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The Journal of Neuroscience, January 1, 1998, 18(1):81-92
The Relation of Exocytosis and Rapid Endocytosis to Calcium Entry
Evoked by Short Repetitive Depolarizing Pulses in Rat Melanotropic
Cells
Huibert D.
Mansvelder and
Karel S.
Kits
Research Institute Neurosciences Vrije Universiteit, Faculty of
Biology, Membrane Physiology Section, 1081 HV, Amsterdam, The
Netherlands
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ABSTRACT |
Melanotropic cells release predocked, large, dense-cored vesicles
containing 
-melanocyte stimulating hormone in response to calcium
entry through voltage-gated calcium channels. Our first objective was
to study the relationship between exocytosis, rapid endocytosis, and
calcium entry evoked by short step depolarizations in the order of
duration of single action potentials (APs). Exocytosis and rapid
endocytosis were monitored by capacitance measurements. We show that
short step depolarizations (40 msec) evoke the fast release of only
~3% of the predocked release-ready vesicle pool. Second, we asked
what the distance is between voltage-gated calcium channels and
predocked vesicles in these cells by modulating the intracellular
buffer capacity. Exocytosis and rapid endocytosis were differentially
affected by low concentrations of the calcium chelator EGTA. EGTA
slightly attenuated exocytosis at 100 µM relative to 50 µM, but exocytosis was strongly depressed at 400 µM, showing that calcium ions have to travel a large
distance to stimulate exocytosis. Nevertheless, the efficacy of calcium
ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related
linearly to the amount and duration of calcium entry during a single
AP. Rapid endocytosis was already strongly depressed at 100 µM EGTA, which shows that the process of endocytosis
itself is calcium dependent in melanotropic cells. Furthermore, rapid
endocytosis proceeded with a time constant of ~116 msec at 33°C,
which is three times faster than at room temperature. There was a
strong correlation between the amplitude of endocytosis and the
amplitude of exocytosis immediately preceding endocytosis. Both this
correlation and the fast time constant of endocytosis suggest that the
exocytotic vesicle is retrieved rapidly.
Key words:
exocytosis; endocytosis; calcium entry; channels; EGTA; BAPTA; pituitary; MSH
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INTRODUCTION |
Neurons and neuroendocrine cells can
release exocytotic vesicles within milliseconds (Augustine et al.,
1985
; Thomas et al., 1993a,b; Heinemann et al., 1994; Borst and
Sakmann, 1996; Mennerick and Matthews, 1996). In both cell types,
vesicles lie predocked to the plasma membrane awaiting the trigger for
release. In melanotropic cells, of the ~3300 vesicles that are
morphologically found to be predocked to the plasma membrane, only
~250 are readily releasable within 40 msec on an immediate uniform
rise in [Ca2+]i (Thomas et al.,
1993a,b; Parsons et al., 1995). The remaining 90% need additional
priming steps, giving rise to slower components of exocytosis.
In synapses, the predocked release-ready vesicles are molecularly
linked to calcium channels (Calakos and Scheller, 1996). Release of
these vesicles occurs immediately on calcium channel opening (Mennerick
and Matthews, 1996), thereby warranting a strong synchronization
between action potential (AP) and secretion of transmitter. In isolated
chromaffin cells, the timing of release is not strictly coupled to
calcium current kinetics, and single APs only infrequently trigger
release (Zhou and Misler, 1995). When longer step depolarizations are
used, exocytosis persists after calcium entry has stopped (Chow et al.,
1992). This time delay of release is thought to arise from the
separation of calcium channel and docking site, thus necessitating
diffusion of calcium between channel and release site (Chow et al.,
1996).
Most data on exocytosis in the melanotropic cells from the
intermediate pituitary were obtained using flash photolysis of caged
calcium, generating high sustained calcium levels (Thomas et al., 1990;
1993a,b; Parsons et al., 1995). Melanotropic cells generate both sodium
and calcium spikes (Douglas and Taraskevich, 1980; Williams et al.,
1990). These phenomena range in duration from a few milliseconds for
sodium spikes to tens of milliseconds for the calcium spikes. The
intracellular calcium profiles thus produced differ strongly from the
long uniform increases by flash photolysis of caged calcium or the
increases produced by long step depolarizations. Here we examine the
coupling between release and short depolarizations in melanotropic
cells. Specifically, we wanted to know whether the predocked
release-ready vesicles are located near calcium channels or whether
calcium has to diffuse a substantial distance to the release site.
Membrane capacitance measurements were used to monitor changes in
cell surface area (Neher and Marty, 1982). We found that in response to
a 40 msec step depolarization only a small portion (~3%) of the
release-ready vesicles is released. Moreover, this rapid release was
depressed at low micromolar concentrations of intracellular EGTA (
400
µM), indicating that the distance separating vesicles
from calcium channels is large in comparison with synapses. Rapid
endocytosis was observed only at low buffering conditions (50 µM EGTA) and was strongly coupled to exocytosis. Despite
this large distance between calcium channel and predocked vesicle, melanotropic cells on average release one vesicle per 2 msec
depolarization. In addition, exocytosis was linearly related to the
amount and duration of calcium entry, suggesting that in melanotropic
cells the amount of release can be regulated by the duration of an
AP.
A preliminary account of some of this work has been published
previously (Mansvelder and Kits, 1997).
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MATERIALS AND METHODS |
Cell culture. Pituitary melanotropic cells of
male Wistar rats (200-300 gm; Harlan CPB, Zeist, Netherlands) were
isolated as described previously (Keja et al., 1991). The cells were
cultured on poly-L-lysine-coated coverslips (7 × 7 mm) at a density of 0.25 intermediate lobe per coverslip. The culture
medium consisted of Biorich I (Flow), NaHCO3 26.2 mM, Ultroser G 5% (Life Technologies, Gaithersburg, MD),
penicillin G 200 U/ml (Sigma, St. Louis, MO), streptomycin 50 µg/ml
(Sigma), and cytosine arabinosine 1 µM (Sigma) adjusted
to pH 7.2 with NaOH. Cells were maintained in a 37°C incubator with a
humidified atmosphere comprising 5% CO2 in air. Recordings
were made up to 4 d after isolation.
