An inhibitory effect of extracellular Ca ²⁺ on Ca ²⁺-dependent exocytosis

Aim: Neurotransmitter release is elicited by an elevation of intracellular Ca ²⁺ concentration ([Ca ²⁺] _i). The action potential triggers Ca ²⁺ influx through Ca ²⁺ channels which causes local changes of [Ca ²⁺] _i for vesicle release. However, any direct role of extracellular Ca ²⁺ (besides Ca ²⁺ influx) on Ca ²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis. Results: Using photolysis of caged Ca ²⁺ and caffeine-induced release of stored Ca ²⁺, we found that extracellular Ca ²⁺ inhibited exocytosis following moderate [Ca ²⁺] _i rises (2-3 μM). The IC ₅₀ for extracellular Ca ²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (~30%) of extracellular Ca ²⁺ concentration ([Ca ²⁺] _o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca ²⁺] _o. The calcimimetics Mg ²⁺, Cd ²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca ²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE. Conclusion/Significance: As an extension of the classic Ca ²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca ²⁺ play dual roles in evoked exocytosis by providing a source of Ca ²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.