Regulation of the fusion pore conductance during exocytosis by cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase involved in synaptogenesis and brain development, and its enzymatic activity is essential for slow forms of synaptic vesicle endocytosis. Recent work also has implicated Cdk5 in exocytosis and synaptic plasticity. Pharmacological inhibition of Cdk5 modifies secretion in neuroendocrine cells, synaptosomes, and brain slices; however, the specific mechanisms involved remain unclear. Here we demonstrate that dominant-negative inhibition of Cdk5 increases quantal size and broadens the kinetics of individual exocytotic events measured by amperometry in adrenal chromaffin cells. Conversely, Cdk5 overexpression narrows the kinetics of fusion, consistent with an increase in the extent of kiss-and-run exocytosis. Cdk5 inhibition also increases the total charge and current of catecholamine released during the amperometric foot, representing a modification of the conductance of the initial fusion pore connecting the granule and plasma membrane. We suggest that these effects are not attributable to an alteration in catecholamine content of secretory granules and therefore represent an effect on the fusion mechanism itself. Finally, mutational silencing of the Cdk5 phosphorylation site in Munc18, an essential protein of the late stages of vesicle fusion, has identical effects on amperometric spikes as dominant-negative Cdk5 but does not affect the amperometric feet. Cells expressing Munc18 T574A have increased quantal size and broader kinetics of fusion. These results suggest that Cdk5 could, in part, control the kinetics of exocytosis through phosphorylation of Munc18, but Cdk5 also must have Munc18-independent effects that modify fusion pore conductance, which may underlie a role of Cdk5 in synaptic plasticity.     

Correlation between vesicle quantal size and fusion pore release in chromaffin cell exocytosis

A significant number of exocytosis events recorded with amperometry demonstrate a prespike feature termed a "foot" and this foot has been correlated with messengers released via a transitory fusion pore before full exocytosis. We have compared amperometric spikes with a foot with spikes without a foot at chromaffin cells and found that the probability of detecting a distinct foot event is correlated to the amount of catecholamine released. The mean charge of the spikes with a foot was found to be twice that of the spikes without a foot, and the frequency of spikes displaying a foot was zero for small spikes increasing to approximately 50% for large spikes. It is hypothesized that in chromaffin cells, where the dense core is believed to nearly fill the vesicle, the expanding core is a controlling factor in opening the fusion pore, that prefusion of two smaller vesicles leads to excess membrane, and that this slows pore expansion leading to an increased observation of events with a foot. Clearly, the physicochemical properties of vesicles are key factors in the control of the dynamics of release through the fusion pore and the high and variable frequency of this release makes it highly significant.     

Temporal separation of vesicle release from vesicle fusion during exocytosis

During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca2+-dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To determine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules released and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber microelectrodes. In external solutions of high osmolarity, release events following entry of divalent ions (Ba2+ or Ca2+) were less frequent. However, the vesicles appeared to be fused to the membrane without extruding their contents, since the maximal observed concentrations of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term "kiss-and-hold," was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demonstrate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation.     

Quantitative investigations of amperometric spike feet suggest different controlling factors of the fusion pore in exocytosis at chromaffin cells

Around 30% of exocytosis events recorded by amperometry at carbon fiber microelectrodes exhibit a pre-spike feature (PSF) termed a “foot”. This wave is associated with the release of the neurotransmitters via a transitory fusion pore, whilst the large, main exocytotic spike is due to complete release. The amperometric data reported herein were obtained using bovine chromaffin cells stimulated with either potassium or barium ions, two commonly-employed elicitors of exocytosis. Identical trends are observed with both activators: (i) they induce the same ratio (close to 30%) of events with a foot in the population of amperometric spikes, and (ii) spikes with a foot can be divided into two primary categories, depending on the temporal variation of the current wave (viz. as a ramp, or a ramp followed by a plateau). Correlations between the characteristics of the whole current spike, and of its observed foot, have been sought; such analyses demonstrate that the maximum current of both foot and spike signals are highly correlated, but, in contrast, the integrated charges of both are poorly correlated. Moreover, the temporal duration of the PSF is fully uncorrelated with any parameter pertaining to the main current spike. On the basis of these reproducible observations, it is hypothesized that the characteristics (dimensions and topology, at least) of each secretory vesicle determine the probability of formation of the fusion pore and its maximum size, whilst molecular factors of the cell membrane control its duration, and, consequently, the amount delivered prior to the massive exocytosis of catecholamines observed as a spike in amperometry.     