Recording solutions. Coverslips bearing melanotropic cells
were transferred to the recording chamber containing ~0.5 ml external solution. The external solution consisted of (in mM): TEACl
142; glucose 10; CaCl2 5; HEPES 10; 4-aminopyridine 1; pH
adjusted to 7.4 with TEAOH. The internal solution contained (in
mM): CsCl 160; MgCl2 2; HEPES 10; MgATP 2; pH
adjusted to 7.4 with CsOH. To this medium the following calcium
chelators were added: 50-800 µM EGTA (Sigma) or BAPTA,
Cs4 salt (Molecular Probes, Eugene, OR), as indicated in
Results. The recording chamber was perfused continuously at a rate of
~1.5 ml/min, driven by air pressure, whereas the bath volume was kept
constant by continuous suction. All experiments were performed at a
temperature of 32-34°C.
Capacitance measurements. Electrodes were pulled on a
Flaming/Brown P-87 (Sutter Instruments, Novato, CA) horizontal
microelectrode puller from thick-walled Clark GC-150 borosilicate glass
(Clark Electromedical Instruments, Pangbourne, UK). To reduce the
pipette capacitance, the tips of the electrodes were covered with
Sylgard. Impedance of the electrodes after fire-polishing was 2-4
M
, and the access resistance after establishment of the whole-cell
configuration was 7.4 ± 0.24 M (n = 75). The
whole-cell membrane current was monitored with an Axopatch 200A
amplifier (Axon Instruments, Foster City, CA) and digitized with a
Digidata 1200 interface (Axon Instruments). Capacitance measurements
were made using a software-based phase-sensitive detector (Joshi and
Fernandez, 1988; Fidler and Fernandez, 1989; Fidler Lim et al., 1990).
The source codes of the acquisition and analysis software, which run in
an Axobasic environment (Axon Instruments), were acquired from Axon
Instruments and modified in our laboratory (by P. F. van Soest and
H. D. Mansvelder). A 40 mV peak-to-peak, 1.2 kHz sine wave was
added to a holding potential of 
80 mV, and the resulting membrane
current was filtered at 2 kHz (4-pole low-pass Bessel filter on the
Axopatch 200A) and sampled at 20 kHz. Before analysis at two orthogonal
phase angles, 10 cycles of the sine wave were averaged. The correct
phase angle was determined every 18 sec by repetitive
computer-controlled switching of a 500 k resistor in series with
ground until changes in the capacitance trace were minimal. An
independent measure of the membrane conductance was obtained by
applying a hyperpolarizing pulse of 20 mV and 6 msec duration to the
membrane between two groups of 10 sine waves (see Fig.
1A). The resulting temporal resolution of each
capacitance, total conductance, and membrane conductance point was 18 milliseconds. In the experiments for Figures 10 and 11, the
hyperpolarizing pulse between sine waves was omitted, thus leading to
an enhanced temporal resolution of 10 msec. Changes in capacitance were
calibrated by a temporary 100 fF change in the compensation circuitry
of the amplifier, and the conductance trace was calibrated by the 500 k dither resistor. The initial membrane capacitance was 5.4 ± 0.18 pF, and the average membrane conductance was 258 ± 113 pS
(average of the mean ± SD).
Cells with a peak calcium current of less than 50 pA at the first
pulse were left out of analysis. The number of calcium ions that
entered the cell during a pulse was determined by:
where F is Faraday's constant (96485 coulomb
mol
1) and NA is Avogadro's
constant (6.022 · 1023 mol1).
Tail currents were included in this integration. Leak currents were not
corrected for.
Data analysis. The amount of exocytosis was calculated as
the difference between the average of the last five membrane
capacitance samples before and the first sample after a particular
depolarizing pulse. The amount of endocytosis was calculated as the
difference between the first sample after a pulse and the average of
the last five samples before the next pulse. Monoexponential decays of the calcium currents and the endocytotic events at 50 µM EGTA in Figure 6A,B were fitted
using the NLREG v3.4 software of P. H. Sherrod (Nashville,
TN). Pairwise comparisons (with Bonferroni adjustment) in Figures
4B,C, 5B,C, and 6B,C
were made using the Systat Software (Evanston, IL). The nonparametric
correlation coefficient of Spearman (Spearman's 
) was calculated
for the data of Figure 6C using the Simstat software of N. Péladeau (Montreal, Canada). Running averages of membrane
conductance traces (see Fig. 1B) were calculated
using software written by P. F. van Soest. Means mentioned in the
text are given with SEM unless mentioned otherwise. Error bars
represent SEM.
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RESULTS |
Changes in membrane capacitance were induced by interrupting the
capacitance measurements and applying 25 step depolarizing pulses of 40 msec from the holding potential 80 mV to +10 mV with an interval of
500 msec (Fig. 1B).
Capacitance measurements were resumed ~15 msec after the end of each
pulse. By that time the membrane conductance had recovered to the level
it had before the pulse (see both conductance traces in Fig.
1B), indicating that the first sample of the membrane
capacitance (
Cm) after the pulse is
reliably measured. The depolarizing pulses were evoked 5 min after
establishment of the whole-cell configuration to allow equilibration of
the intracellular medium and pipette solution. The average peak
whole-cell calcium current generated by the first pulse of each series
was 127 ± 38 pA (mean ± SD; n = 72) (Fig. 1B). Inactivation during these current responses was
adequately described by monoexponential functions fitted to the decay
phase of the current, yielding an average time constant of 13.9 ± 3.6 msec and an asymptote (I
) of 50 ± 26 pA (both mean ± SD; fits not shown). This time constant is
approximately seven times faster than the inactivation time constant of
the Q-type current found in these cells at room temperature and with
barium as charge carrier (101.7 ± 28.4 msec; mean ± SD)
(Mansvelder et al., 1996).
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Figure 1.
Voltage command protocols applied to the cell to
monitor Cm,
Rac, and
Gm and to evoke
ICa. A, Protocol used to
acquire one sample of the capacitance trace
(Cm), conductance trace
(Rac and
Gm), and membrane conductance
trace (Gm). Ten cycles of a sinusoidal voltage command of 1200 Hz were averaged for one sample of
Cm and the combined
Rac and Gm.
