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The Journal of Neuroscience, February 15, 1999, 19(4):1317-1323
Inhibitors of Myosin Light Chain Kinase Block Synaptic Vesicle
Pool Mobilization during Action Potential Firing
Timothy A.
Ryan
Department of Biochemistry, The Weill Medical College of Cornell
University, New York, New York 10021
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ABSTRACT |
During repetitive action potential firing the maintenance of
synaptic transmission relies on a continued supply of synaptic vesicles
for fusion with the presynaptic plasma membrane. The mechanism of
transport by which vesicles are delivered to the site of fusion from a
reserve pool is unknown, as are the biochemical pathways linking
intracellular Ca2+ elevation with vesicle
mobilization. Here, using the fluorescent tracer FM1-43 in hippocampal
synaptic terminals, I show that inhibitors of myosin light chain kinase
can block mobilization of the reserve pool and not the immediately
releasable pool.
Key words:
exocytosis; vesicle recycling; synaptic vesicles; myosin
light chain kinase; hippocampus; presynaptic terminal; FM1-43
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INTRODUCTION |
Although there has been tremendous
progress in understanding the molecular basis of membrane fusion and
vesicle docking (Schiavo et al., 1995 ; Sudhof, 1995; Hay and Scheller,
1997), much less is known about the mechanisms that underlie the
transport of vesicles to their target membranes. In synaptic terminals
neurotransmitter-containing vesicles are maintained in large clusters
in apposition to the site of membrane fusion and must be mobilized in
synchrony with vesicle fusion to sustain synaptic transmission during
repetitive stimulation. It has long been hypothesized that the
elevation of intracellular Ca2+, which promotes
membrane fusion, also might serve as a trigger for vesicle
mobilization and transport (Berl et al., 1973). Recent studies at
different types of central synapses (Stevens and Wesseling, 1998; Wang
and Kaczmarek, 1998) indicate that the resupply of vesicles after
stimulation is accelerated by elevations of intracellular Ca2+. The molecular basis of the movement of
vesicles that would be required to refill the readily releasable pool
remains unknown. Previous studies have established that myosin and the
Ca2+/calmodulin-dependent regulatory enzyme of
actin-myosin interactions, myosin light chain kinase (MLCK), are
required for presynaptic function (Mochida et al., 1994), but their
precise role in the synaptic vesicle life cycle has not been
established. Thus an actin-myosin interaction likely drives vesicle
movements within the terminal in at least one phase of the synaptic
vesicle life cycle.
To pinpoint the possible functional role of MLCK in synaptic vesicle
recycling, I have investigated the impact of known membrane-permeant organic inhibitors of MLCK on presynaptic function in combination with
optical assays of membrane trafficking at individual synaptic boutons
of hippocampal neurons in culture. Optical assays of membrane traffic
that use the fluorescent tracer FM1-43 provide a means of isolating
several different steps in the synaptic vesicle life cycle (Betz and
Bewick, 1993; Ryan and Smith, 1995). In addition, they allow one to
estimate the size of the recycling vesicle pool on a bouton by bouton
basis (Ryan et al., 1996b; Murthy et al., 1997). My results
indicate that inhibition of MLCK reduces the size of the recycling
vesicle pool but does not change the kinetics of vesicle pool turnover
significantly. Finally, I show that the reduction in pool size by the
inhibition of MLCK results from the block of a distal recycling pool
that normally is released with >20 AP and that the turnover of a
readily releasable pool is unaffected.
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MATERIALS AND METHODS |
Cell culture. Hippocampal CA1-CA3 regions were
dissected from 4-d-old Sprague Dawley rats, dissociated, and plated
onto Matrigel (Collaborative Biochemical, Franklin Lakes, NJ) or
polyornithine-coated coverslips inside a 5-mm-diameter cloning cylinder
(100 µl vol; Bellco Glass, Vineland, NJ). Animals used in this study
were cared for in accordance with institutional guidelines. Cells were
maintained in culture media consisting of MEM (Life
Technologies, Gaithersburg, MD), 0.6% glucose, 0.1 gm/l bovine
transferrin (Calbiochem, La Jolla, CA), 0.25 gm/l insulin, 0.3 gm/l
glutamine, 5-10% fetal calf serum (HyClone, Logan, UT), 2% B-27
(Life Technologies), and 8 µM cytosine
 -D-arabinofuranoside. Cultures were maintained at 37°C
in a 95% air/5% CO2 humidified incubator for 14-50 d
before use. Unless otherwise indicated, all chemicals were obtained
from Sigma (St. Louis, MO).
