Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells
2000; Springer Nature; Volume: 19; Issue: 1 Linguagem: Inglês
10.1093/emboj/19.1.84
ISSN1460-2075
Autores Tópico(s)Photoreceptor and optogenetics research
ResumoArticle4 January 2000free access Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells A.W. Henkel A.W. Henkel Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany Search for more papers by this author H. Meiri H. Meiri Hebrew University-Hadassah Medical School, Jerusalem, Israel Search for more papers by this author H. Horstmann H. Horstmann IMCB Institute of Molecular and Cell Biology, 30 Medical Drive, 117609 Singapore Search for more papers by this author M. Lindau M. Lindau Applied and Engineering Physics, Cornell University, 217 Clark Hall, Ithaca, NY, USA Search for more papers by this author W. Almers Corresponding Author W. Almers Vollum Institute, 3181 SW Sam Jackson Park Road, Portland, OR, 97201 USA Search for more papers by this author A.W. Henkel A.W. Henkel Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany Search for more papers by this author H. Meiri H. Meiri Hebrew University-Hadassah Medical School, Jerusalem, Israel Search for more papers by this author H. Horstmann H. Horstmann IMCB Institute of Molecular and Cell Biology, 30 Medical Drive, 117609 Singapore Search for more papers by this author M. Lindau M. Lindau Applied and Engineering Physics, Cornell University, 217 Clark Hall, Ithaca, NY, USA Search for more papers by this author W. Almers Corresponding Author W. Almers Vollum Institute, 3181 SW Sam Jackson Park Road, Portland, OR, 97201 USA Search for more papers by this author Author Information A.W. Henkel1, H. Meiri2, H. Horstmann3, M. Lindau4 and W. Almers 5 1Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany 2Hebrew University-Hadassah Medical School, Jerusalem, Israel 3IMCB Institute of Molecular and Cell Biology, 30 Medical Drive, 117609 Singapore 4Applied and Engineering Physics, Cornell University, 217 Clark Hall, Ithaca, NY, USA 5Vollum Institute, 3181 SW Sam Jackson Park Road, Portland, OR, 97201 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:84-93https://doi.org/10.1093/emboj/19.1.84 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Constitutive exo- and endocytic events are expected to increase and diminish the cell surface area in small spontaneous steps. Indeed, cell-attached patch–clamp measurements in resting chromaffin cells revealed spontaneous upward and downward steps in the electrical capacitance of the plasma membrane. The most frequent step size indicated cell surface changes of <0.04 μm2, corresponding to vesicles of <110 nm diameter. Often downward steps followed upward steps within seconds, and vice versa, as if vesicles transiently opened and closed their lumen to the external space. Transient openings and closings sometimes alternated rhythmically for tens of seconds. The kinase inhibitor staurosporine dramatically increased the occurrence of such rhythmic episodes by making vesicle closure incomplete and by inhibiting fission. Staurosporine also promoted transient closures of large endocytic vesicles possibly representing remnants of secretory granules. We suggest that staurosporine blocks a late step in the endocytosis of both small and large vesicles, and that endocytosis involves a reaction cascade that can act as a chemical oscillator. Introduction Through exo- and endocytosis, cells release and take up soluble material as well as adding and retrieving plasma membrane. The mechanisms of exo- and endocytosis are not completely understood. Much information on exocytosis has been gained by studying single exocytic events in real time by electrophysiological techniques. Regulated exocytosis of large dense-core vesicles (LDCVs) increases the cell surface area in steps, as has been documented by measuring the electrical capacitance of the plasma membrane. Such studies were carried out in a variety of cells, among them mast cells (Fernandez et al., 1984; Breckenridge and Almers, 1987; Alvarez de Toledo et al., 1993), eosinophils (Scepek and Lindau, 1993), neutrophils (Lollicke et al., 1995), melanotrophs (Zupancik et al., 1995) and chromaffin cells (Albillos et al., 1997; Ales et al., 1999). In all these studies, exocytic fusion appeared reversible, in that step increases in cell surface area sometimes were followed by step decreases of essentially equal amplitude. The membrane retrieval following triggered exocytosis can also result in measurable step decreases in cell surface area (Neher and Marty, 1982; Rosenboom and Lindau, 1994; Thomas et al., 1994). Apart from regulated exocytosis and the ensuing endocytosis, endocrine and other cells carry out constitutive exocytosis (Kelly, 1985) and endocytosis (Seaman et al., 1996). Constitutive endocytosis is thought to occur by