Development of ion channels and neurofilaments during neuronal differentiation of mouse embryonal carcinoma cell lines.

Abstract

1. an embryonal carcinoma cell line, PCC4-Aza1-ECA2, was induced to differentiate to neurones by two different procedures: an addition of retinoic acid to the culture medium or a reduction of serum concentration. The changes in membrane currents during differentiation were studied by the whole-cell variation of the patch-clamp technique and the change in neurofilament expression was studied immunohistochemically. 2. Stem cells showed the outward K+ current which inactivated slightly, but no inward currents were observed. These cells did not express neurofilament. 3. Three days after an addition of 10(-7) M-retinoic acid, neurofilament-positive round cells without processes began to appear. The inward currents observed in these cells were the Na+ current and fast-inactivating Ca2+-channel current. Four days after an addition of 10(-7) M-retinoic acid, the cells began to extend processes and showed an intense neurofilament expression. The inward currents were the Na+ current and slow-inactivating Ca2+-channel current, while the fast-inactivating Ca2+-channel current observed previously had almost disappeared. The amplitude of the outward K+ current was larger than that in the stem cell and it did not show clear inactivation. 4. By reducing the serum concentration in the medium from 10 to 0.1%, cells with processes were observed after 6 days. They were neurofilament-positive and had the Na+ current, both fast- and slow-inactivating Ca2+-channel currents, and the outward K+ current which inactivated slightly. 5. The properties of these ionic currents observed in induced neurones were studied. The Na+ current was blocked by 0.1 microM-tetrodotoxin at any stage. The Na+ current was evoked by a depolarization pulse to a level above -40 mV with a maximum amplitude at around -10 mV. The fast-inactivating Ca2+-channel current was evoked by a depolarization to a level above -50 mV with a maximum amplitude at around -15 mV. It was resistant to 50 microM-Cd2+. The slow-inactivating Ca2+-channel current was evoked by a depolarization pulse to a level above -30 mV with a maximum amplitude at around +5 mV. It was blocked by 50 microM-Cd2+. It showed slight inactivation, which was not voltage-dependent but current-dependent. It was enhanced by 1 microM-Bay K 8644. The outward K+ current was blocked by replacing intracellular K+ with Cs+. 6. Another embryonal carcinoma cell line, P19, was induced to differentiate to neurons by adding 10(-6) M-retinoic acid to the medium.(ABSTRACT TRUNCATED AT 400 WORDS)