The astrocyte excitability brief: from receptors to gliotransmission

Astrocytes do not merely serve as the supporting cast and scenery against which starring roles would be played by neurons. Rather, these glial cells are intimately involved in many of the brain's functions by responding to neuronal activity and modulating it. Such interplay between two principle neural cells, neurons and astrocytes, is evidenced in bi-directional glutamatergic astrocyte-neuron signaling. A key feature in this signaling pathway is astrocytic excitability based on variations of cytosolic Ca(2+). It enables astrocytes, through the activation of their glutamatergic receptors, to respond to the same signal used by nearby neurons in synaptic transmission. Furthermore, increases in cytosolic Ca(2+) in astrocytes can subsequently lead to Ca(2+)-dependent exocytotic secretion of gliotransmitter glutamate that in turn can signal to adjacent neurons. Astrocytic secretory machinery includes an assortment of exocytotic proteins which governs a merger of secretory vesicles to the plasma membrane. A cumulative knowledge on astrocytic excitability will aid better understanding of operating procedures in the brain in health and disease.     

Trafficking of astrocytic vesicles in hippocampal slices

The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.     

2-D DIGE analyses of enriched secretory lysosomes reveal heterogeneous profiles of functionally relevant proteins in leukemic and activated human NK cells

As part of the innate immune system, natural killer (NK) cells detect and lyse tumor and virus-infected cells without prior antigen-dependent recognition and expansion. To this end, they utilize dual-function organelles that combine properties of conventional lysosomes and exocytotic vesicles. Upon stimulation, these secretory lysosomes (SLs) release their cytotoxic molecules into the immunological synapse. In addition, several molecules associated with secretory vesicles become exposed on the plasma membrane. Recent studies often took advantage of the few established NK cell lines, for instance to analyze the exocytotic machinery associated with NK cell vesicles. NK cell lines and primary NK cells differ, however, substantially in the expression of "typical" surface receptors and their requirements to induce target cell lysis. Here, we directly compared the lysosomal compartments of different NK cell populations. We enriched SLs of two leukemic cell lines (YTS and NKL) and IL-2-expanded NK cells by subcellular fractionation and characterized their proteome by 2-D difference gel electrophoresis and MS. Although the overall protein composition of the lysosomal preparations was very similar and more than 90% of the proteins were present at comparable levels, we define a cell line-specific setup of functionally relevant proteins involved in antigen presentation and cytotoxic effector function.     

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.     

Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route

Astroglial cells, due to their passive electrical properties, were long considered subservient to neurons and to merely provide the framework and metabolic support of the brain. Although astrocytes do play such structural and housekeeping roles in the brain, these glial cells also contribute to the brain''s computational power and behavioural output. These more active functions are endowed by the Ca²⁺-based excitability displayed by astrocytes. An increase in cytosolic Ca²⁺ levels in astrocytes can lead to the release of signalling molecules, a process termed gliotransmission, via the process of regulated exocytosis. Dynamic components of astrocytic exocytosis include the vesicular-plasma membrane secretory machinery, as well as the vesicular traffic, which is governed not only by general cytoskeletal elements but also by astrocyte-specific IFs (intermediate filaments). Gliotransmitters released into the ECS (extracellular space) can exert their actions on neighbouring neurons, to modulate synaptic transmission and plasticity, and to affect behaviour by modulating the sleep homoeostat. Besides these novel physiological roles, astrocytic Ca²⁺ dynamics, Ca²⁺-dependent gliotransmission and astrocyte–neuron signalling have been also implicated in brain disorders, such as epilepsy. The aim of this review is to highlight the newer findings concerning Ca²⁺ signalling in astrocytes and exocytotic gliotransmission. For this we report on Ca²⁺ sources and sinks that are necessary and sufficient for regulating the exocytotic release of gliotransmitters and discuss secretory machinery, secretory vesicles and vesicle mobility regulation. Finally, we consider the exocytotic gliotransmission in the modulation of synaptic transmission and plasticity, as well as the astrocytic contribution to sleep behaviour and epilepsy.     

Improvement of metabolic competence of isolated nerve terminals by extracellular pyruvate