Granule matrix property and rapid "kiss-and-run" exocytosis contribute to the different kinetics of catecholamine release from carotid glomus and adrenal chromaffin cells at matched quantal size

Catecholamine-containing small dense core granules (SDCGs, vesicular diameter of ~100 nm) are prominent in carotid glomus (chemosensory) cells and some neurons, but the release kinetics from individual SDCGs has not been studied in detail. In this study, we compared the amperometric signals from glomus cells with those from adrenal chromaffin cells, which also secrete catecholamine but via large dense core granules (LDCGs, vesicular diameter of ~200-250 nm). When exocytosis was triggered by whole-cell dialysis (which raised the concentration of intracellular Ca(2+) ([Ca(2+)](i)) to ~0.5 μmol/L), the proportion of the type of signal that represents a flickering fusion pore was 9-fold higher for glomus cells. Yet, at the same range of quantal size (Q, the total amount of catecholamine that can be released from a granule), the kinetics of every phase of the amperometric spike signals from glomus cells was faster. Our data indicate that the last phenomenon involved at least 2 mechanisms: (i) the granule matrix of glomus cells can supply a higher concentration of free catecholamine during exocytosis; (ii) a modest elevation of [Ca(2+)](i) triggers a form of rapid "kiss-and-run" exocytosis, which is very prevalent among glomus SDCGs and leads to incomplete release of their catecholamine content (and underestimation of their Q value).     

Simultaneous capacitance and amperometric measurements of exocytosis: a comparison.

We measured the exocytotic response induced by flash photolysis of caged compounds in isolated mast cells and chromaffin cells. Vesicle fusion was measured by monitoring the cell membrane capacitance. The release of vesicular contents was followed by amperometry. In response to a GTP gamma S stimulus we found that the time integral of the amperometric current could be superimposed on the capacitance trace. This shows that the integrated amperometric signal provides an alternative method of measuring the extent and kinetics of the secretory response. Very different results were obtained when photolysis of caged Ca2+ (DM-nitrophen) was used to stimulate secretion. In mast cells, there was an immediate, graded increase in membrane capacitance that was followed by step increases (indicative of granule fusion). During the initial phase of the capacitance increases, no release of oxidizable secretory products was detected. In chromaffin cells we also observed a considerable delay between increases in capacitance, triggered by uncaging Ca2+, and the release of oxidizable secretory products. Here we demonstrate that there can be large increases in the membrane capacitance of a secretory cell, triggered by flash photolysis of DM-nitrophen, which indicate events that are not due to the fusion of granules containing oxidizable substances. These results show that increases in capacitance that are not resolved as steps cannot be readily interpreted as secretory events unless they are confirmed independently.     

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.     

Regulation of exocytosis by protein kinases and Ca(2+) in pancreatic duct epithelial cells

We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca(2+) using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca(2+) was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca(2+), PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.     

Amisyn regulates exocytosis and fusion pore stability by both syntaxin-dependent and syntaxin-independent mechanisms

Amisyn and tomosyn are related by the possession of a C-terminal vesicle-associated membrane protein-like domain that allows them to bind to syntaxin 1 and assemble into SNARE complexes. The formation of inactive complexes may sequester syntaxin and allow tomosyn and amisyn to act as inhibitors of exocytosis. We aimed to use adrenal chromaffin and PC12 cells to probe this possible mode of action of amisyn and tomosyn in dense core granule exocytosis. Although tomosyn is expressed by adrenal chromaffin and PC12 cells, amisyn expression could not be detected allowing examination of the effect of introduction of amisyn expression onto a neuronal-like background. Overexpression of m-tomosyn1 and expression of amisyn both inhibited Ca2+-induced exocytosis in transfected PC12 cells. Surprisingly, this inhibition was not removed when amisyn and tomosyn constructs were used in which key residues required for efficient binding to syntaxin1 were mutated. The effect of amisyn was further characterized using carbon fiber amperometry in chromaffin cells. Expression of amisyn had no effect on the basic characteristics of the amperometric spikes but reduced the number of spikes elicited. This inhibitory action on the extent of exocytosis was also seen with the amisyn mutant deficient in syntaxin1 binding. In addition, expression of amisyn resulted in an increase in the lifetime of the prespike foot, and this effect was abolished by the mutations. These results show that tomosyn and amisyn can negatively regulate exocytosis independently of syntaxin and also that amisyn can regulate the stability of the fusion pore.     