vesicles much smaller than LCDVs, as does constitutive exocytosis (Bursztajn and Fischbach, 1984; Griffiths et al., 1985). Cell-attached capacitance measurements have sufficient sensitivity to detect the small steps expected to result from single events in constitutive endo- and exocytosis (Lollicke et al., 1995; Kreft and Zorec, 1997). Here we use this method to investigate constitutive exo- and endocytosis in chromaffin cells. We detected a slow constitutive membrane turnover caused by vesicles with a diameter of <110 nm. Both exo- and endocytic events seem reversible, in that a significant portion of exo- and endocytic events appear transient. Surprisingly, exo–endocytic organelles can lapse into a state where they rhythmically make and break connections with the cell surface. Previous results have suggested that staurosporine causes exocytic events to become transient (Henkel and Betz, 1995). Here we show that the drug encourages vesicles to close and re-open rhythmically, consistent with the idea that the drug inhibits a late step in vesicle fission. Results Cell-attached recording reveals stepwise changes in cell surface area Chromaffin cells in standard bath solutions are at rest. Their plasma membrane potential is so negative that calcium channels are shut, and exocytosis of dense-core granules is not expected. Nonetheless, we observed spontaneous events presumably representing single constitutive exo- and endocytic events. Figure 1 illustrates the first 5 min of a typical experiment. Figure 1A plots the capacitance of the patch (ΔC) against time, as well as the conductance (ΔG) and the patch current (I). ΔC and ΔG represent out-of-phase and in-phase components, respectively, of the sinusoidal patch current driven by a sinusoidal voltage. With the phase properly set (see Materials and methods), ΔC assays changes in the area of plasmalemma in the patch. The ΔG trace tracks patch conductance changes due to the opening of ion channels, as well as changes in the electrical resistance of aqueous openings (fusion and fission pores) connecting exo- and endocytic vesicles with the external space. At this resolution, the most prominent features of the ΔC trace are the downward calibration marks at the beginning of the trace (arrowhead) and a slow upward drift. It was gradual, not observed in all recordings and could be increased by making the potential in the pipet negative and thereby depolarizing the membrane patch. Atomic force microscopy has shown that excised membrane patches and vesicles bulge more deeply into the pipet when a negative potential is applied to the pipet (Hörber et al., 1995). Hence the upward drift in Figure 1A probably represents plasmalemma slowly moving into the pipet as an expanding bulge. Figure 1.Capacitance steps in chromaffin cells. (A) Capacitance (ΔC), conductance (ΔG) and current (I) traces in a cell-attached recording. Arrowhead, 3 fF capacitance calibration marks. Arrow, a temporary seal instability or ion channel opening caused an inward current and a simultaneous conductance increase. In the original recording, they were accompanied by slight ΔC deflections; these were removed by phase-shifting the ΔC and ΔG traces by −7° relative to φ0 (see Materials and methods). (B) The ΔC trace in (A) after subtraction of a sloping baseline and at higher magnification. Steps labeled 1–5 passed our step selection criteria, other deflections did not. (C) Steps 1–5 in (B) magnified. C1 shows details of the analysis (see Materials and methods). Regression lines were fitted to the sections of both ΔC and ΔG traces that are marked by horizontal bars. Their vertical distance at the step position defines the amplitude of the ΔC and ΔG steps, if any. Standard bath solution. Download figure Download PowerPoint The ΔG trace remained flat except for a temporary increase in conductance (arrow) caused either by the opening of ion channels in the patch or by a temporary leak in the seal. At the same time, a current flowed from the bath or from the cell into the pipet. In the original trace, there were small simultaneous deflections in the ΔC trace but these had been eliminated here by optimization of the phase setting (see Materials and methods). Recordings as in Figure 1A typically continued for 15–20 min. Figure 1B divides the ΔC trace into segments to show it at higher magnification and time resolution. Several deflections appear that are not readily seen in Figure 1A; five of them (indicated by numbers) passed our criteria for capacitance steps (see Materials and methods) and may represent single endo- and exocytic events. Figure 1C shows each deflection at still higher gain, along with the accompanying ΔG trace. Regression lines (dashed) were fitted to the ΔC segments indicated by horizontal bars in Figure 1C1; they formed the basis for deciding whether or not