A 6 msec hyperpolarization yielded an independent measure of the cell
conductance. The current response of the cell was filtered at 2 kHz
with the low-pass Bessel filter on the amplifier, digitized, and
analyzed on-line. B, Top trace, Voltage
command trace (Vm) shown on a larger
time scale. The sinusoidal was omitted for the sake of simplicity.
Depolarizing pulses were applied every 500 msec from 80 to +10 mV.
Middle traces, The response to the 25 step
depolarizations. Cm, combined Rac and Gm
trace, and membrane conductance trace were obtained with the protocol
in A. The Gm trace is a 5 points running average. Bottom traces, Peak calcium
(ICa peak) current during the 40 msec step
depolarization. Below, the full current traces are given for the 1st and 25th pulse.
[EGTA]i = 200 µM.
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Short step depolarizations trigger fast exocytosis
First we determined what types of exocytosis were evoked by the 40 msec step depolarizations. In the majority of cells (>93%) that we
recorded from, the capacitance increase was already complete when the
step depolarization had ended, and no further increase was observed
(see capacitance trace in Fig. 1B). Most of the time the Cm trace decreased a little after the
initial increase after the pulse (2.3 ± 0.22 fF;
n = 13 cells) (Fig.
2A). In chromaffin cells, Chow et al. (1992) found that although the capacitance trace
ceased to increase as soon as the depolarization was ended, the
amperometric events continued for tens of milliseconds, suggesting that
exocytosis was still taking place without any apparent change in
membrane capacitance. The apparent difference was explained by
transient changes in the capacitance trace that were not related to
exocytosis (Horrigan and Bookman, 1994). When the capacitance traces
were corrected for these events, a slow increase in membrane capacitance appeared that was in agreement with the amperometric record
(Chow et al., 1996). We tested whether a nonsecretory capacitive transient contaminated the capacitance measurements in our experiments, by depolarizing cells in the presence of 100 µM
Cd2+ to block the calcium influx. We assume
that by blocking the calcium influx, depolarization-induced exocytosis
is prevented, and any further change in Cm is
not related to exocytosis or endocytosis. In the presence of 100 µM Cd2+, calcium currents were
completely blocked (not shown). Still, an increase in capacitance of
2.3 ± 0.20 fF (average of 225 depolarizations in three cells) was
detected right after the pulse, and the capacitance successively
decreased within ~35 msec by 1.1 ± 0.21 fF (Fig. 2B). Correction of the Cm
record by subtraction of the records in the presence of
Cd2+ showed that the Cm
trace did not increase any further after the end of the depolarization
(Fig. 2C). This suggests that exocytosis is complete at the
end of the pulse and that a 40 msec depolarization only triggers fast
exocytosis. However, we cannot exclude the occurrence of slow
exocytosis after the pulse that is counterbalanced by the retrieval of
the same amount of membrane (see also Discussion). To quantify
exocytosis in the rest of the paper we have taken the difference
between the membrane capacitance before the depolarization and the
first Cm sample immediately after the
depolarization (see Materials and Methods). Furthermore, all
quantifications of exocytosis and endocytosis that follow below are
corrected for the Cd2+-insensitive transient,
discussed above. This method yields the net amount of exocytosis
occurring during and after the depolarization and will be an
underestimation of the actual amount of exocytosis if any endocytosis
occurs simultaneously.
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Figure 2.
Exocytosis does not persist after calcium entry
has stopped. A, Average of responses to 25 step
depolarizations ([EGTA]i = 200 µM).
B, Average transient capacitance change in the presence of 100 µM Cd2+, to block
the calcium influx (average response to 75 depolarizations of one
cell). Solid line shows a single exponential fit to the decay of the average capacitance transient. Data were obtained from a
cell different from that in A ([EGTA]i = 50 µM). The average of three such experiments on
different cells was used to correct all quantifications of exocytosis
and endocytosis throughout this study. There were no significant
differences between the three cells, and they were all stimulated with
75 depolarizations. C, The solid line of
B is subtracted from the
Cm trace in A, to give the
Cm that is caused by exocytosis.
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The average amount of exocytosis that was elicited by a step
depolarization was 7.8 ± 0.41 fF, which corresponds to ~3% of the immediately releasable pool revealed by flash photolysis of caged
Ca2+ in these cells (Thomas et al., 1993a; Parsons
et al.,. 1995). Obviously, a short opening of calcium channels leads to
a completely different calcium profile underneath the membrane than a
uniform rise throughout the cell, and as a result, single step
depolarizations recruit only a small fraction of the total
release-ready vesicle pool.
During the 25 depolarizing pulses the peak calcium current declined
steadily from 127 ± 38 at the first pulse to 79 ± 24 pA
(mean ± SD) at the last pulse (Fig. 1B). As a
result, the number of calcium ions that came into the cell during a
step depolarization decreased as well. At the first depolarization
9.5 ± 3.4 106 ions entered the cell, whereas
at the last pulse 5.5 ± 2.1 106 ions entered.
At the same time, the amount of exocytosis per pulse decreased steadily
during the pulse train (Fig.
3A). When the amount of
exocytosis per pulse was corrected for the diminished calcium influx,
thus calculating Cm per number of calcium
ions that entered the cell, the decrease was almost absent (Fig.
3B). This indicates that the reduction in exocytosis per
pulse at the end of the pulse train can be largely explained by a
diminished calcium influx at the later pulses.
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Figure 3.
The decrease of Cm
during the pulse train can be attributed to a decrease of calcium
influx. A, Average Cm
response per pulse during the pulse train. The amount of
Cm per pulse decreased during the pulse
train. The pipette solution contained 100 µM EGTA
(n = 13 cells). B, Same data as in
A but now every capacitance jump is divided by the
number of calcium ions that came into the cell during the step
depolarization. The number of calcium ions is determined by integrating
the current trace, as described in Materials and Methods. The efficacy
of calcium ions to stimulate exocytosis showed almost no attenuation
during the pulse train.