Experimental conditions. Coverslips were mounted in a
rapid-switching, laminar-flow perfusion and stimulation chamber on the stage of a laser-scanning confocal microscope. The total volume of the
chamber was ~75 µl and was perfused at a rate of 1-1.5 ml/min.
Action potentials were evoked by passing 1 msec current pulses yielding
fields of ~10 V/cm through the chamber via platinum-iridium electrodes. Except as otherwise noted, cells were superfused
continuously at room temperature (~24°C) in a saline solution
consisting of (in mM) 119 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 25 HEPES
(buffered to pH 7.4), and 30 glucose plus 10 µM
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Research Biochemicals,
Natick, MA) and 50 µM µM
D,L-2-amino-5-phosphonovaleric acid (AP-5; Research
Biochemicals). FM1-43 (Molecular Probes, Eugene, OR) was used at a
concentration of 15 µM. ML-9 and ML-7 were obtained from
Calbiochem and Alexis Pharmaceuticals (San Diego, CA) and prepared as
100 mM stock solutions in DMSO. The final concentration of
DMSO never exceeded 0.05%. Butane-2,3-dione-monoxine (BDM) was
obtained from Sigma and prepared fresh with each use in the perfusion saline.
Optical measurements, microscopy, and analysis.
Laser-scanning fluorescence and differential interference contrast
images were acquired simultaneously at a spatial sampling of 125 nm/pixel and a dwell time of 2 µsec/pixel through a 40× 1.3 numerical aperture Zeiss Fluar objective (Oberkochen, Germany), using a
custom-built laser-scanning microscope. Specimens were illuminated with
~1.5 µW of the 488 nm line of an argon ion laser that was shuttered rapidly during all nondata-acquiring periods by the use of
acousto-optic modulation. Under these conditions, photobleaching of
FM1-43 fluorescence was <0.25% per image. Quantitative measurements
of fluorescence intensity at individual synapses were obtained by
averaging a 4 × 4 area of pixel intensities centered on the
optical center of mass of a given fluorescent punctum. Individual
puncta were selected by hand, and the optical center of mass used to
center the measurement box was computed over a slightly larger area
(typically 6 × 6 pixels). Large puncta, typically representative
of clusters of smaller synapses, were rejected during the selection
procedure as were any puncta that were not clearly discernible in all
test episodes. Errors shown are SEM.
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RESULTS |
Inhibitors of MLCK partially block release of FM1-43 during
continuous action potential stimulation
The strategy used for these studies is depicted in Figure
1. To measure the impact of various
inhibitors of MLCK or myosin on vesicle pool turnover during defined
action potential trains, I used the fluorescent probe FM1-43 in
combination with superfusion, imaging, and stimulation methods. All
measurements proceed in two phases. The loading phase consisted of a
period of vesicle exocytosis stimulated by a defined action potential
train in the presence of FM1-43. FM1-43 was left in the superfusate 1 min beyond the firing of action potentials. The extra 1 min exposure to
dye ensured the labeling of all of the vesicles retrieved during
endocytosis (Ryan and Smith, 1995; Ryan et al., 1996a). The unloading
phase, performed after 10 min of rinsing in dye-free solution,
consisted of at least two episodes of acquiring fluorescence images
before and after a prolonged train of action potentials (900 at 10 Hz). Each unloading episode was separated by a brief rest period (150 sec).
For some experiments a series of images was acquired during the
unloading phase to measure the kinetics of dye release and vesicle pool
turnover. A measure of the vesicle turnover that occurred during the
loading phase was obtained by calculating the sum of the differences in
fluorescence intensity,  F, at individual boutons between
images before and after each unloading episode. The impact of MLCK
inhibitors was assessed in one of two ways: inhibitors were perfused in
during the loading phase, in the presence of the dye, and unloading was
performed in drug-free conditions, or inhibitors were perfused onto
preloaded boutons during one of the unloading episodes. In either case
the drug application condition was interspersed between two bracketing
control runs during which no drug was applied. The average response of
the control runs was used as normalization for the drug-containing episodes.
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Figure 1.