Neuronal activity is tightly coupled with brain energy metabolism. Numerous studies have proved that glucose is not a sole energy substrate for neurons; metabolic monocarboxylate intermediates derived from glucose (pyruvate and lactate) released by astrocytes are shown to be taken up and oxidized by neurons, and, moreover, could serve as neuroprotective agents. Herein, we presented the data that extracellular pyruvate (4 mM) in the presence of glucose caused the increase in synaptosomal ATP content from 3.48+/-0.30 to 4.38+/-0.23 nmol/mg of protein. This correlates with the enhanced accumulation of fluorescent dye acridine orange in the available and the recycling synaptic vesicles within the synaptosomes reflecting the improved generation of proton gradient through the synaptic vesicle membrane. We have also demonstrated the effect of extracellular pyruvate on distribution of [3H]GABA between synaptic vesicles and cytoplasm in loaded synaptosomes. To estimate [3H]GABA accumulation into the synaptic vesicles, Ca 2+-dependent 4-aminopyridine-triggered exocytotic neurotransmitter release was studied. Evaluation of cytosolic 1H]GABA pool was performed by measuring the Ca2+-independent transporter-mediated neurotransmitter release evoked by nipecotic acid or high K+. The presence of pyruvate resulted in doubled exocytotic release of [3H]GABA, and significantly attenuated Ca2+-independent release of cytosolic [3H]GABA. Together, these observations provide insight into the important role of glucose metabolic intermediate, pyruvate, in sustaining activity of vesicular inhibitory amino acid transporter and so normal inhibitory transmission. We propose to use pyruvate for keeping up synaptosomal preparations in state of metabolic stability.     

Calcium sensors in regulated exocytosis

Neurotransmitter release, hormone secretion and a variety of other secretory process are tightly regulated with exocytotic fusion of secretory vesicles being triggered by a rise in cytosolic Ca2+ concentration. A series of proteins that act as part of a conserved core machinery for vesicle docking and fusion throughout the cell have been identified. In regulated exocytosis this core machinery must be controlled by Ca(2+)-sensor proteins that allow rapid activation of the fusion process following elevation of cytosolic Ca2+ concentration. The properties of such Ca2+ sensors are known from physiological studies but their molecular identity remains to be unequivocally established. The multiple Ca(2+)-dependent steps in the exocytotic pathway suggest the likely involvement of several Ca(2+)-binding proteins with distinct properties. Functional evidence for the role of various Ca(2+)-binding proteins and their possible sites of action is accumulating but a definitive identification of the major Ca(2+)-sensor in the final step of Ca(2+)-triggered membrane fusion in different cell types awaits further analysis.     

Rate of protein transport in thyroid follicle cells of normal, thyroxine-treated and TSH-injected rats

The transport of labeled protein in thyroid follicles was studied with quantitative electron microscopic autoradiography in normal and T4-treated rats (2d) injected with 3H-leucine 1 to 6 h before perfusion fixation. During this time interval the total amount of labeled protein in either group was unchanged, although T4-treatment caused a reduction by about 30% of the amount of 3H-leucine incorporated into protein. The autoradiographic data were corrected for the effect of scatter of radioactivity. The relative amounts of labeled, exportable protein in the compartments Er-Golgi and exocytotic vesicles were then estimated. The half-lives of labeled, exportable protein in these compartments were calculated with non-linear regression analysis. In normal rats the half-life of labeled, exportable protein in ER-Golgi was 28 min and in the exocytotic vesicles 18 min. Inhibition of TSH-secretion by injection of thyroxine decreased the rate of protein transport through the follicle cell and increased the half-lives to 63 min (ER-Golgi) and 62 min (exocytotic vesicles). TSH given to thyroxine-treated rats 20 min or 1.5 h before fixation reduced the half-lives of labeled, exportable protein in ER-Golgi to 25 to 33 min and in exocytotic vesicles to 9 min. The findings indicate that TSH regulates the rate of intracellular protein transport in rat thyroid follicle cells at the exocytotic step as well as at an earlier step in the pathway of intracellular protein transport. The mechanism and exact location of the latter TSH regulated step is at present unknown.     

Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.     

Myosin Va transports dense core secretory vesicles in pancreatic MIN6 beta-cells

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic beta-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative-acting globular tail domain of MyoVa decreased by approximately 50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in beta-cells.     

Emerging Role of the Scaffolding Protein Dlg1 in Vesicle Trafficking

Discs large 1 (Dlg1) is a modular scaffolding protein implicated in the control of cell polarity through assembly of specific multiprotein complexes, including receptors, ion channels and signaling proteins, at specialized zones of the plasma membrane. Recent data have shown that in addition to these well‐known interaction partners, Dlg1 may also recruit components of the vesicle trafficking machinery either to the plasma membrane or to transport vesicles. Here, we discuss Dlg1 function in vesicle formation, targeting, tethering and fusion, in both the exocytotic and endocytotic pathways. These pathways contribute to cell functions as major and diverse as glutamatergic activity in the neurons, membrane homeostasis in Schwann cell myelination, insulin stimulation of glucose transport in adipocytes, or endothelial secretion of the hemostatic protein, von Willebrand factor (VWF).     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Sweeping model of dynamin activity. Visualization of coupling between exocytosis and endocytosis under an evanescent wave microscope with green fluorescent proteins

