1 Department of Psychiatry, University of Erlangen, Schwabachanlage 6, 91054 Erlangen, Germany
2 Department of Physiology and Neuroscience, New York University Medical Center, MSB 442, 550 First Avenue, NY 10016, USA
Author for correspondence (e-mail: andreas.henkel{at}psych.imed.uni-erlangen.de)
Accepted September 10, 2001
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SUMMARY |
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Key words: Exocytosis, Kiss-and-run, Staurosporine
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INTRODUCTION |
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In the case of exocytosis, the granular membrane has to be recycled in order to avoid infinite growth of the plasma membrane (Smith and Neher, 1997). A major pathway for synaptic vesicle recycling at the frog neuromuscular junction is endocytosis by clathrin-coated vesicles (De Camilli, 1995; Heuser and Reese, 1973). This concept was supported by several groups, presenting biochemical (Maycox et al., 1992; Südhof and Jahn, 1991; Wigge and McMahon, 1998) and morphological evidence (Takei et al., 1995). A different model was first proposed by Ceccarelli and co-workers (Ceccarelli et al., 1973) and was further developed by Meldolesis group (Meldolesi, 1998). This alternative kiss-and-run mechanism requires the formation of a transient pore, a channel-like protein complex, that mediates the passage of transmitters into the extracellular space. During the secretion process, the vesicle maintains its integrity without collapsing into the plasma membrane. However, the evidence for this pathway was indirect (Artalejo et al., 1995; Betz and Angleson, 1998; Haller et al., 1998; Henkel and Betz, 1995; Klingauf et al., 1998) until the high resolution on-cell capacitance measurement technique was introduced (Henkel and Almers, 1996; Zupancic et al., 1994). This method enabled direct qualitative and quantitative analysis of vesicle turnover with sufficient temporal and spatial resolution. It was shown that a pore transiently connects the lumen of the vesicle with the plasma membrane but prevents its fusion with the membrane (Alés et al., 1999; Henkel et al., 2000; Lollike et al., 1998). Nevertheless, classical exo- and endocytosis of single vesicles is also detected by on-cell capacitance measurements. Since both mechanisms can be observed in chromaffin cells, it is important to analyse how their ratio is controlled.
A common property of both mechanisms is the presence of an aqueous pore observed in the initial state of vesicle fusion and also during kiss-and-run type secretion (Albillos et al., 1997). The pore resembles an ion channel with regard to its conductance, and it was proposed that the pore consists of a ring of several protein subunits (Lindau and Almers, 1995; Lollike et al., 1995). Recently, in a fundamental study, fusion pore proteins were identified in yeast as the V0 sectors of the H+-ATPase, forming a trans-complex that fuses vacuolar membranes together (Peters et al., 2001).
In our study we demonstrate that phosphorylation regulates the mode of secretion by affecting the conductance and stability of the pore. We found evidence that classical exocytosis and kiss-and-run are not independent molecular mechanisms but represent rather different stages of a common secretion machinery.
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MATERIALS AND METHODS |
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STB was used in all stimulation experiments in bath and pipette. Additionally, the pipette contained 0.1 mM carbachol in some experiments as indicated. The kinase inhibitor staurosporine (Calbiochem) was applied to the cells by preincubation at a concentration of 2 µM at least 30 minutes before the experiments. Stimulation by temperature elevation was performed by slowly heating the bath on a temperature-controlled stage (LN-PCT-2, Luigs and Neumann) from room temperature (22°C-24°C) to physiological temperature (35°C).
Pipettes and patch clamp sealing
The patch pipettes (borosilicate glass, wall thickness 0.38 mm, outer diameter 1.5 mm, World Precision Instruments) were pulled with a micropipette puller (Flaming/Brown, Model P-97) and coated with dental wax (Type 2356200, Firma J. Schmalz, Heidelberg) by dipping the tip into molten wax. After fire-polishing, the pipettes had resistances between 1.8-2.5 M. Before immersing into the bath and during the approach towards the cell, a constant positive pressure of 10 cm water column was applied to the pipette tip. The pipette was pushed gently against the membrane of the cell, and the water pressure was suddenly reversed, which caused most cells to seal instantaneously. The pipette potential was kept at 70 mV d.c. until the seal had reached a resistance of more than 2 G
. Then it was switched to +20 mV. This potential was applied to the patch, keeping the patch potential 20 mV more negative than the cells resting potential.
