Antagonists-resistant calcium currents in rat embryo motoneurons

Ca²⁺ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5-20 mM Ba²⁺, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed ≃ 20% L-, ≃ 45% N- and ≃ 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20-200 nM ω-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nM in 5 mM Ba(2+)), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca²⁺ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 μM ω-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the α(1E) channel. The current activates around -20 mV in 20 mM Ba²⁺; inactivates slowly and independently of Ca²⁺; is blocked by low [Cd²⁺] and high [Ni²⁺]; and is larger with Ba²⁺ than Ca²⁺. The uncovered R-type calcium current can account for part of the presynaptic Ca²⁺ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP- and ω-CTx-GVIA, and displays anomalous sensitivity to ω-Aga-IVA and ω-CTx-MVIIC.