Identification and Characterization of Voltage- Sensitive Calcium Channels in Neuronal Clonal Cell Lines1

Voltage-sensitive calcium channels (VSCCs) have been identified in three clonal cell lines. These are the neuroblastoma x Chinese hamster brain hybrid (NCB-20), the neuroblastoma x glioma hybrid (NGlOB-15), and the neuroblastoma (N4TGl). Depolarization of NCB-20 cells with 50 mM KC1 or 50 yM veratridine (VE) produced a 2to 3-fold increase in net 45Ca2+ uptake. In NCB-20 cells, this voltage-sensitive 45Ca2+ uptake was inhibited selectively by organic calcium antagonists such as nitrendipine, cinnarizine, verapamil, and diltiazem (I(& values = 6.4, 750, 1800, and 4500 nM, respectively). High K+-induced uptake was unaffected by 4-aminopyridine, tetraethylammonium, and tetrodotoxin (TTX), whereas VE-induced 45Ca2f uptake was completely blocked by 3 yM TTX. In contrast to NCB-20 cells, NG108-15 cells showed a much smaller response to depolarizing stimuli. Following differentiation of NG108-15 cells by chronic treatment with 10 pM prostaglandin E, and 50 pM 3-isobutyl-1-methylxanthine, depolarization induced a large increase in voltagesensitive 45Ca2f uptake. This induction was apparent after 24 hr and increased linearly for 96 hr. VSCC activity was also induced by 1.5% dimethyl sulfoxide and by other agents that increase intracellular CAMP, such as forskolin (1 pM) and cholera toxin (1 pg/ml). Voltage-sensitive 45Ca2+ uptake in differentiated NG108-15 cells was inhibited by nitrendipine, D-600, and diltiazem (IC& values = 7, 690, and 1600 nM). Our results suggest that VSCCs in neuronal clonal cell lines can be altered by cellular differentiation. In contrast to those VSCCs involved in neurotransmitter release, the VSCCs described here appear to be blocked by organic calcium channel antagonists at very low concentrations. Calcium ions play an important role in the control of cellular function in all tissues. An important feature of this regulation is that the intracellular free calcium ion concentration is kept within low (nanomolar) concentrations compared with the millimolar concentrations present extracellularly. The entry of calcium ions into the cell is rigidly controlled. Calcium influx through voltagesensitive calcium channels (VSCCs) represents one of the major mechanisms for the regulation of calcium entry, particularly in neurons and some muscle and endocrine cells. The entry of calcium through VSCCs is important for initiating contraction of smooth and cardiac muscle, and also for release of neurotransmitters and hormones from neuronal and endocrine cells. r This work was supported by United States Public Health Service Grants PHS DA-02121 and PHS DA-02575. S. B. F. is a North Atlantic Treaty Organization (United Kingdom) Fellow. R. J. M. is an Alfred P. Sloan Fellow. We thank S. Kongsamut and T. Grady for assistance