On-cell capacitance measurements
The capacitance of the membrane patch was continuously monitored for 10 to 20 minutes. An EPC-7 patch clamp amplifier was connected to a SR830-DSP lock-in amplifier (Stanford Research Systems, Stanford, CA), which overlaid an eight kHz sine wave of 240 mV peak-to-peak voltage onto the holding potential. The lock-in amplifier analysed the resulting current that is phase-shifted with respect to the command voltage. This phase-shift relation can be expressed as the cells complex admittance, which has a real part (Re) that corresponds to the conductance and an imaginary part (Im) 90° out of phase. The imaginary part is equivalent to the capacitance of the patch. The real vesicle capacitance can be calculated from Im and Re as: Cvesicle=[(Re_+Im_) / Im] / and the pore conductance as: Gpore="(Re2 + Im2) / Re (Lollike and Lindau, 1999). The data were recorded at 100 Hz or 342 Hz via an A/D converter (Labmaster TL-1, Axon Instruments) into a 166 MHz Pentium computer running Windows95 operating system. The measuring system was described in detail earlier (Henkel et al., 2000). The recording (Labmaster Recorder 2.05) and the analysing (WinPCA 3.80) software were self-written. It is available on the internet at http://www.synosoft.de or by contacting A.W.H.
Capacitance step and flicker analysis
Analysis of solitary capacitance steps and flickers was performed by several semi-automated computer routines. The traces were at first scanned manually for upward capacitance steps. Step amplitudes were measured by fitting regression lines to the Im (capacitance) and the Re (conductance) traces over 150 milliseconds before and after the step and determining their vertical distance at the position of the step. Steps were accepted if they passed the following criteria:
1. The Im step amplitude was at least three times larger than the corresponding Re step amplitude.
2. The pre- and post step rms noise was less the 250 attofarad (aF).
3. The step amplitude was between 0.5 and 10 femtofarad (fF).
As it was found that steps occur isolated, in solitary flickers or during capacitance flicker bursts, we defined these three step categories:
1. Solitary step: no step in the opposite direction with approximately the same amplitude occurred either five seconds before or five seconds after the step.
2. Solitary flicker step: at least one downward step followed an upward step of approximately the same amplitude, and the two steps were not separated by more than five seconds. No time limit was applied if a persisting pore conductance was recorded between the two steps.
3. Burst step: any episode of at least two steps in opposite directions separated by at least four additional transitions. Five seconds was the maximum time given for successive transitions in order to qualify for definition as a burst step.
The step frequency was defined by the total step count of the respective category in a cell, divided by the recording time when the rms noise was less then 250 aF. These values were averaged for each specified stimulation condition.
Calcium and amperometry measurements
The intracellular calcium concentration was determined by Indo-1 acetoxymethyl ester (AM). Chromaffin cells grown on coverslips were loaded with Indo-1 (AM) prior to the experiments. For loading, the cells were incubated for 30 minutes at 37°C with 5 µM Indo-1 AM in SBM. The cells were transferred into Indo-free medium (SBM or STB) and allowed to adjust for at least 15 minutes to the appropriate temperature (23°C or 35°C) on a temperature-controlled microscope stage (LN-PCT-2, Luigs and Neumann). Then single cells were illuminated with 365 nm UV-light for 15 msec periods at a frequency of 1 Hz. Emission fluorescence was measured using two photomultipliers (Hamamatsu H3460) at 405 nm and 485 nm wavelengths. Digital signals were sampled into a AD-converter (Labmaster TL-1, Axon Instruments) into a MS-DOS PC, running self-written software (CapsRec 8.0) available on the internet at http://www.synosoft.de or by contacting A.W.H. Background fluorescence was collected from cell-free regions on the coverslip. The calibration procedure was described in detail earlier (Lee, 1996).
Amperometry measurements were performed as described in detail elsewhere (Chow and von Rüden, 1995). Briefly, 10-µm carbon fibres, insulated with polyethylene were cut before each experiment and positioned 1-3 µm apart from the membrane of a chromaffin cell. Catecholamines were oxidised at a fibre electrode potential of 800 mV and the resulting amperometric current was recorded by a modified EPC-7 amplifier.
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RESULTS |
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Fig. 1B shows that cells incubated with STB plus carbachol had a frequency of 3.1 mHz for solitary steps and 0.75 mHz for solitary flickers. Elevation of temperature from room temperature to 35°C in STB triggered steps and flickers in on-cell patches too. The cells showed an exocytic frequency of 2.9 mHz for solitary steps and 0.7 mHz for solitary flicker steps, values indistinguishable from the carbachol-stimulated cells.
The stimulative effect of STB at 35°C was confirmed by amperometry with a carbon fibre, close to the cells surface. The temperature elevation proved to be sufficient to trigger release of catecholamines without any additional chemical secretagouges. Fig. 2A shows the secretory response of a chromaffin cell stimulated with STB at 35°C. Five cells were tested by amperometry and all showed a sudden onset of catecholamine secretion that persisted as long as the temperature was above 32°C.
