Voltage-gated calcium currents in cultured embryonic Xenopus spinal neurones.
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 microM-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 microM). Both components of HVA current were sensitive to verapamil (100 microM), were almost completely blocked by omega-conotoxin (3 microM) and were insensitive to nifedipine (20 microM). 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 Ca(2+)-dependent embryonic action potential. HVA Ca2+ currents (particularly the relaxing component) influence the plateau phase.
Barish
M E
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Department of Physiology and Biophysics, University of California, College of Medicine, Irvine 92717.
D000200:Action Potentials; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004558:Electric Stimulation; D004744:Enkephalin, Methionine / Q000494:pharmacology; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000502:physiology*; D008978:Mollusk Venoms / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology*; D014700:Verapamil / Q000494:pharmacology; D014981:Xenopus; D020866:omega-Conotoxin GVIA
D015220:Calcium Channels; D008978:Mollusk Venoms; D004744:Enkephalin, Methionine; D020866:omega-Conotoxin GVIA; D014700:Verapamil; D002118:Calcium