Protocols for FM1-43 labeling of vesicle recycling
in the presence of MLCK or myosin inhibitors. Recycling vesicles were
labeled by exposure to 15 µM extracellular FM1-43
(Load) during AP stimulus. The 90 sec of 10 Hz
stimulation in a dye-free solution then released ~90% of the
vesicular dye in control conditions (Unload;
F0). A second round of 900 AP
released the remaining 10% vesicular fluorescence
(Unload; F1). The
impact of inhibitors of MLCK or myosin on vesicle recycling was
determined by application during either the loading or the unloading
phase. In either case, inhibitors were perfused in 150 sec before the
stimulus and washed out after the stimulus.
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Figure 2a is a Nomarski image
illustrating a typical axodendritic network of hippocampal neurons in
culture. Figure 2b shows a pseudocolored fluorescence image
of the same field acquired after FM1-43 loading with a 900 action
potential (AP) stimulus (image A in the schematic). The
image shows the characteristic distribution of fluorescent puncta
corresponding to clusters of labeled synaptic vesicles in individual
presynaptic boutons (Betz and Bewick, 1992; Ryan et al., 1993; Henkel
et al., 1996). Previous studies have shown that the labeling of the
total recycling pool is saturated under these conditions (Ryan and
Smith, 1995). Figure 2c shows a large reduction in
fluorescence produced by a long unloading train of APs (900) in control
conditions (image B). Approximately 90-95% of the total
labeled pool is turned over in a single long stimulus train, whereas a
subsequent long unloading train releases the remaining labeled vesicles
(images C, D; data not shown). The fluorescence
signal F is defined as the difference in bouton
fluorescence measured before and after unloading (that is, images
A-B and C-D in the schematic). The total
F (FT
=F0 + F1)
signal represents the amount of dye taken up into a
releasable vesicular pool during the specified load. To examine the
role of MLCK on the release of FM1-43 from the total recycling pool, I
reloaded the boutons as in Figure 1 and then perfused in 30 µM ML-9, a potent and relatively specific inhibitor of
MLCK (Saitoh et al., 1987) (Fig. 2d). Then the boutons were
stimulated with a long unloading train of action potentials. Figure
2e shows that a large fraction of fluorescence remains in
the terminal after a long unloading stimulus train; further stimulation
with an additional 900 AP in the presence of ML-9 fails to release all
but 10% of the remaining fluorescence. Reperfusion of the boutons in
control saline, followed by additional unloading stimuli, returns the fluorescence to baseline (Fig. 2f). Finally, a third
round of dye loading and unloading in the absence of ML-9 reveals that the boutons recover completely from the drug exposure, indicating that
the effects of ML-9 are completely reversible. Thus ML-9 causes a
reversible, partial block of vesicle pool turnover during action
potential stimulation. The degree of block obtained varies across a
population of boutons. I define the efficacy of inhibition as the ratio
of the fraction of the total pool released during a long stimulus train
in the presence of ML-9
(F0/FT)ML-9
with that in control conditions
(F0/FT)control.
The distribution of inhibition values measured on a bouton by bouton
basis from a single experiment, like that shown in Figure 2, is
displayed in Figure 3 and indicates that
the mean efficacy of inhibition is 68 ± 2.4%. Higher-frequency
stimulation (20 Hz) for the same unloading period gives identical
values of inhibition (data not shown), suggesting that the blockade
cannot be overcome with stronger stimuli.
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Figure 2.
FM1-43 visualization of vesicle turnover block by
MLCK or myosin inhibition. a, A Nomarski image of
hippocampal culture showing a typical axodendritic network. Scale bar,
3 µm. b, Fluorescence of same field after a 900 AP dye
load. c, Same as in b, after a 900 AP
unload. d, Same as in b after a second
900 AP load. e, Same as in c after
unloading with 900 AP in the presence of 30 µM ML-9. A
large fraction of the fluorescence remains in the boutons.
f, The fluorescence retained in e is now
released with 900 additional AP applied after ML-9 washout.
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Figure 3.
Inhibition of vesicle pool mobilization by MLCK or
myosin inhibitors. The degree of inhibition from a typical experiment
as in Figure 1 was measured over a population of 44 synaptic boutons.
