N-TYPE CA2-MEMBRANE DURING REGULATED EXOCYTOSIS( CHANNELS ARE PRESENTIN SECRETORY GRANULES AND ARE TRANSIENTLY TRANSLOCATED TO THE PLASMA)

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 channel s in both IMR32 human neuroblastoma and PC12 rat pheochromocytoma cell s, Intracellular N-type calcium channels were found to be accumulated in subcellular fractions where the chromogranin B-containing secretory granules were also enriched, I-125-omega-Conotoxin GVIA binding assay s on fixed and permeabilized cells revealed that intracellular N-type calcium channels translocate to the plasma membrane in cells exposed t o secretagogues (KCl, ionomycin, and phorbol esters). The kinetics, Ca 2+ and protein kinase C dependence, and brefeldin A insensitivity of N -type calcium channels translocation were similar to the regulated rel ease of chromogranin B, while no correlation was found with the consti tutive secretion of a heparan sulfate proteoglycan. A PC12 subclone de ficient in the regulated but not in the constitutive pathway of secret ion had a small intracellular pool of N-type calcium channels, and no secretagogue-induced translocation occurred in these cells, Calcium ch annel translocation was accompanied by a stronger response of Fura-2-l oaded cells to depolarizing stimuli, suggesting that the newly inserte d channels are functional.