Ca -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 entry through voltage-dependent Ca channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca current and intracellular Ca 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 was estimated using epifluorescence microscopy and fluo-8 (salt form).

RESULTS

Cells stimulated by brief depolatizations in absence of extracellular Ca show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca 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) ω-agatoxin IVA (blocks P/Q-type Ca channel gating), (ii) in cells from knock-out P/Q-type Ca channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association).

CONCLUSION

We demonstrated that Ca -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 channels can operate as voltage sensors of this process.