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Ca 2+ ‐independent and voltage‐dependent exocytosis in mouse chromaffin cells
Author(s) -
MoyaDíaz José,
Bayonés Lucas,
Montenegro Mauricio,
Cárdenas Ana M.,
Koch Henner,
Doi Atsushi,
Marengo Fernando D.
Publication year - 2020
Publication title -
acta physiologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.591
H-Index - 116
eISSN - 1748-1716
pISSN - 1748-1708
DOI - 10.1111/apha.13417
Subject(s) - exocytosis , depolarization , chromaffin cell , endocytosis , membrane potential , biophysics , patch clamp , microbiology and biotechnology , extracellular , chemistry , biology , biochemistry , secretion , adrenal medulla , endocrinology , cell , catecholamine , receptor
Abstract 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) ω‐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.