1Department of Biological Sciences, University of Denver, Denver 80208; and 2Department of Physiology and Biophysics, University of Colorado Medical School, Denver, Colorado 80262
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ABSTRACT |
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Angleson, J. K. and
W. J. Betz.
Intraterminal Ca2+ and Spontaneous Transmitter
Release at the Frog Neuromuscular Junction.
J. Neurophysiol. 85: 287-294, 2001.
We investigated
the relationship between intraterminal Ca2+
concentration ([Ca2+]i)
and the frequency of miniature end plate potentials (MEPPs) at the frog
neuromuscular junction by use of ratiometric imaging of fura-2-loaded
nerve terminals and intracellular recording of MEPPs. Elevation of
extracellular [KCl] over the range of 2-20 mM resulted in increases
in [Ca2+]i and MEPP
frequency. Loading terminals with the fast and slow Ca2+-buffers
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in
equivalent reductions in the KCl-dependent increases in MEPP frequency.
The [Ca2+]i dependence of
MEPP frequency determined by elevation of
[Ca2+]i due to
application of 0.1-10 µM ionomycin was similar to that determined
when [Ca2+]i was raised
by increasing extracellular KCl. Measurements in 10 mM extracellular
KCl revealed that application of the phorbol ester phorbol 12 myristate
13-acetetate (PMA) caused an increase in MEPP frequency while the
inactive analogue, 4-PMA, did not. PMA application also caused an
increase in [Ca2+]i. The
relationship between
[Ca2+]i and MEPP
frequency in PMA was the same as was determined by the other methods of
raising [Ca2+]i. Under
all conditions tested, our data revealed a low
[Ca2+]i threshold for
activation of transmitter release and are consistent with a
Kd for
[Ca2+]i on the order of 1 µM.
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INTRODUCTION |
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The cellular and molecular
complexities of the processes involved in
Ca2+-dependent secretion are beginning to be
unraveled in detail (reviewed in Angleson and Betz 1998;
Neher 1998
; Zucker 1996
).
Ca2+ plays an essential role in triggering evoked
neurotransmitter (Katz 1969
). Influx of
Ca2+ during an action potential through
Ca2+ channels located in the plasma membrane of
the presynaptic terminal raises intraterminal
Ca2+ concentration
([Ca2+]i), which then
triggers exocytosis and transmitter release. A quantitative
understanding of the [Ca2+] dependence of
transmitter release requires knowledge of the Ca2+ affinities of Ca2+
sensors required for transmitter release and the spatial relationships between the site of Ca2+ influx and
Ca2+ sensors. Different models exist for the
spatial relationship between Ca2+ channels and
the Ca2+ sensors for release. The single-channel
domain hypothesis places the transmitter release site close (~10 nm)
to a single Ca2+ channel where the
Ca2+ sensor for release experiences a restricted,
short-lived domain of high [Ca2+] (~100
µM). The overlapping domain hypothesis places the release sites at
locations more remote (100-200 nm) from Ca2+
channels where [Ca2+] reaches concentrations of
<10 µM and where the Ca2+ sensor is influenced
by Ca2+ influx from multiple channels (for
reviews, see Neher 1998
; Stanley 1997
).
Neurons that undergo synchronous or evoked release of transmitter in
response to an action potential also display asynchronous and
spontaneous transmitter release. At the frog neuromuscular junction,
there is evidence that the synchronous, evoked end plate potentials
(EPPs) may have different Ca2+ requirements and
be subject to distinct regulation from asynchronous release or the
spontaneous miniature end plate potentials (MEPPs). For example, there
appears to be different divalent cation requirements for the different
types of release (Zengel and Magleby 1981). In addition
to Ca2+ stimulation of MEPPs, it has been
reported that the frequency of MEPPs can be increased by
Ca2+-independent mechanisms (e.g., Chen
and Grinnell 1997
; Kijima and Tanabe 1988
).
The ability to simultaneously monitor transmitter release and measure
intraterminal [Ca2+] with fluorescent
indicators at the frog neuromuscular junction (Narita et al.
