Ca2+‐Independent and Voltage‐Dependent Exocytosis in Mouse Chromaffin Cells

AIM: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca(2+) entry through voltage-dependent Ca(2+) channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca(2+) current and intracellular Ca(2+) increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca(2+) was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS: Cells stimulated by brief depolatizations in absence of extracellular Ca(+2) show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca(+2) release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) omega-agatoxin IVA (blocks P/Q-type Ca(2+) channel gating), (ii) in cells from knock-out P/Q-type Ca(2+) channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). CONCLUSION: We demonstrated that Ca(2+) -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca(2+) channels can operate as voltage sensors of this process.