New roles of myosin II during vesicle transport and fusion in chromaffin cells

Modified herpes virus (amplicons) were used to express myosin regulatory light chain (RLC) chimeras with green fluorescent protein (GFP) in cultured bovine chromaffin cells to study myosin II implication in secretion. After infection, RLC-GFP constructs were clearly identified in the cytoplasm and accumulated in the cortical region, forming a complex network that co-localized with cortical F-actin. Cells expressing wild type RLC-GFP maintained normal vesicle mobility, whereas cells expressing an unphosphorylatable form (T18A/S19A RLC-GFP) presented severe restrictions in granule movement as measured by individual tracking in dynamic confocal microscopy studies. Interestingly, the overexpression of this mutant form of RLC also affected the initial secretory burst elicited by either high K(+) or BaCl(2), as well as the secretion induced by fast release of calcium from caged compounds in individual cells. Moreover, T18A/S19A RLC-GFP-infected cells presented slower fusion kinetics of individual granules compared with controls as measured by analysis of amperometric spikes. Taken together, our results demonstrate the implication of myosin II in the transport of vesicles, and, surprisingly, in the final phases of exocytosis involving transitions affecting the activity of docked granules, and therefore uncovering a new role for this cytoskeletal element.     

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.     

Brefeldin A increases the quantal size and alters the kinetics of catecholamine release from rat adrenal chromaffin cells.

The fungal metabolite, brefeldin A (BFA), is known to inhibit guanine nucleotide exchange on the ADP-ribosylating factors that are involved in vesicle membrane trafficking. Here, we investigated the action of BFA on Ca2+-regulated exocytosis in single rat adrenal chromaffin cells. Incubation of chromaffin cells with BFA (1 or 10 microM) for 2 h effectively disrupted the Golgi membranes but did not affect the pattern of catecholamine release triggered by high extracellular K+, which was monitored with carbon fiber amperometry along with cytosolic Ca2+ measurement. The BFA treatment, however, increased the mean quantal size of catecholamine-containing vesicles and the occurrence of amperometric events with a "foot" or "stand alone" signal (which reflects sluggish or incomplete dilation of the fusion pore). To examine whether BFA altered the Ca2+-dependence of exocytosis, we employed the whole-cell recording technique in conjunction with the capacitance measurement to measure exocytosis evoked from the entire cell during voltage-gated Ca2+ entry. Our results suggested that BFA treatment did not alter either the initial rate of capacitance increase or the total amount of capacitance increase. Therefore, in chromaffin cells, BFA treatment affects Ca2+-regulated exocytosis predominantly by increasing the quantal size and by slowing the fusion kinetics of some vesicles.     

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Under low stimulation, adrenal chromaffin cells release freely soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.     

Syntaxin/Munc18 interactions in the late events during vesicle fusion and release in exocytosis

The SNARE proteins, syntaxin, SNAP-25, and VAMP, form part of the core machinery for membrane fusion during regulated exocytosis. Additional proteins are required to account for the speed, spatial restriction, and tight control of exocytosis and a key role is played by members of the Sec1/Munc18 family of proteins that have been implicated either in vesicle docking or fusion itself through their interactions with the corresponding syntaxin. Using amperometry to assay the kinetics of single vesicle fusion/release events in adrenal chromaffin cells, the effect of expression of syntaxin 1A mutants was examined. Overexpression of wild-type syntaxin or its cytoplasmic domain had no effect on the kinetics of release during single exocytotic events although the cytoplasmic domain reduced the frequency of exocytosis. In contrast, expression of either an open syntaxin 1A or the I233A mutant resulted in increased quantal size and a slowing of the kinetics of release. The wild-type and mutant syntaxins were overexpressed to a similar extent and the only common defect shown by the syntaxin 1A mutants was reduced binding to Munc18-1. These results are consistent with a role for Munc18-1 in controlling the late stages of exocytosis by binding to and limiting the availability of syntaxin in its open conformation. Modification of the Munc18-1/syntaxin 1A interaction would therefore be a key mechanism for the regulation of quantal size.     

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.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Analysis of exocytotic events recorded by amperometry

Amperometry is widely used to study exocytosis of neurotransmitters and hormones in various cell types. Analysis of the shape of the amperometric spikes that originate from the oxidation of monoamine molecules released during the fusion of individual secretory vesicles provides information about molecular steps involved in stimulation-dependent transmitter release. Here we present an overview of the methodology of amperometric signal processing, including (i) amperometric signal acquisition and filtering, (ii) detection of exocytotic events and determining spike shape characteristics, and (iii) data manipulation and statistical analysis. The purpose of this review is to provide practical guidelines for performing amperometric recordings of exocytotic activity and interpreting the results based on shape characteristics of individual release events.     

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.     

Modifications in the C terminus of the synaptosome-associated protein of 25 kDa (SNAP-25) and in the complementary region of synaptobrevin affect the final steps of exocytosis.

Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis.