Retinoic Acid Induction of Calcium Channel Expression in Human NT2N Neurons

Abstract

Ca2+channel expression and regulation of intracellular Ca2+homeostasis were studied during retinoic acid (RA)-induced differentiation of the human teratocarcinoma cell line Ntera 2/C1.D1 (NT2− cells) into NT2N neurons, a unique model of human neurons in culture. The cytosolic Ca2+level of undifferentiated NT2− cells was low (75 ± 5 nM) and stable under basal conditions, and it was only marginally decreased (by 9%) upon removal of extracellular Ca2+. After 10 μM RA treatment, NT2− cells were irreversibly differentiated into a phenotype of neuron-like NT2N cells. Cytosolic Ca2+level of NT2N neurons was higher (106 ± 14 nM) than that of NT2− cells and spontaneously fluctuated (0.208 ± 0.038 transients/min) under basal conditions. Although K+increased86Rb fluxes in both NT2− cells and NT2N neurons, it only increased cytosolic Ca2+level in NT2N neurons. The K+-induced increase in cytosolic Ca2+in NT2N neurons was antagonized by 0.1–10 μM nifedipine or verapamil, 5 μM ω-CgTx GVIA, but not by 1 μM ω-agatoxin IVA, 1 μM ω-agatoxin TK, 1 μM FTX-3.3, or 100 μM Ni+implicating L- and N-type voltage-dependent Ca2+channels. In L- and N-type channels, but not in P- and Q-types, mRNAs were expressed in NT2N neurons as well as NT2− cells. Quantitative analysis of L- and N-type Ca2+protein levels showed major differences between NT2− cells and NT2N neurons. In NT2− cells, N-type Ca2+channels were undetectable while L-type channels levels were fivefold lower compared to NT2N neurons. Our findings show that L- and N-type channels are expressed during differentiation of NT2− cells into neurons, and that these voltage-dependent Ca2+channels have a major role in regulating intracellular Ca2+homeostasis and neuronal excitability.