Membrane retrieval after Immediately Releasable Pool (IRP) exocytosis is produced by dynamin‐dependent and dynamin‐independent/protein kinase C‐dependent mechanisms

Abstract

The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action potentials at basal physiological frequencies. To accomplish this duty, IRP should be replenished as a function of time. We have previously reported that an action potential‐like stimulus (APls) triggers the release of ~50% IRP, followed by a fast dynamin‐dependent endocytosis and an associated rapid replenishment process. In this work, we investigated the endocytosis and IRP replenishment produced after the exocytosis of variable IRP fractions in mice primary chromaffin cell cultures. Exocytosis and endocytosis were estimated by membrane capacitance measurements obtained in patch‐clamped cells. In addition to the dynamin‐dependent fast endocytosis activated after the application of APls or 5 ms squared depolarizations, we found that depolarizations lasting 25–50 ms, which release >80% of IRP, are related with a fast dynamin‐independent, Ca2+‐ and protein kinase C (PKC)‐dependent endocytosis (time constant <1 s). PKC inhibitors, such as staurosporine, bisindolylmaleimide XI, PKC 19–31 peptide, and prolonged treatments with high concentrations of phorbol esters, reduced and decelerated this endocytosis. Additionally, we found that the inhibition of PKC also abolished a slow component of replenishment (time constant ~8 s) observed after total IRP exocytosis. Therefore, our results suggest that PKC contributes to the coordination of membrane retrieval and vesicle replenishment mechanisms that occur after the complete exocytosis of IRP.

A centronuclear myopathy‐causing mutation in dynamin‐2 disrupts neuronal morphology and excitatory synaptic transmission in a murine model of the disease

“…Furthermore, Dyn2 mutations associated with CNM reportedly impair actin remodelling and actin-dependent processes in skeletal muscle cells [67,68]. Consistent with the role of Dyn2 in the cytoskeleton dynamics, we showed that the expression of the CNM-causing p.R465W mutation induced a reduction in dendritic arborisation and loss of dendritic spines in HTZ hippocampal neurons (Figure 1).…”

“…More presynaptic axons could be recruited through this mechanism to increase the glutamate release probability. Considering that dynamins modulate the SV recycling [21,102,103] and the maintenance of the SV readily releasable pool (RRP) [21,67,104], it is possible that the p.R465W mutation in Dyn2 negatively impacts the RRP size in the hippocampal neurons, impairing synaptic transmission. In that case, a greater FV amplitude would not necessarily reflect a change in the neurotransmitter release probability.…”

“…Different members of the DSF are involved in the morphogenesis of neurites and dendritic spines through mechanisms that probably involve interactions with proteins implicated in cytoskeleton dynamics [4,[64][65][66]. On the other hand, mutations in Dyn2 associated with CNM reportedly impairs actin remodelling and actin-dependent process in skeletal muscle cells [67,68]. Therefore, these mutations might also affect actin-dependent structures and/or mechanisms in other tissues.…”

Dynamin-2 Mutations Linked to Neonatal-onset Centronuclear Myopathy impair exocytosis and endocytosis in adrenal chromaffin cells

“…In addition, Figure 1 represents typical capacitance recordings obtained in response to the application of both types of depolarizations (Fig.1 A), and a summary of the different parameters associated to exocytosis and endocytosis obtained from the same experiments (Fig.1 B to E). In control conditions, similarly to murine cells (Bayonés et al, 2022b), bovine cells did not exhibit appreciable differences in ∫I Ca2+ , exocytosis (ΔCm exo ), or endocytosis (ΔCm endo ). Additionally, also resembling murine cells, there were no effects of Anti-Dyn on I Ca2+ , òI Ca2+ , or ΔCm exo .…”

“…1 B to E). In control conditions, similarly to murine cells (Bayonés et al, 2022b), bovine cells did not exhibit appreciable differences in ∫ICa2+, exocytosis (ΔCmexo), or endocytosis (ΔCmendo). Additionally, also resembling murine cells, there were no effects of Anti-Dyn on ICa2+, ICa2+, or ∆Cmexo.…”