A novel tetrodotoxin-resistant sodium current from an immortalized neuroepithelial cell line

1. Voltage-gated ionic currents were recorded from cells of an immortalized neuroepithelial cell line named V1. The cell line was produced by insertion of the temperature-sensitive tsA58 allele of the SV40 large T-antigen into embryonic day 14 mouse hypothalamic cells. V1. cells display a mixed immature neural-glial phenotype and have two phenotypes, round and flat. 2. Recordings from round V1 cells demonstrate voltage-gated Na+ and K+ currents (n = 297), while no voltage-gated currents were observed in flat V1 cells (n = 45). Voltage-gated currents were recorded from cells cultured at both permissive and restrictive temperatures. 3. Internal Cs+ and external tetraethylammonium (TEA) were used to suppress outward currents. The remaining inward current has rapid activation and inactivation time constants which decreased as the test 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 close to the predicted Nernst equilibrium potential for Na+. Both the magnitude 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 the 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 Hodgkin-Huxley model with an m(3) variable. The time constants of activation and inactivation of I-Na are fast, similar to those of the TTX-resistant and TTX-sensitive Naf currents in central nervous system neurons and glial cells. 6. The divalent and trivalent cations Cd2+, Co2+, Ni2+, Zn2+ and La3+ shifted the activation of I-Na to more positive potentials and decreased the maximal conductance in a dose-dependent manner. The apparent K-d values for blockade of the I-Na by Cd2+, Co2+, Ni2+, Zn2+ and La3+ were 430, 3500, 1900, 83 and 202 mu M, respectively. 7. The addition of phorbol myristate acetate, an activator of protein 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 kinetics and sensitivity to blockade by TTX and cations. We hypothesize that this Na+ current may be expressed transiently during development of the central nervous system at the stage of development represented by the V1 cell line.