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

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.

Flagging fusion: Phosphatidylserine signaling in cell–cell fusion

“…Possibly, after these treatments, the residual levels of accessible exofacial PS and bound PS receptors remain high enough to support background levels of fusion. Alternatively, there may be levels of redundancy built into cell-cell fusion that bypass the need of PS as a regulatory signal to some extent, as is the case in exocytosis, where low levels of fusion are observed even in the presence of high concentrations of cytosolic chelator (170).…”

Rapid vesicle replenishment after the immediately releasable pool exocytosis is tightly linked to fast endocytosis, and depends on basal calcium and cortical actin in chromaffin cells

“…This is in the range of basal frequencies measured in mouse chromaffin cells (de Diego et al., 2008; Holman et al., 1994; Marcantoni et al., 2010; Moya‐Diaz et al., 2016; Wallace et al., 2002). Both ETAP and dynamin‐dependent fast endocytosis become negligible when action potential rate is increased above 2 Hz (Chan & Smith, 2001; Moya‐Diaz et al., 2016, 2020). Therefore, it seems that the chromaffin cell invokes different replenishment mechanisms, depending on the stimulation intensity and/or the exocytotic demand.…”

Recent developments concerning the investigation of exocytosis with amperometry

“…Other biological challenges also need to be addressed, like the relationship between exocytosis and calcium. In this way, a new Ca 2þ -independent and voltagedependent pathway of exocytosis is also demonstrated with CFA that is correlated to electrophysiological and fluorescence techniques in chromaffin cells [16]. The above results were obtained by biologists who appropriated the electroanalytical technique.…”