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Exocytosis begins with formation of a small fusion pore which then expands allowing rapid release of granular contents. We studied the influence of cytoplasmic free Ca2+ ([Ca2+]i) on the conductance of the initial pore and on the dynamics of subsequent expansion in horse eosinophils using the patch clamp technique. The mean initial conductance is approximately 200 pS independent of [Ca2+]i. This value is close to that previously found in beige mouse mast cells. The pore subsequently expands by 18 nS/s at [Ca2+]i10 nM, by 40 nS/s at [Ca2+]i = 1.5 microM and by 90 nS/s at [Ca2+]i = 10 microM. These results show that the structure of the initial fusion pore is independent of cytoplasmic Ca2+. However, the pore expansion is a Ca(2+)-dependent process modulating secretion at a step later than vesicle-plasma membrane fusion.

We measured capacitance changes in cell attached patches of human neutrophils using a high frequency lock-in method. With this technique the noise level is reduced to 0.025 fF such that capacitance steps of 0.1 fF are clearly detected corresponding to exo- and endocytosis of single 60 nm vesicles. It is thus possible to detect almost all known exocytotic and endocytotic processes including exocytosis of small neurotransmitter containing vesicles in most cell types as well as endocytosis of coated and uncoated pits. In neutrophils we demonstrate a stepwise capacitance decrease generated by 60-165 nm vesicles as expected for endocytosis of coated and non-coated pits. Following ionomycin stimulation a stepwise capacitance increase is observed consisting of 0.1-5 fF steps corresponding to the different granule types of human neutrophils from secretory vesicles to azurophil granules. The opening of individual fusion pores is resolved during exocytosis of 200 nm vesicles. The initial conductance has a mean value of 150 pS and can be as low as 35 pS which is similar to the conductance of many ion channels suggesting that the initial fusion pore is formed by a protein complex.

1. We have investigated the granule size distributions in human and horse eosinophils by time-resolved patch-clamp capacitance measurements. 2. During exocytosis of single granules the electrical capacitance of the plasma membrane increases in discrete steps. The steps in horse cells are about six times larger than those in human cells in accordance with the difference in granule size. 3. In both species a multimodal capacitance step size distribution is observed with a first peak at 6-7 fF corresponding to granules with a diameter of about 450-500 nm and a surface area of about 0.7 microns2, which we call the unit granule. The other peaks in the distributions correspond to multiples of the surface area of these units. 4. These results show that the larger granules are formed by fusion of several unit granules and the final size of mature granules is determined by the number of units allowed to fuse with each other. Whereas in human eosinophils most granules consist of one or two units, most granules of horse eosinophils are formed by fusion of seven to fifteen units. 5. The intracellular fusion events associated with vesicular traffic are believed to occur constitutively. In contrast, our results indicate that a cellular mechanism exists which regulates the size of the mature granules by determining the number of units allowed to fuse with each other. In view of our recent report that granule-granule fusion can be activated by GTP gamma S, this regulation may possibly involve GTP-binding proteins.

In rat peritoneal mast cells, we have investigated the influence of the chloride transport blocker 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and the extracellular chloride concentration on the chloride current induced by intracellular application of cyclic AMP (cAMP) and on hexosaminidase secretion from intact cells stimulated with compound 48/80. The inhibition of the Cl-current by extracellular DIDS is voltage and time dependent. Upon depolarization from -10 to +70 mV, the outward current diminishes with millisecond kinetics. The size of the steady state current and the time constant of the decrease both decrease with increasing DIDS concentrations. The steady state current at +70 mV is blocked by DIDS with an IC50 of 2.3 microM. The number of open channels at -10 mV is reduced with an IC50 of 22 microM. The electrophysiological and pharmacological properties of this current are most similar to those of the Cl- current in T lymphocytes activated by osmotic stress (Lewis, R. S., P. E. Ross, and M. D. Cahalan. 1993. Journal of General Physiology. 101:801-826). Extracellular DIDS also inhibits exocytosis. At optimal stimulation with 10 micrograms/ml compound 48/80 secretion is inhibited with an IC50 = 50 microM and a Hill coefficient n = 10. At half optimal stimulation with 1 microgram/ml inhibition occurs with an IC50 = 10 microM and n = 1. Substitution of extracellular chloride by glutamate has only very small effects on secretion stimulated with 10 micrograms/ml compound 48/80. We conclude that activation of the chloride current in mast cells is not essential for stimulation of exocytosis but may enhance secretion at suboptimal stimulation. Alternatively, the channel may play a role in volume regulation following degranulation.

The killing of metazoan parasitic larvae by eosinophils occurs following cell adhesion and the secretion o f their cytotoxic proteins onto the surface o f these targets. In eosinophils, as in mast cells and neutrophils, stimulus-secretion coupling is mediated by GTP-binding proteins. In this article, Susanne Scepek Redwon Moqbel and Manfred Lindou summarize recent results indicating that the granule-fusion events activated by GTP-binding proteins lead to compound exocytosis and cumulative fusion. They propose that these exocytotic processes, following contact with opsonized larvae, may direct secretion to a restricted space defined by the site o f contact Such a focused release may be essential for effective targeting in parasite killing, thus preventing uncontrolled random diffusion of the secreted cytotoxic proteins with the possible undesirable consequences of damage to intact host tissue.

We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion occurs inside the cell forming large compound granules, which then fuse with the plasma membrane (compound exocytosis). The electrical equivalent circuit of the cell during degranulation indicates the formation of a degranulation sac by cumulative fusion events. Fusion of the first granule with the plasma membrane induces fusion of further granules with this granule directing the release of all the granular material to the first fusion pore. The physiological function of eosinophils is the killing of parasites. Compound exocytosis and cumulative fusion enable the cells to focus the release of cytotoxic proteins to well defined target regions and prevent uncontrolled diffusion of this material, which would damage intact host cells.

Intracellular calcium ([Ca2+]i) and exocytosis of human neutrophils were investigated with patch-clamp capacitance and Fura-2 fluorescence measurements. Intracellular application of GTP gamma S induces a calcium transient and exocytosis. The onset of degranulation occurs at the time where the maximal [Ca2+]i is reached. Despite the close correlation in time, buffering [Ca2+]i at the resting level or at approximately 2 microM leaves the extent and the time course of degranulation unchanged. The decay of the calcium transient is due to diffusional equilibration between the cytosol and the pipette volume. GTP gamma S activates no cellular mechanisms for Ca2+ reuptake or extrusion. The endogenous calcium buffer capacity can be estimated to be as low as that of approximately 90 microM Fura-2. Stimulation with fMLP also induces degranulation and a calcium transient. The decay of fMLP-induced calcium transients is much faster than that of GTP gamma S-induced transients and is independent of diffusion indicating that fMLP also induces rapid reuptake or extrusion of Ca2+. Degranulation but not the calcium transient requires the presence of intracellular GTP. Different signalling pathways appear to be involved in GTP gamma S- and fMLP-stimulated calcium signals. The intracellular calcium release is not an essential signal to initiate exocytosis in neutrophils.

We have investigated the granule fusion events associated with exocytosis in degranulating peritoneal guinea pig eosinophils by time-resolved patch-clamp capacitance measurements using the phase detector technique. Intracellular stimulation with micromolar calcium and GTP gamma S induces a 2- to 3-fold capacitance increase. The main phase of the capacitance increase occurs after a delay of 2–7 minutes and is composed of well-resolved capacitance steps. The number of steps is very close to the number of crystalloid granules contained in a resting cell and the step size distribution with a peak at 9 fF is in excellent agreement with the granule size distribution determined by electron microscopy. The individual granules thus fuse sequentially with the plasma membrane. The stepwise capacitance increase is frequently preceded by an apparently continuous capacitance increase which consists of steps smaller than 4 fF, indicating exocytosis of small vesicles as distinct from crystalloid-containing granules. In some cases the time course of the opening of individual fusion pores could be recorded, and this revealed metastable conductance states below 300 pS but random fluctuations at higher conductance levels. This behaviour suggests that the small fusion pore might be a protein structure similar to an ion channel, which becomes a continuously variable lipid pore at higher conductances. In some cells a significant capacitance decrease was observed which is apparently continuous, suggesting a process of membrane uptake by endocytosis of small vesicles.