Vesicle recycling through exocytosis and endocytosis is mediated by a coordinated cascade of protein-protein interactions. Previously, exocytosis and endocytosis were studied separately so that the coupling between them was understood only indirectly. We focused on the coupling of these processes by observing the secretory vesicle marker synaptobrevin and the endocytotic vesicle marker dynamin I tagged with green and red fluorescent proteins under an evanescent wave microscope in pheochromocytoma cells. In control cells, many synaptobrevin-expressing vesicles were found as fluorescent spots near the plasma membrane. Upon electrical stimulation, many of these vesicles showed an exocytotic response as a transient increase in fluorescence intensity followed by their disappearance. In contrast, fluorescent dynamin appeared as clusters increasing slowly in number upon stimulation. The clusters of fluorescent dynamin moved around beneath the plasma membrane for a significant distance. Simultaneous observations of green fluorescent dynamin and red fluorescent synaptobrevin indicated that more than 70% of the exocytotic responses of synaptobrevin had no immediate dynamin counterpart at the same site. From these findings it was concluded that dynamin-mediated recycling is not directly coupled to exocytosis but rather completed by a scanning movement of dynamin for the sites of invaginating membrane destined to endocytosis.     

Lateral drift correction in time-laps images by the particle-tracking algorithm

In long-term time-laps imaging of living cells, a significant lateral drift of the fluorescently labeled structures is often observed due to many reasons including superfusion of solution, temperature gradients, bolus addition of pharmacological agents and cell motility. We have detected lateral drift in long-term time-laps confocal imaging by tracking fluorescent puncta, which represent single exocytotic vesicles expressing synaptopHluorin (spH), a pH sensitive green fluorescence protein. Following the initial increase in fluorescence intensity due to alkalinization of vesicle lumen, the spH fluorescent puncta dimmed, which may be attributed to the resealing of the fusion pore and subsequent slow reacidification of the vesicle, or alternatively the dimming may be due to a significant lateral drift of the vesicle out of the region of interest (ROI). We identified and compensated the lateral drift by tracking particles present in the confocal images, without any additional mechanical and/or optical hardware components. The peak of the Gaussian two-dimensional (2D) curve fitted to the fluorescent particle intensity profile was recorded as the X and Y coordinates of the vesicle in each frame. The resulting coordinates of vesicle positions were averaged and rounded to the nearest pixel value, which was used to correct the drift in the time-laps images. In drift corrected time-laps images, the vesicle remained enclosed by the ROI, and the time dependent changes of spH fluorescence intensity averaged from the ROI remained at a constant level, revealing that endocytosis with subsequent slow reacidification of vesicles was an unlikely event.     

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.     

A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles

Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.     

Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and beta-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-alphaM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1-5 microm) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]d-aspartate and of endogenous glutamate in a Ca(2+)-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca(2+)-dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d-aspartate release in a concentration- (0.1-3 mm) and Ca(2+)-dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin-1, vesicular-associated membrane protein type 2 (VAMP-2), 23-kDa synaptosome-associated protein (SNAP-23) and 25-kDa synaptosome-associated protein (SNAP-25)] co-existing with GFAP immunoreactivity. Moreover, GFAP or VAMP-2 co-expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several approximately 30-nm non-clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate-accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.     

Characterization of sequential exocytosis in a human neuroendocrine cell line using evanescent wave microscopy and “virtual trajectory” analysis

Secretion of hormones and other bioactive substances is a fundamental process for virtually all multicellular organisms. Using total internal reflection fluorescence microscopy (TIRFM), we have studied the calcium-triggered exocytosis of single, fluorescently labeled large, dense core vesicles in the human neuroendocrine BON cell line. Three types of exocytotic events were observed: (1) simple fusions (disappearance of a fluorescent spot by rapid diffusion of the dye released to the extracellular space), (2) "orphan" fusions for which only rapid dye diffusion, but not the parent vesicle, could be detected, and (3) events with incomplete or multi-step disappearance of a fluorescent spot. Although all three types were reported previously, only the first case is clearly understood. Here, thanks to a combination of two-color imaging, variable angle TIRFM, and novel statistical analyses, we show that the latter two types of events are generated by the same basic mechanism, namely shape retention of fused vesicle ghosts which become targets for sequential fusions with deeper lying vesicles. Overall, approximately 25% of all exocytotic events occur via sequential fusion. Secondary vesicles, located 200-300 nm away from the cell membrane are as fusion ready as primary vesicles located very near the cell membrane. These findings call for a fundamental shift in current models of regulated secretion in endocrine cells. Previously, sequential fusion had been studied mainly using two-photon imaging. To the best of our knowledge, this work constitutes the first quantitative report on sequential fusion using TIRFM, despite its long running and widespread use in studies of secretory mechanisms.     

Local electrostatic interactions determine the diameter of fusion pores

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.     

Local electrostatic interactions determine the diameter of fusion pores

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.