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Calcium chelator EGTA affects fast exocytosis at
low concentrations
We asked whether voltage-gated calcium channels are located close
to release-ready vesicles or whether calcium ions have to diffuse a
large distance. To answer this question we increased the intracellular
buffer capacity by increasing the concentration of the calcium chelator
EGTA, thereby reducing the chance that a calcium ion reaches the
vesicle at a certain distance. Changing the concentration of a
diffusable calcium buffer with a slow forward binding constant like
EGTA has only limited effects on the peak calcium concentration just
beneath the site of influx, but is more effective on calcium levels
farther away from this point (Nowycky and Pinter, 1993; Roberts,
1994).
When the EGTA concentration was raised, the total amount of
Cm after 25 pulses was significantly
decreased from 257 ± 63 fF at 50 µM
(n = 8) to 175 ± 22 fF at 100 µM
and 183 ± 37 fF at 200 µM (both n = 13) (Fig. 4A,D). When
the EGTA concentration was further increased to 400 µM,
exocytosis was reduced more dramatically to 83 ± 9 fF at 400 µM (n = 9) and 48 ± 19 fF at 800 µM (n = 5). The amount of exocytosis per
pulse decreased significantly from 10.3 ± 0.54 fF at 50 µM EGTA to 1.9 ± 0.20 fF at 800 µM
(Fig. 4B). We corrected these values for differences
in calcium entry between cells and pulses (Fig. 4C). From
this it became evident that the efficacy of calcium ions to stimulate
exocytosis was only slightly reduced at 100 and 200 µM
EGTA but was especially hampered at 400 and 800 µM EGTA
(p < 0.01). Figure 4D shows
that the membrane capacitance increased approximately linearly with the
cumulative number of calcium ions that came into the cell during the
pulse train. There was no threshold of a minimal number of calcium ions
needed to start exocytosis at 40 msec pulse durations, in contrast to
what was found in chromaffin cells (Seward et al., 1995) and
peptidergic nerve terminals (Seward and Nowycky, 1996). Increasing EGTA
concentrations did not introduce a calcium threshold of secretion;
instead, it mainly decreased the slope of these plots, and this effect
became most prominent at 400 and 800 µM (Fig.
4D). So, fast exocytosis of predocked vesicles is
suppressed at relatively low concentrations of EGTA, suggesting that
calcium ions have to diffuse a large distance from the site of influx to the calcium sensor of the exocytotic complex in melanotropes. Given
this large distance, the calcium concentration that is sensed by a
predocked vesicle most likely results from the entry of calcium through
multiple calcium channels.
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Figure 4.
Exocytosis is affected by low concentrations of
EGTA. A, Examples of responses to 25 step
depolarizations with different EGTA concentrations as indicated next to
the traces. For every experiment a new cell was taken. The noise in the
800 µM trace (SD of 1.72 fF) was somewhat lower than in
the other two traces (SD of 2.26 fF at 200 µM), but the
values are within the normal variation encountered in both groups
[2.25 ± 0.60 fF at 800 µM and 2.24 ± 1.12 fF
at 200 µM (both mean ± SD; P > 0.9)]. B, The average Cm
per depolarization at different EGTA concentrations. C,
Efficacy of calcium ions to stimulate exocytosis at different EGTA
concentrations. Pairwise comparisons showed that the amount of
exocytosis at 100 and 200 µM did not differ
significantly; all other groups differed significantly from each other
(p < 0.01). D, Cumulative
capacitance change plotted versus the cumulative number of calcium ions
that came into the cell during a pulse train. These curves showed
almost straight lines with different slopes for the different EGTA
concentrations. The numbers next to each curve represent
the intracellular EGTA concentration in micromoles. For B,
C, and D: 50 µM,
n = 8; 100 µM, n = 13; 200 µM, n = 13; 400 µM, n = 10; 800 µM,
n = 5.
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BAPTA is only twice as effective as EGTA
The calcium chelator BAPTA has a forward calcium binding constant
that is ~100 times faster than EGTA, while at the same time the
KD for calcium is ~100 nM at pH
7.3 for both (Tsien, 1980; Smith et al., 1984). Given these properties,
BAPTA should be ~100 times more effective in blocking exocytosis in
melanotropes, if calcium only has to diffuse from its site of entry to
the calcium sensor of the exocytotic complex in the immediate vicinity.
However, when the pipette solution contained 50 µM BAPTA
instead of EGTA, exocytosis still was possible (Fig.
5A). The total amount of
Cm after 25 depolarizations was 168 ± 36 fF at 50 µM BAPTA (n = 10) (Fig.
5A,D), which was not significantly different from the amount of exocytosis at 100 and 200 µM EGTA. At 200 µM BAPTA, the membrane capacitance increased by 116 ± 46 (n = 5), which was not significantly different
from the change seen at 400 µM EGTA. The average amount of exocytosis per pulse decreased from 6.7 ± 0.5 fF to 4.6 ± 0.5 fF per pulse (p < 0.01) when BAPTA was
raised from 50 to 200 µM (Fig. 5B). Correction
for the number of calcium ions that came into the cells during a pulse
showed that the average efficacy of calcium ions to stimulate
exocytosis decreased from 1.15 ± 0.07 fF/106
calcium ions to 0.7 ± 0.07 fF/106 calcium ions
(p < 0.01) (Fig. 5C). No threshold
secretion was observed using BAPTA as the mobile calcium chelator (Fig.
5D). As with EGTA, increasing BAPTA concentrations affected
the slope of cumulative Cm versus cumulative
calcium plot. Taken together, these data show that although BAPTA is
~100 times faster than EGTA in binding calcium ions, it is only
approximately twice as effective as EGTA in blocking exocytosis in
melanotropes. Possibly the distance that calcium ions have to travel
before reaching the predocked vesicle is so large that the binding
kinetics of the soluble chelator is less important than the
KD for calcium. These data support the idea that
influx of calcium through multiple calcium channels will contribute to
the concentration sensed by the predocked vesicle.
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Figure 5.
Exocytosis is still possible with 50 and 200 µM intracellular BAPTA. A, Examples of
responses on two different cells with BAPTA concentrations indicated.
B, Average Cm per
depolarization at the two different BAPTA concentrations.