Stimulated exocytotic secretion from nerve endings is initiated by an increase in intracellular free calcium concentration. We summarize here our latest findings regarding the temporal relationship between depolarization, elevation of [Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve endings. In addition, we present surprising findings for a regulatory role of intracellular Na+ on exocytosis.

In vivo electrochemistry has become a fascinating research tool allowing neuroscientists to study the release of oxidizable neurotransmitters, such as dopamine and norepinephrine in the brain of freely moving animals (Garris et al., 1997, J. Neurochem., 68(1): 152–161). The main limitation of this technique is the wired connection from the working electrode at the animal's head to the data acquisition apparatus, thus restricting the animal's freedom of motion. To overcome this limitation, we are designing an electronic device with the capability of performing fast-scan cyclic voltammetry measurements and wirelessly transmitting the recorded data. The device consists of two parts: the base station, which is connected to a PC, and the remote unit, which the rat carries on its back. The base station can wirelessly transmit the potential waveform applied to the working electrode, using the Advanced Audio Distribution Profile (A2DP) protocol. At the remote unit, a capacitance compensation circuit partially removes the capacitive background current present in voltammetric measurements due to charging of the Debye double layer. This increases the device's dynamic range, allowing for the detection of lower neurotransmitter levels. Although the forward telemetry (PC to remote unit) is functional, we have not yet characterized the reverse telemetry (remote unit to PC) in A2DP format. After finalizing the design, the device will be tested in vivo and subsequently employed in behavioral experiments, allowing researchers to obtain data from freely behaving rodents.

The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.

Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (tau)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.

Two methods are described for estimation of passive cell parameters such as membrane capacitance, membrane conductance and access resistance in tight-seal whole cell recording. Both methods are restricted in their application to cases where the cell under study can be approximated by a simple three-component network with linear properties over some voltage range. One method, referred to as the time domain technique, requires only standard electrophysiological equipment and a computer. Parameters are derived from an analysis of capacitive transients during square wave stimulation. It is readily adaptable to wide variations in experimental parameters. Particularly, it is equally applicable to the "slow whole-cell" configuration (access resistance in the range 100 M omega to 1 G omega) and to normal whole-cell measurements (access resistance typically 10 M omega). The other method applies a sine wave command signal to the cell and employs a lock-in amplifier to analyse the resulting current signal. Two modes of operating the lock-in amplifier are described. One mode provides an output signal directly proportional to small changes in capacitance at maximum resolution (1-10 fF). The other mode, in conjunction with a digital computer, supplies estimates of all passive cell parameters, as does the time domain technique, but with a large amount of data reduction performed by the lock-in amplifier itself. Due to the special hardware, however, this method is not as flexible as the time domain technique.

Synaptobrevin 2 (Syb2), syntaxin (Sx1A), and SNAP-25, generate a force to induce fusion pore formation. The v-SNARE, Syb2, is anchored to the vesicle membrane by a single transmembrane domain. Here we show that 2 tryptophans (W89/W90) located in the juxtamembrane domain of Syb2, which stabilize the transmembrane (TM) domain position, control the ratio of spontaneous vs. stimulated membrane fusion events in chromaffin cells. Changing the 2 hydrophobic tryptophans to neutral alanines promotes spontaneous membrane fusion, faster transmitter release kinetics and complete release from individual vesicles. The results indicate that the two tryptophans act as a fusion clamp making fusion stimulus-dependent.

