Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons

The central role of electrical activity and Ca2+ influx in motoneuron development raises important questions about the regulation of Ca2+ signalling induced by voltage-dependent Ca2+ influx. In the purified embryonic rat motoneuron preparation, we recorded barium currents through voltage-activated Ca2+ channels using the whole-cell configuration of the patch-clamp technique. We found that motoneurons express at least four types of high-voltage-activated Ca2+ channels, based on their kinetics, voltage-dependences and pharmacological properties. Of the sustained Ca2+ current activated at 0 mV from a holding potential of -100 mV, approximately 45% was omega-conotoxin-GVIA (1 microM) sensitive, 25% was omega-agatoxin-IVA (30 nM) sensitive and 20% was nitrendipine (250 nM) sensitive. The residual current, after applying these three antagonists, was an inactivating current that differs from classical T-type Ca2+ currents. Based on this pharmacology, changes in intracellular free Ca2+ concentrations were then monitored by Fura 2 digital imaging microspectrofluorimetry. Upon K+ depolarization, the intracellular Ca2+ transient induced by the activation of each type of Ca2+ channel appeared to be quantitatively proportional to their Ca2+ influx. The existence of a calcium-induced calcium release mechanism through activation of caffeine-, ryanodine-sensitive intracellular stores was then investigated. High doses of caffeine and low doses of ryanodine failed to increase intracellular free calcium concentrations and low concentrations of caffeine and high concentrations of ryanodine did not affect K+-induced intracellular free calcium concentration transients indicating both the absence of Ca2+-gated Ca2+-release channels and of a Ca2+-induced Ca2+ release mechanism. Together, these data provide evidence that embryonic motoneurons express multiple Ca2+ channels that function as important regulators of intracellular Ca2+ signalling and may be involved in their development.