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DISCUSSION |
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High intracellular Ca2+ concentration and Ca2+ transients trigger steps and flickers
In order to facilitate intracellular enzymatic reactions and phosphorylation/dephosphorylation cycles for priming of the vesicles (Ashery et al., 2000; Heidelberger, 1998), the temperature was increased to physiological level in a subset of experiments. We showed that stimulation at 35°C raised the intracellular Ca2+ concentration almost four-fold and triggered spontaneous calcium transients, and these effects facilitated LDCV exocytosis. The capacitance-step frequency in stimulated chromaffin cells was 39-times higher than in unstimulated controls. Reduction of extracellular calcium to 156 nM completely prevented capacitance steps and calcium transients (data not shown).
The ratio between complete exocytosis and the kiss-and-run mechanism was about 4:1 in cells stimulated with carbachol at room temperatre (23°C). A similar ratio was reported in amperometric measurements of spike-like secretion events and stand-alone-foot secretion events of catecholamines in stimulated chromaffin cells (Zhou et al., 1996). Amperometric spikes rise from complete fusions of LDCVs, whereas stand-alone-foot events originate from the transient openings of flickering pores (Alvarez de Toledo et al., 1993). The ratio between the two secretion modes is also strongly dependent on the Ca2+ concentration in the patch pipette. De Toledos group reported that flickers triggered by higher Ca2+ concentrations had short mean open times of 0.4 seconds (Alés et al., 1999). This is in the same range as our data (0.55 seconds) from cells that were stimulated at 35°C. We found that staurosporine pre-treated cells had a subpopulation of flickers with mean open times of 0.25 seconds, and one other with 3.2 seconds mean open time. These significant differences (Students t-test: P<0.01) are probably caused by independent mechanisms (Scepek et al., 1998). Since our STB contained 10 mM Ca2+, we suggest that LDCV flickers with short open times are elicited by elevated extracellular Ca2+ and these with longer mean open-times are triggered by staurosporine action.
Burst flickers indicate sites of secretion hot spots
In addition to solitary flickers, we found capacitance flicker bursts, where up and down steps followed each other in rapid succession. STB at 35°C and STB/carbachol/ staurosporine at 23°C induced strong flicker-burst activity in chromaffin cells. Bursts were never seen in carbachol/STB-stimulated cells at 23°C, if staurosporine was omitted from the carbachol/STB. Like temperature elevation in chromaffin cells, staurosporine triggers Ca2+ transients in smooth muscle cells, an effect that could elicit these bursts (Himpens et al., 1993). This observation suggests that the elevated temperature activates an additional metabolic pathway that is controlled by phosphorylation. Amperometric measurements, displayed in Fig. 2A, confirmed the capacitance measurement results by registering strong catecholamine secretion after temperature elevation. As an increased pore conductance was detected during flicker bursts, most of the steps could have been kiss and run events. It can not be excluded that solitary steps were hidden in such a burst, since the amperometric recording in Fig. 2A shows some tall, sharp spikes of more 10 pA. The majority of the spikes had currents of less than 5 pA, a value that indicates low catecholamine release through a transient pore (Albillos et al., 1997; Alés et al., 1999). Zhou and colleagues reported much higher amperometric currents of more than 100 pA for genuine exocytic events (Zhou et al., 1996). Our observation that 43% of all steps occurred during bursts at 35°C suggests that stimulated secretion in mammals utilises the kiss-and-run pathway at more physiological temperatures as an important alternative to classical exocytosis.
It appears likely that flicker bursts occur at so called hot spots of secretion (Travis and Wightman, 1998). Interestingly, similar bursts were reported in Zea mays coleoptiles protoplasts, where they accounted for 71% of all steps when measured at the physiological temperature for maize (Weise et al., 2000).