to accept the step and for determining its amplitude. Three downward and two upward steps suggest three endo- and two exocytic events, respectively. Often a ΔC step was followed by another of similar amplitude but opposite direction (‘capacitance flicker’), as if either an exocytic vesicle fused with the plasmalemma but then disconnected (Figure 2A) or an endocytic vesicle budded off the plasmalemma and then reconnected (Figure 2B). Note that both the transient upward and downward ΔC steps in Figure 2B were associated with a transient increase in the ΔG trace, indicating that the fusion pore connecting the vesicle in Figure 2A remained narrow and failed to dilate fully, and that the ‘fission pore’ connecting the endocytic vesicle in Figure 2B constricted but did not close completely (see later). ΔG changes as large as these were not always observed. Figure 2.Capacitance flicker. (A) Transient upward step; (B) transient downward step; (C) repeated cycles of up- and downward steps characteristic of a flicker burst. (A–C) are each from a different cell. Download figure Download PowerPoint More rarely, upward and downward ΔC steps alternated rhythmically. Since it is highly unlikely that different vesicles result in alternating up- and downward steps of such similar amplitudes, we suggest that a single vesicle repeatedly broke and re-made an electrical connection with the cell surface (Figure 2C). A more thorough description of such capacitance flicker is given later. Bursts of flicker were seen in five of 106 cells. Although times between up and down steps varied considerably from episode to episode, individual open and close times were closely similar within the same flicker burst (see later, Figure 4). Figure 3.Step amplitude distribution in chromaffin cells. From recordings as in Figures 1 and 2. (A) Steps <0.5 fF, detected as described in Materials and methods. Open bars include all steps passing the selection criteria, including those during flicker bursts as in Figure 2C. Black bars: steps in flicker bursts were excluded except for the first cycle of up–down or down–up pairs. Negative values indicate downward and positive values upward steps. Error bars give the square root of the step count in each bin, scaled to units of frequency. To obtain frequency, counts were divided by total recording time (29.4 h at an r.m.s. noise level of 0.05 fF or better in 106 cells). (B) Steps between 0.5 and 4 fF sorted into 0.25 fF bins; ordinates were divided by 5 to make frequencies applicable to 0.05 fF bins as in (A). Download figure Download PowerPoint Spontaneous exo- and endocytosis involve small vesicles Recordings were made from 106 patches, each on a different cell and yielding from 177 to 1670 s of recording (total 29.4 h) at an r.m.s. noise level of <0.05 fF. The number of exo- and endocytic events was plotted as a histogram in Figure 3A (white bars) with ΔC steps sorted into 0.05 fF bins. Amplitudes were plotted positive for exo- and negative for endocytic steps. Since steps <0.15 fF could not be detected reliably, they were not counted even though their frequency may well be high. It is clear nonetheless that most steps in Figure 3A must represent structures smaller than large dense-core granules. Dense-core granules have a mean diameter of ∼300 nm in electron micrographs (Parsons et al., 1995; Plattner et al., 1997), and each contributes an average of ∼2 fF (Neher and Marty, 1982; Albillos et al., 1997; Moser and Neher, 1997). The great majority of exo- and endocytic steps were 1.0 fF were seen in this data set. Figure 4.Staurosporine-induced capacitance flicker. Standard bath solution with 2 μM staurosporine. (A) Probable instance of frustrated endocytosis. Upper trace, ΔC; lower trace, ΔG. The trace starts 398 s after the beginning of the recording; prior to the episode shown, there were no episodes of flicker by other vesicles. (B) As in (A) but magnified and from a different cell. Note the regular rhythm of up- and downward steps. (C) Histograms of open times (ΔC trace high) and closed times (ΔC trace low) from an episode including the trace in (B). The timing of ΔC steps was determined as described in Materials and methods, except that the presence or absence of a step was decided by inspection. Download figure Download PowerPoint Even though flicker bursts were rare, they accounted for a significant portion of the steps in Figure 3A (white bars). The black bars result from eliminating steps in flicker bursts. They include the first opening or closing of a vesicle as well as the first subsequent step, if any, even if it was in the opposite direction. However, they exclude all subsequent actions of a flickering vesicle. All black bars together correspond to frequencies of one endocytic step every 14 min (1.2 mHz) and one exocytic step every 9 min (1.8 mHz). Next, steps