1998; Robitaille et al. 1996
; Wu and Betz
1996
) can allow for direct assessment of the
Ca2+ dependence of MEPPs. We have used
ratiometric imaging of fura-2 in presynaptic terminals together with
intracellular recordings of MEPPs to investigate the relationship
between presynaptic
[Ca2+]i and spontaneous
transmitter release at the frog neuromuscular junction. Various
treatments that alter both MEPP frequency and [Ca2+]i gave the same
[Ca2+]i dependence of
transmitter release. The results suggest that MEPP frequency is
stimulated by a low-threshold, high-affinity Ca2+
sensor that senses Ca2+ from multiple neighboring
Ca2+ channels during mild depolarization. A
preliminary report of this study has appeared in abstract form
(Angleson and Betz 1997
).
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METHODS |
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Preparation and solutions
Frog (Rana pipiens) cutaneous pectoris nerve-muscle
preparations were dissected and mounted in silicone elastomer
(Sylgard)-lined chambers containing normal frog Ringer [which
contained (in mM) 115 NaCl, 1.8 CaCl2, 2 KCl, 5 HEPES-NaOH, pH 7.2]. High-KCl solutions were prepared by elevating
extracellular KCl concentration ([KCl]o) to the
indicated level with an offsetting reduction in [NaCl] to keep all
solutions isotonic. Ionomycin (Calbiochem), prepared as a 5 mM stock in
dimethyl sulfoxide (DMSO) was added to normal frog Ringer at a
concentration of 0.1-10 µM immediately before use. The phorbol
esters phorbol 12 myristate 13-acetetate (PMA) and 4-phorbol 12 myristate 13-acetetate (4
-PMA; Biomol, Plymouth Meeting, PA) and the
protein kinase inhibitor staurosporine (Sigma) were prepared as
concentrated stock solutions in DMSO. Neostigmine (1 µg/ml; Marsam
Pharmaceuticals, Cherry Hill, NJ) was added to some experiments to
increase MEPP amplitude. It did not effect MEPP frequency. All
solutions used for electrophysiology or Ca2+
imaging contained 1 µM tetrodotoxin (Sigma) to block
Na+-dependent action potentials.
The membrane permeable acetoxymethyl ester form of the
Ca2+ chelators EGTA-AM and
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM, Molecular Probes, Eugene, OR) were stored in stock
solutions (5 mM) in DMSO. The standard conditions for loading neuromuscular preparations for our studies were based on those described by Robitaille et al. (1993) and consisted of
incubation in normal frog Ringer with 25 µM EGTA-AM or BAPTA-AM for
60 min at room temperature (20-23°C). The preparation was then
washed with normal frog Ringer before use. Increasing the concentration to 100 µM, the incubation time to 2 h or the temperature to
~30°C did not improve the apparent effectiveness of the loading.
Control incubation with 0.5% DMSO did not significantly alter MEPP
frequency in our experiments.
Ca2+ imaging
The method for loading nerve terminals with membrane impermeant
pentapotassium salt of fura-2 (Molecular Probes) was similar to that
first described for loading bullfrog sympathetic nerve terminals
(Peng and Zucker 1993) and has been described in detail for this preparation (Wu and Betz 1996
). A short piece
of freshly cut nerve trunk was placed into a drop of 50 mM of fura-2
salt. It was important to place the nerve into the fura-2 solution
within ~5 min of cutting to allow exchange into the nerve, longer
time intervals prevented efficient loading, presumably due to resealing of the nerve. Fura-2 was then allowed to diffuse down the cut nerve for
2-3 h at room temperature (20-23°C) in normal Ringer. The
preparation was rinsed and incubated overnight in normal frog Ringer at
4°C and used the following day for imaging and electrophysiology. The
loading and incubation procedures had no detectable effect on MEPP
frequency or amplitude either at rest or in elevated KCl solutions and
the loading conditions were previously found not to have an effect on
EPP amplitude (Wu and Betz 1996
).
Fura-2-loaded terminals were imaged with a Sensys-cooled CCD camera
(Photometrics, Tucson, AZ) on a Nikon Optiphot upright microscope using
a Zeiss long working distance ×40 water-immersion lens (0.75 N.A.).
