VOLTAGE-GATED CALCIUM CURRENTS IN CULTURED EMBRYONIC XENOPUS SPINAL NEURONS

1. Voltage-gated Ca2+ currents were studied in cultured embryonic Xenopus spinal neurones using whole-cell gigaohm seal techniques. Cultures of neural plate cells were established from stage 15-17 embryos (see Methods), and were studied for up to 80 h in vitro. During this period neural precursor cells morphologically differentiate and commence expression of multiple types of voltage- and ligand-gated ion channels. 2. Embryonic Xenopus neurones studied during the first 20-40 h in culture display Ca2+ currents that correspond to the low-voltage-activated (T-type) and high-voltage-activated forms described in other neurones and excitable cells. These Ca2+ current types could be separated based on voltage dependencies and pharmacological sensitivities. 3. T-type Ca2+ current was activated at voltages positive to -50 mV, and was selectively blocked by 200-mu-M-Ni2+. Curves describing the voltage dependencies of activation and steady-state inactivation overlapped in a region centred on -40 mV. A small sustained Ca2+ current could be recorded within this voltage region. 4. High-voltage-activated (HVA) Ca2+ currents were observed at voltages positive to -10 mV, and could be separated into relaxing and sustained components (denoted as HVA-relaxing and HVA-sustained). HVA-relaxing current was selectively reduced by Met-enkephalin (17.5-mu-M). Both components of HVA current were sensitive to verapamil (100-mu-M), were almost completely blocked by omega-conotoxin (3-mu-M) and were insensitive to nifedipine (20-mu-M). 5. The data indicate that T-type Ca2+ current is present in the membrane during the initial period of channel and receptor expression, process outgrowth, and synaptogenesis, and is the dominant influence on voltage-gated Ca2+ influx during subthreshold voltage excursions. Further, at more positive voltages, T-type Ca2+ current contributes to inward Ca2+ current during the first 5-10 ms after depolarizing voltage steps, and thus to inward Ca2+ current during the rising phase of the long-lasting Ca2+-dependent embryonic action potential. HVA Ca2+ currents (particularly the relaxing component) influence the plateau phase.