Eva Alés

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the ‘kiss-and-run’ mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.

Galantamine prevents apoptosis induced by β-amyloid and thapsigargin: involvement of nicotinic acetylcholine receptors

Galantamine is currently used to treat Alzheimers disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimers disease.

Myosin II contributes to fusion pore expansion during exocytosis.

During exocytosis, the fusion pore expands to allow release of neurotransmitters and hormones to the extracellular space. To understand the process of synaptic transmission, it is of outstanding importance to know the properties of the fusion pore and how these properties affect the release process. Many proteins have been implicated in vesicle fusion; however, there is little evidence for proteins involved in fusion pore expansion. Myosin II has been shown to participate in the transport of vesicles and, surprisingly, in the final phases of exocytosis, affecting the kinetics of catecholamine release in adrenal chromaffin cells as measured by amperometry. Here, we have studied single vesicle exocytosis in chromaffin cells overexpressing an unphosphorylatable form (T18AS19A RLC-GFP) of myosin II that produces an inactive protein by patch amperometry. This method allows direct determination of fusion pore expansion by measuring its conductance, whereas the release of catecholamines is recorded simultaneously by amperometry. Here we demonstrated that the fusion pore is of critical importance to control the release of catecholamines during single vesicle secretion in chromaffin cells. We proved that myosin II acts as a molecular motor on the fusion pore expansion by hindering its dilation when it lacks the phosphorylation sites.

ERG K+ channel blockade enhances firing and epinephrine secretion in rat chromaffin cells: the missing link to LQT2-related sudden death?

The ether‐a‐go‐go‐related genes (erg) are expressed in tissues other than heart and brain, in which human erg (HERG) K+ channels are known to regulate the repolarization of heart action potentials and neuronal spike‐frequency accommodation. We provide evidence that erg1 transcripts and ERG proteins are present in rat chromaffin cells in which we could isolate a K+ current that was biophysically and pharmacologically similar to the ERG current. Firing frequency and catecholamine release were analyzed at the single‐cell level by means of perforated patch‐clamp and carbon fiber electrochemical detection. It was found that the blocking of ERG, KATP, and KCa channels led to hyperexcitability and an increase in catecholamine release. Combined immunocytochemical experiments with antibodies directed against phenylethanolamine N‐methyltransferase and ERG channels suggested expression of these channels in epinephrine‐ but not in norepinephrine‐containing cells. It is concluded that, in addition to being crucial in regulating the QT period in the heart, ERG channels play a role in modulating epinephrine, a fundamental neurotransmitter shaping cardiac function. This finding suggests that the sudden death phenotype associated with LQT2 syndrome mutations may be the result of an emotionally triggered increase in epinephrine in a long‐QT running heart.

Push-and-pull regulation of the fusion pore by synaptotagmin-7

In chromaffin cells, Ca2+ binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca2+ and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7–mutant chromaffin cells to analyze the role of Ca2+ binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca2+ binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca2+ binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.

A choline-evoked [Ca2+]c signal causes catecholamine release and hyperpolarization of chromaffin cells

In bovine chromaffin cells fast‐superfused with Krebs‐HEPES solution containing 1–2 mM Ca2+, 5 s pulses of choline (1–10 mM), elicited catecholamine secretory responses that were only ~10% of those evoked by ACh (0.01–0.1 mM). However, in high‐Ca2+ solutions (10–20 mM) the size of the choline secretory responses approached those of ACh. The choline responses (10 mM choline in 20 mM Ca2+, 10Cho/20Ca2+) tended to decline upon repetitive pulsing, whereas those of ACh were well maintained. The confocal [Ca2+]c increases evoked by 10Cho/20Ca2+ were similar to those of ACh. Whereas 10Cho/20Ca2+ caused mostly hyperpolarization of chromaffin cells, 0.1ACh/20 Ca2+ caused first depolarization and then hyperpolarization; in regular solutions (2 mM Ca2+), the hyperpolarizing responses did not show up. In Xenopus oocytes injected with mRNA for bovine α7 nicotinic receptors (nAChRs), 10Cho/20 Ca2+ fully activated an inward current; in oocytes expressing α3β4, however, the inward current elicited by choline amounted to only 4% of the size of α7 current. Our results suggest that choline activates the entry of Ca2+ through α7 nAChRs; this leads to a cytosolic concentration of calcium ([Ca2+]c) rise that causes the activation of nearby Ca2+‐dependent K+ channels and the hyperpolarization of the chromaffin cell. This response, which could be unmasked provided that cells were stimulated with high‐Ca2+ solutions, may be the underlying mechanism through which choline exerts a modulatory effect on the electrical activity of the chromaffin cell and on neurotransmitter release at cholinergic synapses.

A rapid exocytosis mode in chromaffin cells with a neuronal phenotype.

We have used astrocyte‐conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM‐promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre‐spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7–9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM‐treated cells correspond to the exocytosis of large dense‐core vesicles (LDCV). On the other hand, experiments performed in EGTA‐loaded cells suggest that ACM treatment promotes a better coupling between voltage‐gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.

Mechanisms of granule membrane recapture following exocytosis in intact mast cells

Background: When vesicles undergo exocytosis, the vesicle membrane must be retrieved by endocytosis to maintain a constant cell surface area. Results: Exocytosis in intact mast cells is followed by endocytosis. Conclusion: Mechanisms of granule membrane recapture in intact mast cells include kiss-and-run and “compound endocytosis.” Significance: Compound endocytosis may be a novel mechanism for efficiently compensating for the membrane excess caused by exocytosis. In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.

Depolarization evokes different patterns of calcium signals and exocytosis in bovine and mouse chromaffin cells: the role of mitochondria

This study was planned on the assumptions that different high‐voltage activated calcium channels and/or the ability of mitochondria to take up Ca2+ could be responsible for different cytosolic Ca2+ concentrations ([Ca2+]c) and catecholamine release responses in adrenal chromaffin cells of bovine and mouse species. Short K+ pulses (2–5 s, 70 mm K+) increased [Ca2+]c to a peak of about 1 µm; however, in bovine cells the decline was slower than in mouse cells. Secretory responses were faster in mouse but were otherwise quantitatively similar. Upon longer K+ applications (1 min), elevations of [Ca2+]c and secretion were prolonged in bovine cells; in contrast [Ca2+]c in mouse cells declined three‐fold faster and failed to sustain a continued secretion. Confocal [Ca2+]c imaging following a 50‐ms depolarizing pulse showed a similar Ca2+ entry, but a rate of [Ca2+]c increase and a maximum peak significantly higher in bovine cells; the rate of dissipation of the Ca2+ wave was faster in the mouse. The mitochondrial protonophore CCCP (2 µm) halved the K+‐evoked [Ca2+]c and secretory signals in mouse cells, but had little affect on bovine responses. We conclude that the relative densities of L (15% in bovine and 50% in mouse) and P/Q Ca2+ channels (50% in bovine and 15% in mouse) do not contribute to the observed differences; rather, the different intracellular distribution of Ca2+, which is strongly influenced by mitochondria, is responsible for a more sustained secretory response in bovine, and for a faster and more transient secretory response in mouse chromaffin cells. It seems that mitochondria near the plasmalemma sequester Ca2+ more rapidly and efficiently in the mouse than in the bovine chromaffin cell.

A New Role for Myosin II in Vesicle Fission

An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.