We have investigated the temporal relationship between depolarization, elevation of [Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve terminals. The change of [Ca2+]i and vasopressin release were measured with a time resolution of less than 1 s in response to K(+)-induced depolarization. Exocytosis was also monitored in the whole-terminal patch-clamp configuration by time resolved capacitance measurements while [Ca2+]i was simultaneously followed by fura-2 fluorescence measurements. In intact as well as patch-clamped nerve terminals sustained depolarization leads to a sustained rise of [Ca2+]i. The rate of vasopressin release from intact nerve terminals rises in parallel with [Ca2+]i but then declines rapidly towards basal (t1/2 approximately 15 s) despite the maintained high [Ca2+]i indicating that only a limited number of exocytotic vesicles can be released. We demonstrate that in nerve terminals exocytosis can be followed during step depolarization by capacitance measurements. The capacitance increase starts instantaneously whereas [Ca2+]i rises with a half time of several hundred milliseconds. An instantaneous steep capacitance increase is followed by a slow increase with a slope of 25–50 fF/s indicating the sequential fusion of predocked and cytoplasmic vesicles. During depolarization the capacitance slope declines to zero with a similar time course as the vasopressin release indicating a decrease in exocytotic activity. Depolarization per se in the absence of a sufficient rise of [Ca2+]i does not induce exocytosis but elevation of [Ca2+]i in the absence of depolarization is as effective as in its presence. The experiments suggest that a rapid rise of [Ca2+]i in a narrow region beneath the plasma membrane induces a burst of exocytotic activity preceding the elevation of bulk [Ca2+]i in the whole nerve terminal.

The secretory process requires many different steps and stages. Vesicles must be formed and transported to the target membrane. They must be tethered or docked at the appropriate sites and must be prepared for fusion (priming). As the last step, a fusion pore is formed and the contents are released. Release of neurotransmitter is an extremely rapid event leading to rise times of the postsynaptic response of less than 100 μs. The release thus occurs during the initial formation of the exocytotic fusion pore. To understand the process of synaptic transmission, it is thus of outstanding importance to understand the molecular structure of the fusion pore, what are the properties of the initial fusion pore, how these properties affect the release process and what other factors may be limiting the kinetics of release. Here we review the techniques currently employed in fusion pore studies and discuss recent data and opinions on exocytotic fusion pore properties.

Exocytosis was studied in single rat peritoneal mast cells. Granule fusion was monitored by time-resolved capacitance measurements using the patch-clamp technique. Intracellular stimulation of mast cells with 20 microM GTP-gamma-S stimulates exocytosis with a calcium-dependent time course. Secretion in response to receptor-mediated stimulation with compound 48/80 was completely abolished by treatment with pertussis toxin (IAP) at 180 ng/ml for 4 h. The time course of exocytosis in response to GTP-gamma-S remained unaffected in IAP-treated cells supporting the involvement of a second GTP-binding protein in stimulus-secretion coupling.

We have investigated the dynamics of exocytosis in single human neutrophils. The increase of membrane area associated with granule fusion was followed by time-resolved patch-clamp capacitance measurements. Intracellular application of 20 microM guanosine-5'-O(3-thiotriphosphate) (GTP gamma S) in the presence of 2.5 mM ATP stimulated exocytosis and led to an increase of membrane capacitance from 3.0 to integral of 8.4 pF corresponding to a 540 micron 2 increase of membrane area. This capacitance change is very close to the value expected from morphological data if all primary and secondary granules fuse with the plasma membrane. High resolution measurements revealed stepwise capacitance changes corresponding to the fusion of individual granules. GTP gamma S-stimulated exocytosis did not require pretreatment with cytochalasin B and the amplitude was independent of the intracellular-free calcium concentration between 10 nM and integral of 2.5 microM. In the absence of GTP gamma S elevation of intracellular-free calcium concentration to the micromolar range led to the fusion of only a limited number of granules. Degranulation stimulated with GTP gamma S started after a lag phase of 2-7 min and was usually complete within 5-20 min. The time course was affected by the intracellular ATP and calcium concentration. Exocytosis was markedly accelerated by pretreatment with cytochalasin B. Our results demonstrate that the final steps leading to primary and secondary granule fusion are controlled by a guanine nucleotide-binding protein and do not require an elevation of intracellular calcium. Calcium and other factors are, however, involved in the regulation having pronounced effects on the dynamics of exocytosis.

