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J. Biol. Chem., Vol. 279, Issue 49, 51107-51121, December 3, 2004

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A Dynamic Pool of Calcium in Catecholamine Storage Vesicles

EXPLORATION IN LIVING CELLS BY A NOVEL VESICLE-TARGETED CHROMOGRANIN A-AEQUORIN CHIMERIC PHOTOPROTEIN^*

Nitish R. Mahapatra, Manjula Mahata, Partha P. Hazra¶, Patrick M. McDonough||, Daniel T. O'Connor**, and Sushil K. Mahata¶¶

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⁺ (20–40 mM total), compared with 100 nM in the cytosol. Aequorin, a jellyfish photoprotein with Ca²⁺-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 (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 µM 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 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 (Ca²⁺-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.

Received for publication, August 2, 2004 , and in revised form, September 3, 2004.

^* This work was supported by grants from the Department of Veterans Affairs and the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Biocon Limited, 20th K. M. Hosur Rd., Electronic City P. O. Bangalore-560100, India.

^|| Present address: Dept. of Biology, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182.

To whom correspondence may be addressed: Dept. of Medicine, University of California at San Diego, and VASDHS (0838), 9500 Gilman Dr., La Jolla, CA 92093-0838. Tel.: 858-5528585 (ext. 7373); Fax: 858-642-6425; E-mail: doconnor{at}ucsd.edu. ¶¶ To whom correspondence may be addressed: Dept. of Medicine, University of California at San Diego, and VASDHS (0838), 9500 Gilman Dr., La Jolla, CA 92093-0838. Tel.: 858-5528585 (ext. 2637); Fax: 858-642-6425; E-mail: smahata{at}ucsd.edu.

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