The development and pharmacological characterization of calcium channel currents in cultured embryonic rat septal cells

Abstract

We characterized the development and pharmacology of Ca 2+ channel currents in NGF-treated embryonic day 21 cultured rat septal cells. Using standard whole-cell voltage clamp techniques, cells were held at −80 mV and depolarized to construct current–voltage relations in conditions that eliminated Na + or K + currents. Barium (10 mM) was used as the charge carrier. Maximum current was produced when cells were depolarized to 0 or +10 mV. Recordings from 77 cells revealed that Ca 2+ channel current density increases over time in culture from nearly 0 pA/pF on day 2 in vitro (0.65±0.65 pA/pF) to (6.95±1.59 pA/pF) on days 6–8. This was followed by a period where currents became nearly 3 times more dense (21.05±7.16 pA/pF) at days 9–17. There was little or no evidence for low voltage activated currents. Bath application of 50–100 μM CdCl 2 abolished ∼95% of the current. Application of 10 μM nimodipine produced a 50.5±3.22% reduction in current, 2 μM ω-CTx-GVIA produced a 32.4±7.3% reduction, and application of 4 μM ω-Aga-IVA produced a 29.5±5.73% reduction in current. When all three inhibitors (10 μM nimodipine, 2 μM ω-CTx-GVIA, and 4 μM ω-Aga-IVA) were applied simultaneously, a residual current remained that was 18.0±4.9% of the total current and was completely abolished by application of CdCl 2. This is the first report to characterize Ca 2+ channel currents in cultured embryonic septal cells. These data indicate that there is a steady increase in Ca 2+ channel expression over time in vitro, and show that like other cultured neuronal cells, septal cells express multiple Ca 2+ channel types including L, N, P/Q and R-type channels.