To control for possible rundown, I performed an additional control run
of loading and unloading. Boutons were selected for measurement on the
basis of their appearance in both control runs. A,
Histogram of ML-9 inhibition in a single experiment. Inhibition is
defined as (1  FML-90/Fcontrol0),
where Fcontrol0 is the
average fluorescence signal of the two control runs. B,
Percentage of vesicle pool remaining after 900 AP in 30 µM ML-9, 15 µM ML-7, 25 mM BDM,
or control saline. All three inhibitory conditions reduce the number of
vesicles turned over during prolonged AP stimuli. Slightly longer
wash-in and washout times (4 min) were used for experiments with ML-7,
a more hydrophobic compound than ML-9. Each concentration was measured
in at least two experiments over 40-120 boutons. C,
Dose-response relationship of ML-9 inhibition shown as the fraction of
the recycling vesicle pool remaining after 900 AP unloading stimuli.
The maximal block by ML-9 saturates for [ML-9] >25 µM
and shows a half-maximal inhibition of ~12.5 µM. The
inhibition at each concentration was measured in at least three
separate experiments over a total of 75-95 boutons.
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Similar results were obtained with a higher-affinity inhibitor of MLCK,
ML-7, as well as butanedione-monoxine (BDM), a known inhibitor of both
myosin II and myosin V ATPases (Cramer and Mitchison, 1995) (Fig.
3B). The inhibition of vesicle pool turnover by ML-9 is
dose-dependent (Fig. 3C), exhibits an IC50 value
(the concentration that produced 50% of maximal inhibition) of ~12.5
µM, and saturates for concentrations >30
µM. This is identical to the IC50 value of
ML-9 inhibition of myosin-dependent smooth muscle contraction (Saitoh
et al., 1987) as well as of Ca2+-dependent
phosphorylation of myosin light chain in human platelets (Saitoh et
al., 1986). The IC50 value for ML-7 inhibition in this assay is ~5 µM (data not shown). Together, these data
imply that MLCK and myosin act to control vesicle pool mobilization at
synaptic terminals.
The t1/2 for endocytosis is
unchanged by ML-9
The potential role of MLCK and myosin in controlling endocytosis
was investigated by measuring the time course of FM1-43 uptake after
action potential stimuli in the presence or absence of ML-9 or BDM. The
protocol used for these studies is depicted in the schematic of Figure
4 and is similar to previously published
protocols (Reuter and Porzig, 1995; Ryan and Smith, 1995; Wu and
Betz, 1996; Isaacson and Hille, 1997). As in Figures 1
and 2, the measurement proceeds in two phases: a loading phase,
followed by an unloading phase, in which maximal stimulation was used
to quantify F, the amount of dye taken up during the
loading. Dye loading was performed by stimulating with a train of 100 AP (10 sec at 10 Hz) and presenting FM1-43 after a delay
t with respect to the start of the stimulus. Sequential
measurements were performed, interspersing runs with t = 25 sec between bracketing runs with
t = 0 sec, correcting for possible fluctuations in
the response. The relative amount of loading with t = 25 sec was normalized with the mean values of the bracketing runs,
which measured the total amount of vesicular turnover for the given
stimulus at a single bouton. Measurements on different coverslips then
were repeated in the presence of ML-9 or BDM. I chose to compare the
time that corresponds with the approximate
t1/2 for completion of endocytosis in
control conditions for cultured hippocampal neurons (Reuter and Porzig, 1995; Ryan et al., 1996a; Isaacson and Hille, 1997). The
results, displayed in Figure 3, show that neither ML-9 nor BDM
significantly alters the time course of endocytosis. These results
imply that neither MLCK nor myosin activity regulates the endocytic
branch of synaptic vesicle recycling and indicate that inhibition of vesicle cycling likely acts upstream of endocytosis in the synaptic vesicle cycle. The time scale of endocytosis after longer loading stimuli (300 AP) gave identical results in the same three conditions (data not shown).
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Figure 4.
ML-9 and BDM do not block endocytosis.
Schematic, The protocol used to measure the time course
of endocytosis is shown. FM1-43 uptake at t = 25 sec after the beginning of a 100 AP stimulus train (10 Hz) normalized
to uptake at t = 0 sec is shown for three
conditions: 30 µM ML-9 (n = 38; two
experiments), 25 mM BDM (n = 66; two
experiments), and control saline (n = 44; two
experiments). FM1-43 was applied for 1 min and rinsed for 10 min before
the uptake was measured by using a long unloading train of AP as in
Figure 1.