C, Efficacy of calcium ions to stimulate exocytosis per
pulse. Pairwise comparisons showed that the efficacy at 50 µM BAPTA was not significantly different from the
efficacy at 100 and 200 µM EGTA
(p > 0.1) (Fig. 4C). The
efficacy at 200 µM BAPTA was not significantly different
from the efficacy at 400 µM EGTA (Fig.
4C). D, Cumulative capacitance change
plotted versus the cumulative number of calcium ions that came into the
cell during a pulse train. These curves deviated slightly from straight
lines, with a lower slope at later pulses; still, the different BAPTA
concentrations clearly show different slopes. The
numbers represent the intracellular BAPTA concentration in micromoles. For B, C, and D: 50 µM, n = 10; 200 µM,
n = 5.
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Rapid endocytosis is more sensitive to calcium buffering capacity
than exocytosis
When the EGTA concentration in the pipette solution was set to 50 µM, a rapid decrease of membrane capacitance was seen
right after each depolarization (Fig.
6A). We interpreted
these deflections as rapid membrane retrieval as described by Thomas et
al. (1994). The amount of endocytosis was determined as the difference
between the first sample after the pulse and the average of the last
five samples before the next pulse (see Material and Methods). Again, as with quantifying exocytosis, this method calculates the net amount
of endocytosis and therefore might underestimate the actual values if
exocytosis occurs concomitantly. The total amount of membrane that was
retrieved after 25 step depolarizations was 237 ± 68 fF
(n = 8) (Fig. 6D), and only slightly
more (257 ± 63 fF; see above) was added during the same 25 pulses. As a result, after a few pulses the net membrane capacitance
did not increase any further (Fig. 6A). Excess
retrieval, which was observed by Thomas et al. (1994) using flash
photolysis of caged calcium, was only rarely observed, and when it
occurred it exceeded the amplitude of exocytosis at the same pulse by
only a few femtofarads.
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Figure 6.
Rapid endocytosis is blocked by a lower
concentration of EGTA than exocytosis. A, Examples of
Cm responses of two different cells to 25 step depolarizations with 50 and 100 µM intracellular EGTA. At 100 µM intracellular EGTA, with each pulse
Cm increases, and only small decreases are
seen after each pulse. At 50 µM EGTA, Cm decreases rapidly after each increase
(see inset), so that the net
Cm increases only ~50 fF. The combined
Rac and Gm
traces and the Gm traces are depicted to
show that there was no significant cross-talk between the traces. The
top panels illustrate the method that was used to
calculate the amount of exocytosis (Exo) and endocytosis
(Endo). The same method was used for Figures 7 and 9.
B, Average amount of Cm that
is retrieved after each step depolarization. Rapid endocytosis is
depressed at 100 µM EGTA. C, The efficacy of calcium ions to stimulate rapid endocytosis. Obviously, the efficacy
is also depressed at 100 µM EGTA
(p < 0.01). D, Cumulative Cm that is retrieved during a pulse train
versus the cumulative number of calcium ions that came into the cell
during the pulse train. The slope of the 100 µM EGTA
curve (labeled 100) is much lower than the slope at 50 µM (labeled 50). For B, C,
and D, the data from the same cells as in Figure 4 were
used.
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When the intracellular EGTA concentration was raised to 100 µM, rapid membrane retrieval almost completely
disappeared (p < 0.01) (Fig.
6A,D). At this EGTA concentration only 61 ± 27 fF was retrieved during the pulse train. The amount of endocytosis per
pulse decreased from 9.5 ± 0.57 fF at 50 µM EGTA to
2.4 ± 0.19 fF at 100 µM (p < 0.01), and this was more or less the same for all higher
concentrations of EGTA up to 800 µM (Fig.
6B). When these amounts of membrane retrieval were
corrected for the amount of calcium influx during the pulse, the
picture was the same (Fig. 6C). At all EGTA concentrations
of 100 µM and higher endocytosis is depressed. These
results confirm first of all that endocytosis in melanotropic cells is
calcium dependent. Second, because exocytosis was depressed at
approximately five times higher EGTA concentrations than endocytosis
(compare Figs. 4 and 6), these results indicate that either the
KD for calcium is higher for endocytosis than
for exocytosis or that calcium ions have to travel a larger distance
from the site of influx to the sensor for the endocytotic process than
for the exocytotic process.
Efficacy of calcium ions to stimulate endocytosis increases slowly
during the pulse train
As the example with 50 µM EGTA in Figure
6A showed, the amplitude of endocytosis increased
during the pulse train, and after a few depolarizations the amplitude
of endocytosis matched the amplitude of exocytosis. This is shown for
the average amplitude of endocytosis per pulse in Figure
7A. At the first pulse, <5 fF
membrane was retrieved but the amplitude increased, and at the fourth
pulse already more than 10 fF was retrieved. The fitted line is a
single exponential with a time constant of 0.5 sec. At the same time,
the amount of calcium that entered the cell during the pulse decreased
with increasing pulse number (Fig. 1B). As a result,
the efficacy of calcium ions to stimulate endocytosis continued to
increase after the amplitude of endocytosis reached its maximum during
the pulse train (Fig. 7B). The single exponential fitted to
the data in Figure 7B had a time constant of 2.3 sec. It
took ~10 pulses to reach an efficacy that corresponded to the efficacy of exocytosis at the same pulse (1.7 ± 0.3 vs 1.5 ± 0.3 fF/106 calcium ions) (Fig. 4C).
So, the amount of endocytosis that is triggered by a given number of
calcium ions that came into the cell during a pulse increases during
the pulse train with a time constant of seconds. This might suggest
that a slow step is involved in membrane retrieval that takes a few
seconds.
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Figure 7.
Efficacy of calcium ions to stimulate endocytosis
increases slowly during the pulse train. A, Average
amplitude of rapid endocytosis per pulse number. Data from the cells as
in Figures 4 and 6, with an intracellular EGTA concentration of 50 µM (n = 8). The amplitude of rapid
endocytosis was measured as indicated in Materials and Methods. The
solid line represents a fitted single exponential function with a time constant of 0.5 sec. B, Average
efficacy of calcium ions to stimulate rapid endocytosis per pulse
number. The efficacy increases during the pulse train, with a time
constant of 2.3 sec (solid line).
