A NOVEL TETRODOTOXIN-RESISTANT SODIUM CURRENT FROM AN IMMORTALIZED NEUROEPITHELIAL CELL-LINE

1. Voltage-gated ionic currents were recorded from cells of an immorta lized neuroepithelial cell line named V1. The cell line was produced b y insertion of the temperature-sensitive tsA58 allele of the SV40 larg e T-antigen into embryonic day 14 mouse hypothalamic cells. V1. cells display a mixed immature neural-glial phenotype and have two phenotype s, round and flat. 2. Recordings from round V1 cells demonstrate volta ge-gated Na+ and K+ currents (n = 297), while no voltage-gated current s were observed in flat V1 cells (n = 45). Voltage-gated currents were recorded from cells cultured at both permissive and restrictive tempe ratures. 3. Internal Cs+ and external tetraethylammonium (TEA) were us ed to suppress outward currents. The remaining inward current has rapi d activation and inactivation time constants which decreased as the te st potential increased. In different cells, the amplitude of the peak inward current varied from about 50 pA to as large as 4500 pA (in 120 mM external Na+). The reversal potential for the inward current was cl ose to the predicted Nernst equilibrium potential for Na+. Both the ma gnitude and reversal potential of the inward current were dependent on the external Na+ concentration. It is therefore considered to be a Na + current, I-Na. 4. I-Na was found to be TTX resistant. About 5% of th e I-Na was blocked by 200 nar TTX and 20 mu M TTX fully suppressed the Na+ current. The apparent K-d for TTX blockade was estimated to be 1. 49 mu M. 5. The activation kinetics of I-Na could be described by a Ho dgkin-Huxley model with an m(3) variable. The time constants of activa tion and inactivation of I-Na are fast, similar to those of the TTX-re sistant and TTX-sensitive Naf currents in central nervous system neuro ns and glial cells. 6. The divalent and trivalent cations Cd2+, Co2+, Ni2+, Zn2+ and La3+ shifted the activation of I-Na to more positive po tentials and decreased the maximal conductance in a dose-dependent man ner. The apparent K-d values for blockade of the I-Na by Cd2+, Co2+, N i2+, Zn2+ and La3+ were 430, 3500, 1900, 83 and 202 mu M, respectively . 7. The addition of phorbol myristate acetate, an activator of protei n kinase C, consistently produced a reduction in the amplitude of I-Na without affecting the time course of activation or inactivation. 8. I -Na in V1 cells expresses a unique combination of voltage and time kin etics and sensitivity to blockade by TTX and cations. We hypothesize t hat this Na+ current may be expressed transiently during development o f the central nervous system at the stage of development represented b y the V1 cell line.