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The size of the recycling vesicle pool is reduced significantly but
the kinetics of release of the pool is unchanged in ML-9
Two types of experiments were performed to characterize further
the impact of MLCK inhibition on vesicle pool turnover. Previous measurements of the release of FM1-43 from labeled vesicle pools during
action potential trains at 10 Hz indicate that kinetics of dye loss
exhibits single exponential behavior with a relaxation constant of
~200 AP (Ryan and Smith, 1995; Isaacson and Hille, 1997).
Figure 5 shows the kinetics of release of
FM1-43 from previously loaded synaptic terminals during 10 Hz of action
potential stimulation in varying concentrations of ML-9. The data are
normalized to the total fluorescence released in this run and a
subsequent run after dye washout, as in Figures 2 and 3. The time
course of turnover of the pool is very similar for the four conditions
shown (see figure legend); however, the total fraction of dye released
during a single 900 AP train is reduced gradually by increasing MLCK inhibition. At the highest concentration of ML-9 (30 µM),
a significant fraction of the fluorescence signal is retained, which
lowers the signal-to-noise ratio of this measurement of release
kinetics. However, in all conditions the primary impact of MLCK
inhibition appears to be in reducing the size of the total releasable
pool and not the kinetics of release of this pool. The reduction in pool size, without significant change in kinetics of release, implies
that the amount of dye released on a per-action-potential basis must be
proportional to the pool size. This is expected for any process obeying
first-order kinetics, as is the case here.
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Figure 5.
Kinetics of vesicle pool turnover during MLCK
inhibition. Synaptic boutons were loaded with FM1-43, using long AP
stimuli, before measuring the kinetics of release of the dye in the
presence or absence of varying concentrations of ML-9 (amplitude,
F0 in schematic of Fig. 1). A second
round of stimulation released the remaining fluorescence after
inhibitor washout (amplitude, F1 in
schematic of Fig. 1). For each bouton the fluorescence was normalized
to FT, the total releasable
fluorescence signal from both rounds of unloading. Shown here are
population averages of dye release kinetics from populations of boutons
of individual experiments in control saline, 10 µM ML-9,
15 µM ML-9, and 30 µM ML-9. The 15 µM and control experiments were performed sequentially on
the same boutons, whereas the data for each of the other concentrations
were obtained from different experiments. The accompanying control runs
for [ML-9] = 10 µM and [ML-9] = 30 µM
were identical to that for the 15 µM run (data not
shown). Fluorescence intensity kinetics for each bouton
i were fit by using the equation
Fi(t) = F0
e (tt0)/ i + F1, where
t0 marks the start of the stimulus and
F0 and F1
were fixed as the total fluorescence loss during each run. The
following values were obtained for the decay constants: control saline,
< > = 17.7 ± 0.4 sec (n = 39); [ML-9] = 10 µM, <> = 21.3 ± 1.2 sec
(n = 39); [ML-9] = 15 µM, <> = 20.1 ± 1.3 sec (n = 40); [ML-9] = 30 µM, <> = 22.8 ± 1.6 sec (n = 25), where the brackets indicate the average value
obtained over all i, and n is the
number of boutons measured for each experiment. At [ML-9] = 30 µM, certain boutons release too little fluorescence to
extract kinetic parameters (some boutons are near complete inhibition
at this concentration; see Fig. 3A). These were excluded
from the kinetic analysis performed here. The average inhibition in
these data is 22 ± 1.8% ([ML-9] = 10 µM),
35 ± 1.2% ([ML-9]= 15 µM), and 58 ± 2.4%
([ML-9] = 30 µM). The solid line shows
F(t), using the average values of
obtained in each case.
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The data in Figure 3C indicate that under conditions
of maximal inhibition 30-35% of the total pool is still released.
This partial block can be explained by a limited efficacy of ML-9
inhibition on MLCK, by the existence of a pathway for vesicle fusion
that is independent of MLCK, or by a combination of the two.
