Identification and characterization of voltage-sensitive calcium channels in neuronal clonal cell lines

Abstract

Voltage-sensitive calcium channels ( VSCCs ) have been identified in three clonal cells. These are the neuroblastoma X Chinese hamster brain hybrid ( NCB -20), the neuroblastoma X glioma hybrid (NG108-15), and the neuroblastoma ( N4TG1 ). Depolarization of NCB -20 cells with 50 mM KCl or 50 microM veratridine (VE) produced a 2- to 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 (IC50 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 45Ca2+ uptake was completely blocked by 3 microM 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 microM prostaglandin E1 and 50 microM 3-isobutyl-1-methylxanthine, depolarization induced a large increase in voltage-sensitive 45Ca2+ 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 microM) and cholera toxin (1 microgram/ml). Voltage-sensitive 45Ca2+ uptake in differentiated NG108-15 cells was inhibited by nitrendipine, D-600, and diltiazem (IC50 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.