passing our selection criteria were re-examined visually and selected only when there was no sign of a step in the opposite direction in 3 s intervals either before or afterwards. On average, there was one such solitary exocytic step every 11 min (1.5 mHz) and one solitary endocytic step every 22 min (0.8 mHz). Comparing these frequencies with those given above suggests that 15% of exo- and 35% of endocytic steps were transient. The low step frequencies are averages, include recordings with no steps at all (35 of 106 patches) and contrast with Figure 1B where five steps occurred in ∼5 min. Most probably exo- and endocytic events occur preferentially at discrete locations on the cell surface, as in clathrin-mediated endocytosis (Gaidarov et al., 1999). Thirty-two out of 106 patches had only exocytic and 10 only endocytic steps. At a local level, therefore, exo- and endocytosis are not necessarily in balance. Constitutive membrane turnover is slow in resting chromaffin cells We calculated the surface represented by the steps included in Figure 3A (black bars). In 29.4 h of recording, the total amount of membrane exocytosed during events between 0.15 and 0.5 fF was 38.5 fF, and that endocytosed was 17.4 fF. These values must be divided by 0.5 since most steps <0.5 fF are detected with ∼50% efficiency (see later). The result is 77 fF for exo- and 35 fF for endocytosis. Larger vesicles exocytosed 18 fF and endocytosed 25 fF in the same patches; we expect to have detected them all. Hence we estimate that a total of 95 fF were added by exocytosis and 60 fF withdrawn by endocytosis. Exo- and endocytosis were not exactly in balance, possibly because mechanical effects of the cell-attached pipet favored exo- over endocytosis. The average of the two values is 77 fF. The analysis suggests that a patch containing 6 μm2 of membrane (see Materials and methods) with 60 fF capacitance turns over its membrane ∼77/60 = 1.3 times in 29 h, or about once a day at 20–23°C. The figure could be somewhat larger because steps <0.15 fF are missed in our recordings. Turnover is also expected to be faster at 37°C (Matlin and Simons, 1983). We calculated the volume associated with each downward step (black bars in Figure 3A) assuming that endocytic vesicles are spheres surrounded by membrane of 10 fF/μm2 specific capacitance. After correcting for our detection efficiency, the sum from all recorded steps of <0.5 fF in Figure 3A (black bars) was 0.112 fl. The relatively few larger steps took up as much volume, 0.12 fl, bringing the total to 0.23 fl/patch in 29.4 h. If a patch is representative of the entire plasma membrane and contains 1% of a chromaffin cell's surface, then each cell endocytoses 23 fl in 29.4 h, or 0.9 fl/h. Our estimate includes transient endocytic events that would not cause uptake of extracellular marker, and it fails to include transient exocytic events that would. Apart from flicker bursts, about one-third of exo- and endocytic events are transient. Our estimate is inaccurate in so far as the additional volume imported in transient fusion cannot be expected to equal exactly that attributed erroneously to transient fission events. Staurosporine causes endocytic events to become transient In the presence of staurosporine (2 μM), the occurrence of prolonged episodes of capacitance flicker became more frequent. Figure 4A shows an example. A downward step occurred about midway through the trace and initiated a long sequence of alternating up- and downward steps. As in Figure 2C, we suggest that a single vesicle repeatedly attempted to disconnect its lumen from the external space but succeeded only for short periods. Bursts of such ‘capacitance flicker’ alternated with periods of silence. In our data set of 14 staurosporine-treated cells, eight cells (or 57%) showed flicker bursts, compared with only 6% in the absence of the drug. For analysis, a flicker burst was defined as containing at least four steps alternating in direction, each step following its predecessor within 3 s. Table I summarizes the properties of bursts both with and without staurosporine. The drug strongly increased the fraction of time where at least one vesicle is engaged in a burst. It had lesser or no effects on the other variables in Table I. The mean step amplitudes are similar or identical to that of solitary steps. With staurosporine, there are fewer solitary steps, as if at least some bursts of transient closings and openings occur at the expense of the complete fusions and fissions seen without drug. The last two findings are consistent with the idea that the same types of vesicles successfully complete exo- and endocytosis in the absence of the drug. However, they fail to do so in the presence of staurosporine and instead they lapse into a flickering mode. Flicker with the drug appears similar to the flicker