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The rate of rapid endocytosis is constant during a pulse train
To determine whether the rate of membrane retrieval increased
during the pulse train as well, we fitted monoexponential decay functions to the decreases in membrane capacitance immediately after
each depolarization (see example in the inset of Fig.
8). Not all endocytotic responses could
be fitted with an exponential decay function; some of them showed a
more linear decay. Moreover, because the pulses were set apart in time
by only 500 msec, fits of exponential decays with a time constant
larger than 250 msec were considered as not reliable. Still, ~64% of
the endocytotic responses (127 of 200 in eight cells) showed a
monoexponential decay with a time constant below 250 msec. Figure 8
shows the average time constant per pulse number, with each data point
showing the average of three to eight responses obtained from different cells. The time constant of membrane retrieval did not change with
increasing pulse number, and the overall average was 116 ± 5 msec
(Fig. 8). This is approximately three times faster than the 350 msec
time constant of fastest retrieval measured at room temperature by
Thomas et al. (1994). Although the amplitude and efficacy of
endocytosis grow with increasing pulse number (Fig. 7), the fact that
the average time constant did not change with increasing pulse number
suggests that once the process of endocytosis is triggered it proceeds
at a fixed rate.
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Figure 8.
The time constant of rapid endocytosis is constant
during a pulse train. A single exponential function was fitted to the
decay in membrane capacitance occurring immediately after the
depolarizations (inset). Each decrease after a given
pulse was fitted this way, and the time constants were averaged for
each particular pulse number (n = 8 cells, each
subjected to 1 pulse train). When a decrease could not be reliably
fitted, it was left out of the average. As mentioned in the text, 127 of the 200 endocytotic responses could be reliably fitted with an
exponential decay function. There was no relation found between the
number of acceptable fits underlying each average and the number of the
pulse in the pulse train. The plot shows the average
time constant of membrane capacitance decrease as a function of pulse
number.
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Fast exocytosis and rapid endocytosis are strongly
coupled processes
During the pulse train the amplitude of endocytosis
increased, and after a few pulses it matched the amplitude of
exocytosis (Fig. 6A). As a result the net
Cm did not increase further. In Figure
9 the amplitude of endocytosis after a
given pulse is plotted against the amplitude of exocytosis caused by
the same pulse for all 200 pulses. The amplitude of endocytosis showed
a strong correlation with the amplitude of exocytosis (Fig. 9), with a
correlation coefficient of 0.80 (Spearman's ; p < 0.01). This suggests that the cell retrieves just as much membrane as
has been added during a step depolarization, thereby actively keeping
its membrane area constant. Together with the fast time constant of
membrane retrieval, the strong correlation between the amplitudes of
exocytosis and endocytosis supports the idea that it is the exocytotic
vesicle itself that is retrieved rapidly, immediately after fusion.
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Figure 9.
Fast exocytosis and rapid endocytosis are strongly
coupled. The amplitude of exocytosis and rapid endocytosis at the same pulse show a high correlation (Spearman's = 0.80;
p < 0.01). Amplitude of exocytosis and endocytosis
was determined as illustrated in Figure 6A. These
values were corrected for changes in Cm
not related to exocytosis and endocytosis. The solid
line represents y = x.
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Exocytosis increases linearly with the duration of
calcium influx
Because the mobile calcium buffering capacity of the intracellular
medium affects exocytosis at low concentrations, the distance between
the site of calcium influx and the site of release must be considerably
large. From this it might be inferred that a single spike lasting a few
milliseconds is probably not capable of triggering any secretion, and
that the melanotropic cell is only able to regulate to a limited extent
the timing of release in comparison with neuronal synapses. On the
other hand, if calcium channel and vesicle are far apart, one would
expect that melanotropic cells would show threshold secretion, as was
found for chromaffin cells and peptidergic nerve terminals (Seward et
al., 1995; Seward and Nowycky, 1996); however, this was not found at 40 msec depolarizations (Figs. 4D, 5D). Thus,
we asked how well exocytosis couples to the calcium current in the
melanotropic cell. We applied 15 step depolarizations at 2 Hz with
varying pulse durations from 2 msec up to 40 msec. For these
experiments we used 50 µM EGTA in the pipette solution,
thus allowing rapid endocytosis, because Thomas et al. (1994) showed
that rapid retrieval occurs in perforated patch recordings from
melanotropes, in which the endogenous buffer system is intact.
With increasing pulse durations the total capacitance increase became
larger (Fig. 10). With 2 msec
depolarizations the membrane capacitance increased by 0.83 ± 0.15 fF per pulse (average of 120 depolarizations on eight cells). This is
approximately the estimated capacitance of one large dense-cored
vesicle in melanotropes (Parsons et al., 1995), suggesting that, on
average, at 2 msec step depolarizations one vesicle is released. The
bottom plots of Figure 10 show the average cumulative
Cm of eight cells against the cumulative
number of calcium ions that came into the cell during the pulse train.
As for the 40 msec pulses shown above (Fig. 4D), the
curves show an approximately straight line for shorter pulse durations
as well. Interestingly, the slope of the curves is almost the same for
all pulse durations.
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Figure 10.
Exocytosis increases linearly with the
amount of calcium influx at different pulse durations. The top
traces (Cm) show an example of an increasing amount of Cm at
trains of 15 pulses, with increasing pulse duration on the same cell.
Pulse durations are indicated above the
traces. The amount of exocytosis and endocytosis per
pulse at the 40 msec pulse duration are somewhat smaller than for the
cells in Figure 6. This might be attributable to the fact that the
cells used for the experiments in Figure 6 were subjected to only one
pulse train, whereas the cells for these experiments were all
stimulated with 5 pulse trains with different pulse durations. Middle traces (ICa)
show the current traces at the 1st and 15th step depolarization at the
different pulse durations. Bottom plots show the
cumulative capacitance change versus the cumulative number of calcium
ions that entered the cell during the step depolarizations at different
pulse durations. Each data point represents the average of eight
different cells. Note that the values at axes change for each plot, but
that the ordinate and abscissa values change proportionally, to show
that the slope is roughly constant for all pulse durations.