ML-9 inhibits release only after 20 AP
The data in Figure 5 indicate that MLCK inhibition reduces the
amount of FM1-43 released over a wide range of stimulus numbers (60-900 AP). I designed experiments to measure the impact of MLCK inhibition on vesicle pool turnover for lower numbers of AP. Two factors contribute to limiting the sensitivity of dye release measurements. First, the dissociation rate of FM1-43 from membranes is
too slow (~3 sec; Ryan et al., 1996a; Klingauf et al., 1998) to
measure accurately the release kinetics for times less than several
seconds. Second, fluctuations arising from photon-counting statistics
from fully loaded terminals limit the size of the detectable decreases
in fluorescence intensity. To maximize the detection sensitivity of
vesicle pool turnover for brief stimuli, I performed dye uptake
measurements (Ryan et al., 1997) to characterize the impact of MLCK
inhibition in this stimulus range. In this case a varied number of APs,
n, is used to load the dye into the vesicle pool, and a long
unloading train (900 AP) is used to measure the total uptake,
Fn (see top, Fig. 1), for a
particular load. As in Figure 1, the dye is left in the perfusate for
60 sec beyond the stimulus period to ensure that all vesicles that
undergo recycling in response to the action potential train become
labeled. Thus, although this method more directly measures dye uptake,
it should faithfully track the number, but not the timing, of
successful exocytotic events. A second round of loading and release
with 900 AP is used to normalize the uptake obtained for n
AP as a fraction of the total pool turned over, expressed as
Fn/F900. Previous measurements have shown that dye uptake and release
measurements agree quantitatively (Ryan and Smith, 1995), even at the
level of single vesicle turnover (Murthy and Stevens, 1998). Figure 6A shows the kinetics
of vesicle pool turnover derived from dye uptake measurements in the
0-75 AP range in control conditions. These data show two distinct
kinetic regimes for vesicle pool turnover as a function of action
potential number. In the range 0-20 AP vesicle pool turnover proceeds
at a rate of 0.75%/AP (± 0.08%/AP), whereas in the range of 20-75
AP the rate slows to 0.33%/AP (± 0.007%/AP).
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Figure 6.
MLCK inhibition increases with the number of
stimuli. Dye loading assays were used to measure the impact of ML-9
inhibition for low numbers of APs. Synaptic boutons were loaded with
varying numbers of AP, n, and unloaded with 900 AP to
determine Fn. A second run that used 900 AP loading and unloading was used to determine the fraction of the
vesicle pool turned over by n AP as
Fn/F900.
A, Fractional vesicle pool turnover in control
conditions measured over many experiments at various n
displays two kinetic regimes. Each regimen, 0-20 AP and 20-75 AP, was
fit with a linear dependence on n (dashed
lines). The total number of boutons for each n
varied between 26 and 148 obtained from at least two experiments for
each n. B, Inhibition produced by 30 µM ML-9 for different n. The degree of
inhibition was determined by measuring the amount of dye taken up by
n AP in the absence
(Fncontrol) or
presence (FnML-9) of
ML-9 sequentially in the same boutons. Inhibition is calculated as
(1 FnML-9/Fncontrol).
Shown here is the average over a population that varied between 30 and
61 boutons measured in at least two experiments at each
n. Inhibition is near zero for n  20 AP and gradually increases with the increasing number of AP. A
simple model to explain the use dependence of inhibition whereby
vesicle pool turnover is 85% inhibited for n > 20 AP, and not inhibited for n 20 AP, is shown
(thick dashed line).
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To determine the impact of MLCK inhibition on vesicle turnover in these
two kinetic regimes, I measured dye uptake sequentially in control
saline and in 30 µM ML-9 for varying numbers of action potentials, n. The uptake obtained in ML-9,
FnML-9, was normalized to
measurements of uptake obtained in the absence of ML-9,
Fncontrol, at the same
boutons. The degree of inhibition for a given number of action
potentials is given by (1 FnML-9/Fncontrol).
The amount of inhibition obtained for long action potential trains ( 200 AP) is identical to that obtained in direct release experiments, as
in Figures 2 and 3. These data show that, for stimulus trains <20 AP,
ML-9 does not inhibit vesicle pool turnover. The block caused by ML-9
gradually increases for stimulus trains >20 AP. As in the case with
the overall inhibition of dye release, BDM has a qualitatively similar
effect. Lower numbers of stimuli are less inhibited by BDM (33%
inhibition for 40 AP) than longer action potential trains (48%
inhibition for 600 AP) in triggering vesicle pool turnover (Fig.
7).
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Figure 7.
Inhibition by BDM is use-dependent. Dye uptake
measurements in control saline or 25 mM BDM for 40 and 600 AP were measured sequentially at 46 individual boutons. The total
amount of releasable dye taken up during the dye exposure period,
F, is shown. As in Figure 6, the inhibition is
greater for longer stimuli. The inhibition for 40 AP is 33 ± 7%.
