N-type Ca2+ channels are present in secretory granules and are transiently translocated to the plasma membrane during regulated exocytosis.
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-omega-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 secretagogue-induced 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.
Passafaro
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M
CNR Institute of Biotechnologies Applied to Pharmacology, 88021 Roccelletta di Borgia (CZ), Italy. sher@farma6.csfic.mi.cnr.it
Rosa
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Sala
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Clementi
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Sher
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D000818:Animals; D001692:Biological Transport; D015220:Calcium Channels / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005089:Exocytosis; D006801:Humans; D007457:Iodine Radioisotopes; D008978:Mollusk Venoms / Q000378:metabolism; D016716:PC12 Cells; D011869:Radioligand Assay; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D007457:Iodine Radioisotopes; D008978:Mollusk Venoms