A caffeine- 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 Ca(2+)-activated Cl- current (ICl(Ca)) in approximately two-thirds of the neurons. In these cells, acute application of 10 mM caffeine activated a similar ICl(Ca) and this effect was inhibited by 10 microM ryanodine. The activation of ICl(Ca) by caffeine was not dependent on extracellular Ca2+. These data suggest that caffeine raises intracellular free Ca2+ (Cai2+) by activating the release of Ca2+ from an intracellular store and that this Ca2+ activates the membrane conductance responsible for ICl(Ca). 4. The magnitude of ICl(Ca) activated by depolarization was not affected by ryanodine, implying that the Ca2+ that activates ICl(Ca) in this protocol is supplied by the Ca2+ current without amplification by a ryanodine-sensitive mechanism such as Ca(2+)-induced Ca2+ release. 5. We also used indo-1 to measure Cai2+ in DRG neurons. Ten millimolar caffeine caused a transient increase in Cai2+ that was inhibited by 10 microM ryanodine. 6. The ability of caffeine to elevate Cai2+ and activate ICl(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 Cai2+ and modulate membrane conductances.