The inhibition for 600 AP is 48 ± 7%. Similar results were
obtained in three of three experiments.
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DISCUSSION |
I have shown measurements of the impact of inhibitors of MLCK,
primarily the organic inhibitor ML-9, on vesicle pool mobilization during action potential trains. A major concern with such
pharmacological studies is the specificity of action of the drug of
use. ML-9 and ML-7 are naphthalene sulfonamide derivatives that inhibit MLCK by competing with ATP binding (Saitoh et al., 1987). Although both
compounds also will inhibit protein kinase C (PKC) and protein kinase A
(PKA), the IC50 values determined here for MLCK inhibition are well below the in vitro values of the
Ki of inhibition for these kinases (Saitoh et
al., 1986). The Ki for inhibition of MLCK
in vitro by ML-9 (~4 µM) is eightfold lower
than that for inhibition of PKA and 14-fold lower than that for
inhibition of PKC. Similarly ML-7 discriminates against PKA by a factor
of 70 over MLCK and by a factor of 140 over PKC. In the experiments shown here, the effects of ML-9 and ML-7 have reached saturation below
the concentration at which it is expected to block PKC or PKA (see Fig.
3C). ML-9 also has been shown to inhibit L-type Ca2+ channels with an IC50 value of
~50 µM (Reig et al., 1993); however, vesicle pool
turnover is not mediated by this channel subtype in hippocampal neurons
(Reuter, 1995). Furthermore, the finding that ML-9 does not alter
release kinetics, but instead blocks the size of the releasable pool
(see Fig. 5), is not explained easily by the simple block of
Ca2+ entry. The concentration range for which ML-9
is effective is most consistent with inhibitory action on MLCK. I
cannot rule out the possibility that ML-9 and ML-7 are inhibiting as
yet unknown kinases. However, the fact that very specific
autoinhibitory peptides of MLCK block a step in vesicle recycling
(Mochida et al., 1994), taken together with the effect of ML-9 in an
appropriate concentration range, strongly suggests that the predominant
substrate for ML-9 action in these experiments is MLCK. I also have
shown that the myosin ATPase inhibitor BDM behaves in a qualitatively
similar manner, albeit at much higher concentrations. At present there are no high-potency inhibitors of myosin, and the specificity of action
of BDM in these assays is much less certain. Nonetheless, the general
agreement with the MLCK inhibitor data is consistent with a role of
both actin-myosin interactions and MLCK in the mobilization of
synaptic vesicles. Higher concentrations of ML-9 ( 50 µM) cause a modest increase in spontaneous vesicle
recycling in addition to the block of vesicle pool mobilization (data
not shown). In this concentration range, however, both PKA and PKC are
expected to be inhibited.
Previous studies have indicated that kinase inhibition by staurosporine
also blocks the efficiency of fluorescence destaining in nerve
terminals labeled with endocytic tracers (Henkel and Betz, 1995;
Klingauf et al., 1998). In that case, however, staurosporine failed to
block significantly either the dye uptake or the neurotransmitter release. These data led to the hypothesis that staurosporine inhibition resulted in a drastic reduction in the time scale of endocytosis and
not simply in the amount exocytosis. In this scenario, the unbinding
time of FM dyes would exceed the time frame of endocytosis, but the
time of dye binding would not. In the experiments shown here, the
degree of inhibition obtained with ML-9 in dye uptake is identical to
that achieved with dye release for prolonged trains (see Fig.
5B). Furthermore, the t1/2 for
labeling vesicles after a stimulus was unaltered by ML-9 exposure (see
Fig. 4). Thus ML-9 does not appear to act on the endocytic branch of
vesicle recycling but, rather, upstream of these events. Recently,
Kuromi and Kidokoro (1998) demonstrated that during endocytosis a
distal reserve pool could be replenished in a cytochalasin D-dependent manner. The equivalent of this pool in the hippocampal synaptic terminals studied here is still unclear, because it is accessed only in
the Drosophila neuromuscular junction by first blocking endocytosis and then depleting the total pool by stimulation of the
presynaptic terminal. Here I find no evidence for myosin dependence during endocytosis; however, this does not rule out the possibility that such a pathway can be engaged under some yet-to-be-uncovered conditions.