[EGTA]i = 50 µM.
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With increasing pulse duration the amplitude of exocytosis increased,
as well as the inactivation of the calcium current during a pulse and
over 15 pulses (Fig. 10). The amplitude of exocytosis per pulse
increased from 1.95 ± 0.25 fF at 5 msec to 7.21 ± 0.38 fF
at 40 msec (n = 8). The total amount of exocytosis
after 15 pulses is plotted against pulse duration in Figure
11A. Exocytosis did
not increase linearly with pulse duration and showed saturation at the
longer pulse durations. However, when the cumulative number of calcium
ions that came into the cell during the 15 pulses is plotted against
pulse duration (Fig. 11B), this also showed
saturation at the longer pulse durations caused by increased
inactivation at longer pulse durations (Fig. 10). As a result, the
efficacy, the amount of capacitance change as a fraction of the number
of calcium ions, is constant for all pulse durations up to 40 msec (Fig. 11C). Per one million calcium ions approximately 1 fF,
that is one vesicle, is released. This indicates that in melanotropes release of readily releasable vesicles is directly proportional to the
number of calcium ions that enters the cell. So, secretion in
melanotropic cells is regulated quite precisely by the time that
calcium channels are open and calcium is entering the cell.
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Figure 11.
The efficacy of calcium ions to stimulate
exocytosis is constant for different pulse durations. A,
The cumulative capacitance change increases with increasing pulse
duration. B, The cumulative number of calcium ions that
entered the cell during the pulse train increases similarly with
increasing pulse duration. C, The efficacy of calcium
ions to stimulate exocytosis is constant for all pulse durations. Data
were taken from the same cells as in Figure 10.
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DISCUSSION |
Short step depolarizations evoke fast exocytosis
Exocytosis occurs in multiple phases, not only in neuroendocrine
cells such as melanotropes (Thomas et al., 1990; Thomas et al.,
1993a,b; Parsons et al., 1995), chromaffin cells (Neher and Zucker,
1993; Heinemann et al., 1994; Horrigan and Bookman, 1994; Seward and
Nowycky, 1996), and PC12 cells (Kasai et al., 1996), but also in
peptidergic nerve terminals of the posterior pituitary (Seward et al.,
1995) as well as central synapses (Goda and Stevens, 1994; Mennerick
and Matthews, 1996). Fast exocytosis, defined as exocytosis occurring
within 40 msec at 30-34°C after a uniform rise in
[Ca2+]i in melanotropes, is thought to
result from the release of vesicles that are predocked on the cell
membrane (Thomas et al., 1993a; Horrigan and Bookman, 1994; Parsons et
al., 1995; Mennerick and Matthews, 1996). The slower phases of
exocytosis are probably also the result of fusion of predocked
vesicles, but these vesicles have to undergo one or more priming steps
before they can actually be released (von Rüden and Neher, 1993;
Thomas et al., 1993a,b; Parsons et al., 1995). The immediately
releasable pool of vesicles (available for fast exocytosis) is ~250
fF in melanotropic cells, whereas the total docked vesicle pool is
~3300 fF (Parsons et al., 1995). We found that during a 40 msec step
depolarization with 50 µM intracellular EGTA only ~3%
of the immediately releasable pool is released. Thus, although
immediate calcium concentration increases evoked by flash photolysis of
caged calcium cause the release of a rapid pool of vesicles of ~250
fF within 40 msec (Thomas et al., 1993a), these vesicles may have
actually only a 3% chance to be released on the entry of
~107 calcium ions through voltage-gated calcium
channels during a 40 msec step depolarization.
The methods we used to calculate the amount of exocytosis and
endocytosis neglected the fact that endocytosis might already have
started during the depolarization and that exocytosis might have
continued after the depolarization had ended. Therefore, all the values
we obtained for exocytosis and endocytosis might be underestimations of
the actual values. An independent measure of exocytosis, avoiding
contamination by endocytosis, is to be found in the amperometric
detection of the melanotropic cell hormone MSH, as has been shown by
Paras and Kennedy (1995). A definite quantification awaits such
experiments.
Predocked release-ready vesicles and voltage-gated calcium channels
are far apart
The present experiments examined the question of whether calcium
channels are closely apposed to predocked release-ready vesicles in
melanotropic cells. In a nano- or microdomain (Schweizer et al., 1995)
around the channel mouth, the calcium concentration rises to tens or
hundreds of micromoles within 1 msec (Simon and Llinás, 1985;
Roberts, 1994). Release of a vesicle present in this domain cannot be
impaired by a slow calcium buffer such as EGTA, not even in high
millimolar concentrations (Adler et al., 1991; Nowycky and Pinter,
1993; Roberts, 1994; von Gersdorff and Matthews, 1994; Mennerick and
Matthews, 1996). In contrast, in our experiments EGTA already slightly
affected fast exocytosis at 100 µM and largely impaired
exocytosis at concentrations of 400 µM. This shows that
calcium channels and the predocked release-ready vesicles in
melanotropes are not close together and calcium ions will have to
diffuse a large distance (i.e., >100 nm) to the release site.
Probably, the influx of calcium through multiple calcium channels will
contribute to the calcium concentration buildup near one predocked
vesicle.
BAPTA, which has a ~100 times faster kon for
calcium than EGTA, was only twice as effective in blocking fast
exocytosis. This is an unexpected result if the calcium buffers are
thought to be only in kinetic competition for calcium with the calcium
sensor of the vesicle. Smaller differences in the effectiveness of
BAPTA and EGTA in depressing exocytosis than predicted from calcium binding kinetics were also found for threshold secretion in peptidergic nerve terminals (Seward et al., 1995) and chromaffin cells (Seward and
Nowycky, 1996). Both chelators were equally effective in shifting the
calcium threshold for exocytosis, suggesting that the concentration of
the chelator rather than the binding kinetics determines the threshold
(Seward et al., 1995; Seward and Nowycky, 1996). However, in chromaffin
cells, rapid release of predocked release-ready vesicles was not
affected by 300 µM BAPTA (Seward and Nowycky, 1996;
Klingauf and Neher, 1997). This contrasts with the chelator sensitivity
of secretion of the predocked release-ready vesicles we found in
melanotropic cells, where at 200 µM BAPTA fast exocytosis was depressed. Similar results were found in a neuronal preparation. In
the synapse of Held, neurotransmission is depressed by 1 mM EGTA, whereas BAPTA was only 4-10 times more effective (Borst et al.,
1995; Borst and Sakmann, 1996). The latter authors took this as support
for their finding that opening of multiple calcium channels is
necessary for transmitter release in this synapse (Neher, 1996).