Excitation was alternated between 360- and 380-nm band-pass
filters (Chroma Technology) using a Sutter Lambda-10 filter
wheel. Emitted light was collected through 510/40 dichroic and 400-nm
long pass filter. Images (16 bit) were collected on a PC running
program written in Vwindows (Photometrics) and analyzed off-line on a
Silicon Graphics O2 workstation running software from G. W. Hannaway (Boulder, CO). The outline of a terminal from one image was
drawn by hand and the integral fluorescence intensity for the pair of
images was determined. No noticeable or consistent "hot spots" of
elevated [Ca2+]i were
detected. Care was taken to exclude regions of preterminal axon that do
not demonstrate depolarization-dependent changes in fura-2 signal
(Wu and Betz 1996). Background fluorescence from a
region without nerve terminal was subtracted from each image before
ratio fluorescence calculations. Intraterminal
Ca2+ concentration
([Ca2+]i) was estimated
from the fura-2 ratios (Gryzienski et al. 1985
) using
the procedures and in vitro calibrations described in Wu and
Betz (1996)
. The fura-2 concentration in the terminals was estimated to be 5-50 µM using methods detailed in Wu and Betz (1996)
.
Electrophysiology
Intracellular recordings of MEPPs from surface muscle fibers
were performed with micropipettes (15-20 m ) filled with 3 M potassium acetate using standard techniques (Wu and Betz
1996
). Sweeps of between 25- and 2,024-ms duration were
collected with an acquisition time of between 0.1 and 0.25 ms and
stored and analyzed on a PC running software written in Axobasic. For
conditions with stable and low MEPP frequencies (2 or 10 mM
[KCl]o), MEPPs were acquired intermittently
over a ~120-s period. For high-MEPP frequency conditions (16 or 20 mM
[KCl]o or ionomycin), MEPPs were acquired
during a sufficient number of sweeps of 25- to 250-ms duration to yield
200-300 MEPPs to be counted to determine a frequency. When recordings
were made in 2 or 10 mM [KCl]o, the MEPP
frequency was constant over the entire period sampled (
30 min). MEPPs
in 16 or 20 mM [KCl]o were acquired at points
between 20 and 40 s after switch to the indicated solution. This
time interval was chosen to avoid muscle contractions that often
occurred in 20 mM [KCl]o during the first ~15
s after switch to this solution and to avoid recording at after
prolonged exposure to high [KCl]o; something
that has been reported to cause run down of MEPP frequency.
Fura-2 images were recorded at the same time interval as MEPP recording
for each experimental condition. For ionomycin experiments, MEPP
recording and fura-2 imaging were performed simultaneously from the
same neuromuscular junction throughout the first ~60 s of ionomycin
treatment. Because both MEPP frequency and
[Ca2+]i increase
constantly during this period (Ravin et al. 1997), a
single data point represents the MEPP frequency and fura-2 signal recorded simultaneously during a given 3-s period. Between 1 and 5 such
points were acquired from a terminal in a typical ionomycin experiment.
The higher concentrations of ionomycin (~10 µM) often resulted in
contraction of the muscle fiber after 30-60 s, so data were collected
only briefly after addition of high ionomycin concentrations.
MEPP frequency was analyzed off-line with the aid of software written in Axobasic. The software employed a peak-detection routine that aided in detection of a MEPP. Each peak was displayed on a time scale that allowed visual inspection to determine whether an event had the characteristic rise and decay times of a MEPP or was a noise artifact. Superimposed MEPPs in which a separate peak could be clearly distinguished riding on another were counted as separate events.
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RESULTS |
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MEPP frequency and intraterminal [Ca2+] measured at various extracellular [KCl]
Both the frequency of MEPPs and the average [Ca2+]i demonstrated a strong dependence on [KCl]o (Fig. 1, A and B) presumably due to the opening of voltage activated Ca2+ channels. The relationship between the average values of [Ca2+]i and MEPP frequency measured between 2 and 16 mM [KCl]o demonstrated greater than a 100-fold increase in neurotransmitter release rates with relatively small increases in [Ca2+]i (Fig. 1). If the [Ca2+]i estimated with fura-2 is the same as that present at the release sites, then this result suggests a low threshold for the Ca2+ requirement for activation of transmitter release.