I present data indicating that vesicle pool turnover for 20 or fewer AP
is more efficient on a per-action-potential basis than for longer
stimulus trains (see Fig. 6A). This is consistent with recent findings (Dobrunz and Stevens, 1997) that the vesicles recruited for exocytosis by the first 10-20 AP represent a readily releasable pool and correspond closely to the total number of docked
vesicles (Stevens and Tsujimoto, 1995). In the data presented here, the
vesicles recruited for exocytosis by the first 20 AP correspond to 17%
of the total recycling pool. This exceeds the estimated number of
docked vesicles in cultured hippocampal neurons by a factor of two
(Schikorski and Stevens, 1997); however, differences in culture age and
preparation might cause this number to vary. My findings that ML-9
inhibition begins only after the first 20 AP suggest that MLCK is
involved directly in replacing docked vesicles after their depletion.
Further experiments, however, correlating the vesicles consumed during
this period of stimulation with the ultrastructurally docked vesicles
will be required to determine whether these two pools of vesicles are
identical. The initial pool released during the first 20 AP represents
17 ± 1.2% of the total recycling pool, yet during maximal
inhibition with long action potential trains ~30-35% of the labeled
pool is turned over. Thus, during the slower phase of vesicle pool
turnover, an additional 13-18% of the pool appears to be mobilized by
an MLCK-independent pathway or escapes blockade by ML-9. At present, my
experiments cannot distinguish between these two possibilities. The
dashed line in Figure 6B is derived from a simple
model in which vesicle fusion proceeds for the first 20 AP via an
MLCK-independent pathway and thereafter via a pathway that can be 85%
inhibited by block of MLCK.
These data are consistent with several previous findings. In
sympathetic neurons (Mochida et al., 1994) the injection of an inhibitory MLCK peptide-pseudosubstrate resulted in an
activity-dependent block of neurotransmitter release. In chromaffin
cells MLCK inhibitors appear to block a step before the priming of
exocytotic vesicles (Kumakura et al., 1994). The experiments presented
here suggest that MLCK activity is required specifically to deliver
vesicles to a readily releasable pool in synaptic terminals. The
pharmacologically induced phenotype described here is reminiscent of
the effects of ablation of the synapsin I gene. In synapsin I-deficient
synaptic terminals the size of the recycling pool is reduced as
compared with wild type, but the kinetics of release of the pool is not (Ryan et al., 1996b). Thus, MLCK and synapsin may be involved in
closely related steps in the synaptic vesicle cycle; alternatively, ML-9 directly might block the action of a different unknown kinase important for synapsin function.
Because MLCK is regulated tightly by
Ca2+/calmodulin, this enzyme potentially provides a
critical link between the elevation of intracellular
Ca2+ leading both to membrane fusion and consumption
of synaptic vesicles with replacement of those vesicles from a reserve
pool. The specific myosin substrate for MLCK in synaptic terminals
currently is unknown, although both myosin II and myosin V have been
localized to this cellular region (Espreafico et al., 1992; Mochida et
al., 1994; Prekeris and Terrian, 1997).
Although these data are most consistent with a role of MLCK in the
movement of vesicles through the cluster during sustained stimulation,
my experiments do not rule out a possible additional role of MLCK in
the postendocytotic movement of newly formed vesicles back to the
synaptic vesicle cluster (repriming).
Myosin-based transport of synaptic vesicles thus likely appears to be
required for the mobilization of a large fraction of the recycling
vesicle pool during repetitive action potential firing in synaptic
terminals. The data presented here indicate that engagement of this
pool is regulated by MLCK, the activity for which has been shown to be
modulated by a number of signal transduction pathways (Gallagher et
al., 1997). This MLCK pathway thus is poised to serve as a sensitive
substrate for the modulation of presynaptic function.
| |
FOOTNOTES |
Received Oct. 1, 1998; revised Nov. 23, 1998; accepted Dec. 1, 1998.
This work was supported by National Institutes of Health Grant NS
36942. I thank F. Maxfield, T. McGraw, P. Greengard, and H. Reuter for
useful discussions during the course of this work and M. Delemos for
excellent technical assistance.
Correspondence should be addressed to Dr. Timothy A. Ryan, Department
of Biochemistry, Room E-107, The Weill Medical College of Cornell
University, 1300 York Avenue, New York, NY 10021.
| |
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Top
Abstract
Introduction