Exocytosis is tightly regulated
If calcium channels and predocked release-ready vesicles are not
closely adjacent, can fast exocytosis still be effectively regulated by
the melanotropic cell? In melanotropes and chromaffin cells the rate of
exocytosis has a third- to fourth-power dependence on the global
intracellular calcium concentration (Thomas et al., 1990, 1993b;
Heinemann et al., 1993). It was suggested therefore that the last steps
in exocytosis of large dense-cored vesicles require the binding of
three or more calcium ions. In addition, when calcium enters through
calcium channels the resulting concentration profiles are nonuniform
and highly dynamic. Despite these complexities in the relationship
between calcium influx, calcium concentration, and the last steps in
exocytosis, our results show that the amount of exocytosis was linearly
proportional to the number of calcium ions that entered the cell during
a pulse (Figs. 10, 11). So, although calcium channels and release-ready
vesicles are not closely apposed to each other, the amount of
exocytosis is tightly linked to the amount and duration of calcium
entry.
A similar strong dependence of the probability of release on the flux
of calcium and the duration of the flux was found in chromaffin cells
(Engisch and Nowycky, 1996). In these cells too there is a considerable
distance between calcium channels and predocked vesicles (Chow et al.,
1992, 1994, 1996; Klingauf and Neher, 1997). Still, different types of
calcium channels couple with different efficacies to exocytosis in
these cells (Artalejo et al., 1994). Our results combine with these
data to demonstrate that exocytosis of predocked large dense-cored
vesicles in neuroendocrine cells is subject to regulation by timed and
selective activity of voltage-gated calcium channels.
Retrieval of exocytotic vesicles is rapid and
calcium dependent
Thomas et al. (1994) proposed a close coupling between exocytosis
and endocytosis in melanotropes. After fusion, the vesicle does not
flatten out into the plasma membrane but is retrieved again within a
few seconds. At room temperature, retrieval of the exocytotic vesicle
had two time constants, one of 3.5 sec and one of 350 msec. In our
experiments at 33°C, retrieval had a time constant of ~116 msec,
and this was constant during the pulse train. An even faster time
constant of membrane retrieval of ~60 msec was reported for
chromaffin cells (Heinemann et al., 1994; for review, see Henkel and
Almers, 1996). In addition, we found that the amplitude of endocytosis
increased during a pulse train. After a few pulses the amplitude of
endocytosis matched the amplitude of exocytosis at the same pulse quite
well, giving rise to a strong correlation between exocytosis and
endocytosis. This suggests that exocytotic vesicles are retrieved again
with a time constant of ~116 msec immediately after the fusion step. A strong correlation between exocytosis and endocytosis was also found
in PC12 cells (Kasai et al., 1996).
In chromaffin cells, rapid endocytosis is calcium dependent, and
calmodulin is the receptor of calcium for this process (Artalejo et
al., 1995, 1996). The fact that endocytosis was more sensitive to the
calcium buffering capacity of the intracellular medium than exocytosis
in our experiments shows that endocytosis is calcium dependent in
melanotropic cells as well. The increase of the efficacy of calcium
ions to stimulate rapid endocytosis during the pulse train might
suggest that a slow calcium-dependent factor is involved in rapid
endocytosis that gets increasingly activated during the pulse train.
When this factor is activated, endocytosis proceeds with a time
constant of ~116 msec. This factor did not wash out of the cells
during the first 5 min of the whole-cell recording, as was the case for
slow endocytosis in saccular hair cells (Parsons et al., 1994). It
remains to be established whether calmodulin is involved in
melanotropic cells. Alternatively, an increased efficacy could result
from a buildup of calcium underneath the membrane caused by summation
between pulses. However, simulations of calcium concentration dynamics
with parameters found in the present experiments showed that with a 500 msec interpulse interval, the calcium concentration directly below the
membrane drops rapidly to 100 nM after the pulse ends
(T. A. de Vlieger and H. D. Mansvelder, unpublished results).
An alternative interpretation of the observed increase in endocytosis
during a pulse series, however, might be that slow exocytosis
(occurring after each pulse) does play a role but diminishes during the
pulse train. To resolve this issue, additional experiments with
simultaneous amperometric detection of MSH are needed.
Thomas et al. (1994) showed by making perforated-patch recordings that
rapid endocytosis does occur when the intracellular buffer system is
kept intact. In our experiments, rapid endocytosis occurred only at
very low concentrations of the slow calcium buffer EGTA. Thus, rapid
endocytosis as well as exocytosis turn out to be very sensitive to
mobile calcium chelators when trains of short step depolarizations are
used. This suggests that the endogenous mobile buffer capacity of
melanotropic cells must be relatively weak, compared with other systems
(e.g., Roberts, 1993), in allowing processes such as exocytosis and
rapid endocytosis to occur.
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FOOTNOTES |
Received June 10, 1997; revised Oct. 13, 1997; accepted Oct. 15, 1997.
This work was supported by an NWO-Medical Research Council Grant
(900-553-035). We thank Jacqueline Leyting-Vermeulen for preparing the
cell culture and Paul van Soest for help with programming. We thank
Drs. Arjen Brussaard, Paul van Soest, Hind van Tol, and Theo de Vlieger
for comments on this manuscript.
Correspondence should be addressed to Karel S. Kits, Research Institute
Neurosciences Vrije Universiteit, Faculty of Biology, Membrane
Physiology Section, De Boelelaan 1087, 1081 HV, Amsterdam, The
Netherlands.
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Top
Abstract
Introduction
