A CAFFEINE-SENSITIVE AND RYANODINE-SENSITIVE CA2+ STORE IN AVIAN SENSORY NEURONS

1. We identified and studied the function of ryanodine receptors in neurons isolated from dorsal root ganglia (DRG) of 10-day-old chick embryos. 2. A monoclonal antibody (mAb 34C) that recognizes all known ryanodine receptor isoforms in skeletal and cardiac muscle and CNS identified ryanodine receptor-like immunoreactivity in cultured DRG neurons. 3. Using the permeabilized patch technique to record membrane currents, we found that calcium currents were followed by a current with characteristics of a Ca2+-activated Cl- current (I(Cl(Ca))) in approximately two-thirds of the neurons. In these cells, acute application of 10 mM caffeine activated a similar I(Cl(Ca))) and this effect was inhibited by 10 muM ryanodine. The activation of I(Cl(Ca)) by caffeine was not dependent on extracellular Ca2+. These data suggest that caffeine raises intracellular free Ca2+ (Ca(i)2+) by activating the release of Ca2+ from an intracellular store and that this Ca2+ activates the membrane conductance responsible for I(Cl(Ca)). 4. The magnitude of I(Cl(Ca)) activated by depolarization was not affected by ryanodine, implying that the Ca2+ that activates I(Cl(Ca)) in this protocol is supplied by the Ca2+ current without amplification by a ryanodine-sensitive mechanism such as Ca2+-induced Ca2+ release. 5. We also used indo-1 to measure Ca(i)2+ in DRG neurons. Ten millimolar caffeine caused a transient increase in Ca2+ that was inhibited by 10 muM ryanodine. 6. The ability of caffeine to elevate Ca(i)2+ and activate I(Cl(Ca)) was reduced at higher temperatures, suggesting increased Ca2+ sequestration. 7. These data demonstrate the existence of an intracellular store of Ca2+ that can be mobilized by a caffeine- and ryanodine-sensitive mechanism. The release of Ca2+ from this store can elevate Ca(i)2+ and modulate membrane conductances.