Tunicamycin Dissociates Depolarization‐induced Calcium Entry From Transmitter Release. Involvement of Glycosylated Protein(s) in the Process of Neurosecretion in PC‐12 Cells

The process of regulated secretion in PC-12 cells is tightly coupled to calcium entry, which is absolutely dependent on extracellular Ca2+([Ca2+]ex). Tunicamycin treatment of the cells dissociated depolarization-triggered Ca2+ influx from depolarization (high K+)-induced transmitter release into two distinct and independent phases. Deplarization-evoked Ca2+ influx was not affected by tunicamycin treatment (1 microg/ml, 72 h), whereas depolarization-evoked transmitter release was strongly inhibited (> 60%), suggesting at least a two-step process, and the participation of glycosylated protein(s) in the actual fusion/secretion step. Similarly, bradykinin-mediated transmitter release was linearly related to and absolutely dependent on Ca2+ entry, and was inhibited by tunicamycin treatment (> 80%), whereas bradykinin-evoked Ca2+ entry was not impaired, indicating that glycosylated protein(s) are essential for bradykinin-evoked release at a step subsequent to Ca2+ influx. The heavily glycosylated alpha2 subunit of the dihydropyridine-sensitive channel, which was used to monitor tunicamycin inhibition of glycosylation, was not expressed in the tunicamycin-treated cells, as shown by Western blot analysis. This observation allowed us to conclude that the alpha1 subunit of the heteromeric dihydropyridine voltage-sensitive Ca2+ channel, which is responsible for Ca2+ entry, is also fully functional when not assembled with its corresponding alpha2 subunit. The molecular properties of the alpha2 subunit, whose role in the complex structure of the channel is not yet understood, are shown for the first time for the L-type Ca2+ channel of PC-12 cells. Similar to cardiac and skeletal muscle cells, the alpha2 subunit appears to be a glycosylated polypeptide of molecular weight 170 kD and to display a characteristic mobility shift to 140 kD under reducing conditions.