Staurosporine inhibits and activates different protein kinases
Our results show that chromaffin cells release catecholamines by both exocytosis and by the kiss-and-run mechanism under physiological stimulation conditions. The dominance of either one mechanism depends, most probably, on the level of phosphorylation within the cell. The main reason for this hypothesis is that staurosporine increases the capacitance flicker frequency in stimulated cells and reduces the total number of solitary steps. This effect of staurosporine is concentration-dependent, as less then 1 µM staurosporine is not effective (data not shown). This is a clear indication that inhibition of protein kinase C (PKC) is not the only cause for the observed effects because PKC activity is blocked at nanomolar concentrations of staurosporine (Hamamoto et al., 1990). In initial pilot experiments, we also tested other PKC inhibitors H7 and Ca2+-calmodulin-kinase II (trifluoperazine), as well as PMA as a stimulator for PKC. None of these drugs were effective in regard to altering the ratio between flickers and solitary steps (data not shown). Although we can not exclude small effects on flickering probability, only staurosporine was unambiguously the most potent agent. It is also unlikely that staurosporine acts exclusively by inhibiting protein kinases, as this effect requires only low nanomolecular concentrations of the drug. Besides its long-known role as a potent inhibitor of several kinases, staurosporine also induces the activation of p125FAK kinase, pp44/42 MAP kinase and pk60 kinase (Mangoura and Dawson, 1998; Rasouly et al., 1996; Yamaki et al., 2000). Because no further targets apart from kinases have been identified so far, it is likely that staurosporine drastically alters the degree of phosphorylation of proteins by affecting different protein kinases and accessory proteins, such as munc18, in a complex way (Fisher et al., 2001). We propose from the data presented in this study and results of former indirect experiments on the frog neuromuscular junction (Henkel and Betz, 1995) and hippocampal neurons (Klingauf et al., 1998) that staurosporine alters the secretion mechanism in favour of kiss-and-run release rather than classical exocytosis. However, there is also evidence that staurosporine, by blocking PKC specifically, inhibits the kiss-and-run mechanism in glutaminergic rat brain synaptosomes, emphasising the complex regulation of transmitter release in different secretory systems (Cousin and Robinson, 2000).
Kiss-and-run mechanism is not simply fast exo-endocytosis
Kiss-and-run is characterised by secretion through a pore that does not dilate but remains intact during the whole secretion process (Zhou et al., 1996). In contrast, endocytosis that follows exocytosis rapidly requires the disintegration of the pore structure and the merging of the vesicular and plasma membrane. But how can we distinguish between these two mechanisms? We observed a fluctuating, but persisting, conductance during most capacitance flicker events that can only be explained by a pore-like connection between the lumen of the vesicle and the extracellular space. The upper limit for the pore diameter can be estimated to less than 3 nm (Albillos et al., 1997). Larger diameters would produce a infinite conductance in our measurement system, giving the same result as a complete collapse of the vesicle into the plasma membrane. This system-inherent property allowed us to distinguish between full fusion and the kiss-and-run mechanism, regardless of the time interval between opening and closing of the LDCV. The persisting conductance in Fig. 3A demonstrates that an LDCV stays continuously connected for more than 20 seconds via a narrow pore to the extracellular space. The experiments also showed that pores that eventually dilated (fusion pores) had virtually identical electric conductances and catecholamine-release kinetics during their initial expansion phase as pores of flickering vesicles (Albillos et al., 1997; Lollike et al., 1995). This can be explained most easily by the suggestion that the pore is composed of identical molecular components.
The model: molecular secretion mechanism is regulated by phosphorylation
We propose that the kiss-and-run mechanism and exocytosis use the same molecular machinery because we demonstrated that fusion pores and flicker pores have similar conductances. Two concepts exist in the literature, one favours a lipophilic pore (Nanavati et al., 1992) and one a proteinaceous pore (Almers and Tse, 1990). Althought our own data can not provide direct molecular evidence for either hypothesis, we suggest a model (Fig. 6) for a proteinaceous pore, as this model gained strong support recently (Peters et al., 2001; Zimmerberg, 2001): vesicle proteins and plasma-membrane proteins bind to each other and form a ring, but vesicular phospholipids do not mix with the plasma membrane. The pore is formed from a hexamer of V0 sectors (proteolipids) of the H+-ATPase, and this structure fuses yeast vacuoles in vitro by creating a channel-like structure from two hexamers (Peters et al., 2001). Earlier, it was also shown that the V0 sector was tightly associated with the mammalian synaptic vesicle proteins synaptophysin and synaptobrevin, putting it in close vicinity to the SNARE-vesicle docking complex (Galli et al., 1996). Ductin, a highly conserved gap junction protein, was also identified as the V0 sector of the H+-ATPase that could be oriented in the plasma membrane in both directions (Findlay et al., 1997). Therefore it is likely that the fusion pore consists of two sets of trans-complex proteolipid hexamers, one in the vesicle and one in the plasma membrane (Peters et al., 2001). The concept is also supported by our previous work on synaptic vesicles, where we demonstrated that FM1-43 could not escape through a flickering proteinacious pore. If the pore would be composed of phospholipids, the dye could have left the vesicle by lateral diffusion (Henkel and Betz, 1995).
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Our observations support the hypothesis that exocytosis and the kiss-and-run mechanism use the same core molecular machinery and the ratio between exocytosis and kiss-and-run is controlled by protein phosphorylation.
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ACKNOWLEDGMENTS |
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