vesicle remains in bound form, that vesicular [Ca2ϩ] changes promptly in response to secretory stimulation, and that several discrete ion transporters in the granule membrane mediate these processes
We measured sucrose concentration, norepinephrine radioactivity, and aequorin luminescence in different fractions of the gradient. [3H]norepinephrine and aequorin luminescence co-localized in the same gradient fractions with peaks at 1.2 M sucrose (Fig. 1B), suggesting that the human CgA (hCgA)-Aeq chimera is trafficked into chromaffin granules in PC12 cells
Summary
From the ‡Department of Medicine, the §Division of Biology, and **Molecular Genetics, University of California, and the ‡‡Veterans Affairs San Diego Healthcare System, San Diego, California 92093-0838. Chromaffin vesicles contain very high concentration of Ca2؉ (ϳ20 – 40 mM total), compared with ϳ100 nM in the cytosol. Because of the presence of vesicular trafficking domain within CgA we engineered sorting of an expressed human CgA-Aequorin fusion protein (hCgAAeq) into the vesicle compartment as confirmed by sucrose density gradients and confocal immunofluorescent co-localization studies. Calibrating the pH dependence of hCgA-Aeq luminescence allowed estimation of [Ca2؉]ves at granule interior pH (ϳ5.5). 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 M under basal conditions indicating that >99% of granule total Ca2؉ is in a “bound” state. The basal free [Ca2؉]ves/[Ca2؉]cyto ratio was ϳ10.8-fold, indicating active, dynamic Ca2؉ uptake from cytosol into the granules. Changes in cytoplasmic [Ca2ϩ] can be monitored by introducing Ca2ϩ-chelating fluorescent dyes such as Indo-1 [5, 14] or
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