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Volume 271, Number 47, Issue of November 22, 1996 pp. 30096-30104
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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N-type Ca²⁺ Channels Are Present in Secretory Granules and Are Transiently Translocated to the Plasma Membrane during Regulated Exocytosis

(Received for publication, June 26, 1996)

Maria Passafaro , Patrizia Rosa ^§ , Carlo Sala ^§ , Francesco Clementi ^§ and Emanuele Sher ^§

From the ^§ Consiglio Nazionale delle Ricerche (CNR) Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milano, 20129 Milano and the  CNR Institute of Biotechnologies Applied to Pharmacology, 88021 Roccelletta di Borgia (CZ), Italy

An intracellular pool of N-type voltage-operated calcium channels has recently been described in different neuronal cell lines. We have now further characterized the intracellular pool of N-type calcium channels in both IMR32 human neuroblastoma and PC12 rat pheochromocytoma cells. Intracellular N-type calcium channels were found to be accumulated in subcellular fractions where the chromogranin B-containing secretory granules were also enriched. ¹²⁵I--Conotoxin GVIA binding assays on fixed and permeabilized cells revealed that intracellular N-type calcium channels translocate to the plasma membrane in cells exposed to secretagogues (KCl, ionomycin, and phorbol esters). The kinetics, Ca²⁺ and protein kinase C dependence, and brefeldin A insensitivity of N-type calcium channels translocation were similar to the regulated release of chromogranin B, while no correlation was found with the constitutive secretion of a heparan sulfate proteoglycan. A PC12 subclone deficient in the regulated but not in the constitutive pathway of secretion had a small intracellular pool of N-type calcium channels, and no secretagogueinduced translocation occurred in these cells. Calcium channel translocation was accompanied by a stronger response of Fura-2-loaded cells to depolarizing stimuli, suggesting that the newly inserted channels are functional.

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