occasionally recorded without the drug, except for a difference described later. We cannot rule out that staurosporine also induces a completely new exo–endocytic mechanism. Table 1. Effect of staurosporine on flicker bursts and solitary steps No drug Staurosporine Flicker bursts: burst time (%) 0.58 ± 0.4 106 cells 14.5 ± 7.3 14 cells burst duration (s) 20.1 ± 5.4 8 bursts 54.4 ± 27.8 21 bursts open time (s) 0.44 ± 0.13 8 bursts 0.55 ± 0.08 21 bursts closed time (s) 1.39 ± 0.29 8 bursts 1.5 ± 0.25 21 bursts step size (fF) 0.21 ± 0.02 8 bursts 0.31 ± 0.05 21 bursts Solitary steps: size (fF) 0.27 ± 0.01 241 steps 0.32 ± 0.05 11 steps Step frequency (mHz) 2.3 ± 0.1 241 steps 1.1 ± 0.3 11 steps In a burst, at least three steps of alternating direction must follow their predecessor within 3 s and satisfy the criteria outlined in Materials and methods. Any period longer than 3 s between qualifying steps was taken to terminate a burst. Solitary steps must neither follow, nor be followed by, a visually detected step of opposite direction within 3 s. Burst time is the percentage of time in each cell where at least one vesicle is engaged in a burst. Open refers to the time the capacitance is high, closed to when it is low in recordings as in Figures 2C or 4B. The step sizes given are the average of mean step amplitudes in each burst, or the average of solitary steps in each condition. Vesicles close and re-open rhythmically Figure 4B shows a segment of a burst at higher magnification. Again, alternating up- and downward ΔC steps of very similar amplitude suggest that the fission pore of a single vesicle repeatedly opened and closed, or dilated and constricted. Figure 4C shows histograms for the ‘open time’ of this burst (where the ΔC level was high and the vesicle presumably connected to the extracellular space) and for the closed time. The open time histogram in particular showed a pronounced peak. Both histograms differ strongly from those obtained for the stochastic opening and closing of ion channels (summarized, for example, in Colquhoun and Hawkes, 1981). Evidently fission pores in endocytic vesicles may open and close rhythmically rather than stochastically. Staurosporine hinders complete vesicle closure Do flicker bursts result from exocytic vesicles failing to open fully, from endocytic vesicles failing to close fully, or both? Flicker bursts caused by an endocytic vesicle start with a downward step, and those caused by an exocytic vesicle start with an upward step. However, this distinction cannot be made in most recordings in staurosporine as these start in the middle of a burst. The calculation plotted in Figure 5A illustrates an alternative method to distinguish a vesicle trying to undergo fusion from one trying to undergo fission. Figure 5.Incomplete fusions and fissions cause ΔG steps. (A) Real (ΔG) and imaginary parts of the electrical admittance (ΔC) are calculated for a typical 0.3 fF vesicle and an excitation frequency of 8 kHz, and plotted against the conductance of the aqueous pore connecting the vesicle lumen with the external space. The equations are ΔC = 2πfCv/z and ΔG = [(2πfCv)2/g]/z where Cv is the capacitance of the vesicle, f the frequency of the sinusoid, g the pore conductance and z = [(2πfCv/g)2 + 1] (see, for example, Breckenridge and Almers, 1987). A vesicle fusing with the plasma membrane (rightward arrow) would start with both ΔG and ΔC equal to zero. As its fusion pore opens to, for example, 100 pS, ΔC increases by nearly 0.3 fF while a ΔG deflection of ∼2 pS remains. Both ΔC and ΔG vary in parallel. A vesicle budding off the plasma membrane and forming a fission pore of 2pS (leftward arrow) would start with ΔG = 0 and cause a drop in ΔC by ∼0.3 fF, but as long as its pore conductance remains finite, a ΔG deflection also remains. ΔG and ΔC vary in opposite directions. (B) Average time course of down- and upward steps in the episode shown in Figure 4B, calculated as follows. We divided the ΔC and ΔG traces into 300 ms segments, one ΔC–ΔG pair for each upward step and one pair for each downward step. Segments containing downward steps were aligned to the falling phases of ΔC and averaged, those with upward steps were treated similarly. The result (left) shows ΔC and ΔG deflections at greatly diminished noise levels. (C) Possible morphological basis of the electrical events in (B). Download figure Download PowerPoint A vesicle fusing with the plasma membrane but opening incompletely is expected to cause an upward ΔC step accompanied by an upward ΔG step (right-pointing arrow). The ΔG step represents the appearance of an electrical conductance connecting the vesicle lumen to the outside, namely the conductance of the fusion pore while it has not dilated fully (e.g. Breckenridge and Almers, 1987; Zimmerberg et al., 1987). ΔG vanishes when the pore