Dynamin-2 mutations linked to neonatal-onset centronuclear myopathy impair exocytosis and endocytosis in adrenal chromaffin cells.

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.

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

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.

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.

The maintenance of the secretory response requires a continuous replenishment of releasable vesicles. It was proposed that the immediately releasable pool (IRP) is important in chromaffin cell secretion during action potentials applied at basal physiological frequencies, because of the proximity of IRP vesicles to voltage-dependent Ca2+ channels. However, previous reports showed that IRP replenishment after depletion is too slow to manage such a situation. In this work, we used patch-clamp measurements of membrane capacitance, confocal imaging of F-actin distribution, and cytosolic Ca2+ measurements with Fura-2 to re-analyze this problem in primary cultures of mouse chromaffin cells. We provide evidence that IRP replenishment has one slow (time constant between 5 and 10 s) and one rapid component (time constant between 0.5 and 1.5 s) linked to a dynamin-dependent fast endocytosis. Both, the fast endocytosis and the rapid replenishment component were eliminated when 500 nM Ca2+ was added to the internal solution during patch-clamp experiments, but they became dominant and accelerated when the cytosolic Ca2+ buffer capacity was increased. In addition, both rapid replenishment and fast endocytosis were retarded when cortical F-actin cytoskeleton was disrupted with cytochalasin D. Finally, in permeabilized chromaffin cells stained with rhodamine-phalloidin, the cortical F-actin density was reduced when the Ca2+ concentration was increased in a range of 10-1000 nM. We conclude that low cytosolic Ca2+ concentrations, which favor cortical F-actin stabilization, allow the activation of a fast endocytosis mechanism linked to a rapid replenishment component of IRP.

Ca2+ -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 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.