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. 2020 Apr;228(4):e13417.
doi: 10.1111/apha.13417. Epub 2019 Dec 11.

Ca2+ -independent and voltage-dependent exocytosis in mouse chromaffin cells

José Moya-Díaz  1 , Lucas Bayonés  1 , Mauricio Montenegro  1 , Ana M Cárdenas  2 , Henner Koch  3   4 , Atsushi Doi  5 , Fernando D Marengo  1
Affiliations

Ca2+ -independent and voltage-dependent exocytosis in mouse chromaffin cells

José Moya-Díaz et al. Acta Physiol (Oxf). 2020 Apr.

Abstract

Aim: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ 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 Ca2+ 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) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association).

Conclusion: We demonstrated that Ca2+ -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 Ca2+ channels can operate as voltage sensors of this process.

Keywords: amperometry; ca2+ channels; endocytosis; membrane capacitance; secretion; secretory vesicle.

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References

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