Exocytosis was followed in single rat peritoneal mast cells, by measuring the cell membrane capacitance using circuit analysis and patch-clamp techniques. After antigenic stimulation or intracellular perfusion with guanine nucleotides, exocytosis followed a time course characterized by a lag period d, area expansion factor A, and a time constant τ. We suggest that A depends entirely on the cell's morphology, d reflects the properties of a GTP-binding regulatory protein that appears to rate limit the response and τ is due to an independent and yet unknown process. In contrast, cells stimulated by compound 48/80 can respond without a measurable delay and degranulate within 2 s, suggesting that this compound acts at a site after the GTP-binding regulatory protein.

The number of transmitter molecules released in a quantal event can be regulated, and recent studies suggest that the modulation of quantal size is associated with corresponding changes in vesicle volume (Colliver et al., 2000; Pothos et al., 2002). If so, this could occur either by distension of the vesicle membrane or by incorporation and removal of vesicle membrane. We performed simultaneous measurements of vesicle membrane area and catecholamine release in individual quantal events from chromaffin cells using cell-attached patch amperometry. Cells were treated with reserpine, a vesicular monoamine transport blocker that decreases quantal size, orl-dopa, a catecholamine precursor that increases quantal size. We show that decrease and increase in quantal size are associated with a respective decrease and increase in vesicle membrane area. These results point to a novel mechanism of vesicle membrane dynamics by which vesicles physically change their membrane area in response to changes in transmitter content such that the intravesicular concentration of transmitter is maintained.

In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at

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.

The molecular mechanisms behind the tethering of secretory vesicles to the plasma membrane are not well understood. To study these mechanisms, we use a combined AFM/TIRF method applying forces to GFP-tagged secretory granules tethered to PC-12 cell membrane sheets. In the experiment shown in the figure, the AFM tip captured a granule, and was then lowered onto the sheet (a-b). Pulling forces were applied, ranging from ∼100pN to ∼700pN (c-e). Extension and rupture events are apparent as transient variations in the pulling force, as well as increases in the scanner z position sensor. Change in sensor position is consistent with changes in vesicle height as observed in the TIRF, based on the calibrated excitation decay constant (c-d). Rapid transients with small amplitude reveal partial tether extensions by 10-20nm, while large transients with slow decay reveal more gradual extensions, exceeding 1μm, that appear to be composed of multiple steps. These observations suggest that the dynamics of vesicle tether extensions are much more complex than previously thought. A large data set of recordings like that in the figure is being analyzed. Supported by NIH grant R21NS072577.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Coarse grain molecular dynamics simulations have become an important tool to study the membrane insertion and dynamic behavior of membrane proteins in lipid membranes. To generate a physiological membrane composition we used the lipid and cholesterol composition approximating that of synaptic vesicles (Takamori et al. 2006 Cell 127:831-46). Since the synaptic vesicle is a recycling organelle, its leaflet asymmetry is expected to reflect that of plasma membranes with the cytoplasmic leaflet mainly composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) and the extracellular (or intravesicular) leaflet mainly composed of phosphatidycholine (PC) and sphingomyelin (SM). Cholesterol also is distributed asymmetrically with 60-70% located in the cytoplasmic leaflet (Mondal et al. 2009 Mol.Biol.Cell 20:581–8). To generate a corresponding coarse grain membrane model we used GROMACS 4 and the MARTINI lipids POPE, POPC, POPS, PPCS (for SM), and CHOL. A 16x16x5 nm3 simulation box was generated and filled with the components of the intravesicular (IV) leaflet (36 PPCS, 180 POPC, 144 CHOL) and another simulation box with the same dimensions with those of the cytoplasmic (CP) leaflet (64 POPS, 210 POPE, 256 CHOL). The CP box was placed on top of the IV box such that there was an overlap of 0.5nm. Sterical overlaps between molecules were removed by running an energy minmization. After addition of water particles as needed, the simulations led reproducibly to self-assembly of a membrane retaining the essential asymmetric leaflet features of the membrane. About ∼82% of the IV lipids were retained in the IV leaflet and ∼70% of the CP lipids remained in the CP leaflet. Cholesterol partitioned equally between the two leaflets. Self-assembly also occurred reproducibly when a membrane protein was included. Supported by NIH grants R01GM085808, R21-NS072577, and the Wellcome Trust.