conductance becomes too large to support a significant voltage difference under sinusoidal excitation. By contrast, when a vesicle buds from the cell surface or when it constricts an initially wide connection with the external space, the result is a downward ΔC step accompanied by a ΔG increase (left-pointing arrow in Figure 5A). Now the ΔG step represents the resistance of the aqueous connection, called the fission pore (Rosenboom and Lindau, 1994). ΔG vanishes when the fission pore has closed completely. Hence ΔC and ΔG will vary in parallel when a vesicle fuses without opening completely, and in opposite directions when a vesicle tries to undergo fission without closing completely. ΔG and ΔC changes during flicker bursts were compared. In most recordings, deflections in the ΔG trace seemed lost in noise. To make them more visible, all segments containing steps were excised from a burst, aligned to the times of capacitance steps, and both the ΔC and ΔG segments thus aligned were averaged. The result of such an analysis for the vesicle in Figure 4C is shown in Figure 5B. ΔG increased while ΔC fell. The ΔG deflection is not the result of an incorrect phase setting. To make the ΔG deflection disappear, the phase would have to be ∼17° more negative. However, when the ΔC and ΔG traces were re-calculated with this new phase, the ΔC trace showed deflections that were correlated temporally with the opening and closing of ion channels elsewhere in this trace (not shown). Since ion channels change only the conductance and not the capacitance, the more negative phase setting must be incorrect. With the new phase, the ΔC changes were also correlated temporally with the ΔG changes. To find out by how much the new phase was in error, we plotted ΔC against ΔG while ion channels opened and closed (not shown). The regression line formed an angle of −20 ± 2° with the abscissa (p <0.0001); hence the new phase was −20° too negative and the phase originally used in Figure 5B was within 3° of being correct. Evidently, ΔG and ΔC deflections of this vesicle were genuinely in opposite directions, consistent with a vesicle closing transiently and incompletely. The traces in Figure 5B are consistent with a vesicle of 0.27 fF capacitance constricting a fission pore down to a conductance of only 4 pS. Figure 5C shows a possible morphological basis for the vesicle in Figure 5B. In other vesicles, no ΔG deflection could be detected even after signal averaging multiple steps. These vesicles must either have closed completely or their fission pore conductance was <1 pS. In 15 of 22 flicker bursts with staurosporine, ΔG and ΔC varied in the opposite direction, in six ΔG did not vary measurably and in only one did ΔG and ΔC vary in parallel. In the absence of the drug, ΔG and ΔC changes were parallel in three of eight vesicles, opposite in four of eight vesicles and in one ΔG did not vary measurably. Apparently, the predominant effect of staurosporine is to prevent vesicles from closing their fission pore completely. Hence the drug may be viewed as an inhibitor of endocytosis. Our step selection erroneously rejects half of the small capacitance steps To avoid contaminating our ΔC step count by noise, we applied stringent step selection criteria (see Materials and methods) that most probably made us reject some genuine steps. Flicker episodes provide an opportunity to test how many steps are falsely rejected since, during flicker, rhythmically repeating steps of similar or identical amplitudes are easily recognized visually even if they do not pass all criteria of automated selection. We inspected traces as in Figures 2C or 4A and B and with step amplitudes from 0.15 to 0.3 fF. Steps were detected visually and their number was compared with the number of steps detected by our selection algorithm. The algorithm accepted 64 ± 13% of transitions in staurosporine (mean step amplitude 0.19 ± 0.01 fF in nine bursts) and 45 ± 7% without the drug (eight bursts; mean step amplitude 0.21 ± 0.02 fF). Staurosporine also induces flicker in large vesicles Unlike the recordings discussed so far, those of Albillos et al. (1997) showed frequent large upward steps that represented exocytosis of dense-core granules as they were accompanied by quantal release of catecholamine. This was confirmed when we made recordings under conditions similar to those of Albillos et al. (1997) in an external solution that encouraged exocytosis of dense-core granules as it contained elevated [Ca2+] and the K channel blocker tetraethylammonium. In a total of 2 h of recordings from 43 cells, exocytic steps with amplitudes between 0.5 and 4 fF appeared at 10 times higher frequency than even the smallest steps in standard bath solution. Recordings from two cells showed particularly high exo
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