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Slow Ca2+ chelator EGTA reduces the [KCl]o dependence of MEPP frequency
If intracellular microdomains of elevated
[Ca2+] near the mouth of open
Ca2+ channels overlapped with a
Ca2+ sensor responsible for synaptic vesicle
fusion, then the fura-2 estimates of
[Ca2+]i, which reflect
global [Ca2+] in the terminal, may not be an
appropriate measure for determining the Ca2+
dependence of MEPP frequency. To test this possibility, we compared the
effect of the slow Ca2+ buffer EGTA with the
faster buffer BAPTA on the [KCl]o dependence of
MEPP frequency. These two Ca2+ chelators, which
have a similar Kd for
Ca2+, differ greatly in their rates of binding
Ca2+ ions, BAPTA being ~150-fold faster than
EGTA under physiological conditions (Naraghi 1997).
Therefore BAPTA should reduce Ca2+ more than EGTA
does in transient microdomains. Terminals were loaded with the membrane
permeant esters EGTA-AM or BAPTA-AM or with the control solution (0.5%
DMSO) under otherwise identical loading conditions (see
METHODS). Both Ca2+ chelators reduced
the [KCl]o dependence of MEPP frequency by a
similar amount (Fig. 2), suggesting that
EGTA and BAPTA are equally effective at reducing
[Ca2+]i at transmitter
release sites. This result argues that single-channel domains of high
[Ca2+] are not required to overlap the
transmitter release sites since, in this case, the faster buffer would
more effectively reduce a Ca2+ microdomain.
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While the Ca2+ chelators EGTA and BAPTA did weakly reduce MEPP frequency under our "resting" conditions (2 mM [KCl]o), these chelators did not abolish MEPPs. These residual MEPPs could be due to Ca2+-independent events. However, because [Ca2+]i measured at rest is 100 ± 40 (SE) nM and the Kd for Ca2+ of both chelators (present at unknown concentrations due to AM-ester loading) is on the order of 100 nM, it is not possible to definitively state that these are Ca2+-independent events.
Most importantly, the strong inhibition of transmitter release measured
at 16 mM [KCl]o by these chelators indicates
that our fura-2 estimates of
[Ca2+]i are a reliable
measure of Ca2+ concentration at release sites
for the experiments using at least
16 mM
[KCl]o. The weaker inhibition of transmitter
release by the two chelators at 20 mM [KCl]o
either may reflect a partial contribution of restricted
Ca2+ domains under these stronger depolarizing
conditions that may activate additional channel types or may reflect
Ca2+ loading that surpassed the buffering
capacity of the exogenous chelators. The equivalent effect of the two
chelators even at 20 mM [KCl]o supports the
latter possibility.
These conclusions depend on the amounts of EGTA and BAPTA in the
terminals being approximately equal. If the final concentration of free
EGTA in the terminals was ~150 times higher than the free BAPTA
concentration, then the similar effects of these buffers on the
[KCl]o dependence of MEPP frequency (Fig. 2)
would not necessarily support the lack of a
high-Ca2+ microdomain overlapping release sites.
Several observations argue against the observed effects being due to
such preferential loading of EGTA over BAPTA. Increasing the time of
BAPTA-AM loading (2 h), the concentration of BAPTA-AM (up to 100 µM), or the temperature of loading (to ~30°C) did not increase
the effectiveness of BAPTA reduction in MEPP frequency (see
METHODS). In addition, Robitaille et al.
(1993)
studied the spatial relationship between voltage-gated Ca2+ channels and
Ca2+-activated potassium channels in frog motor
nerve terminals using EGTA-AM- and BAPTA-AM-loading conditions that
were essentially identical to those reported here. They found that
loading terminals with BAPTA-AM blocked
Ca2+-activated K+ channels
while EGTA-AM was completely ineffective (Robitaille et al.
1993
). This result indicates that loading of membrane permeant BAPTA and EGTA can be used to detect the presence of processes dependent on spatially restricted domains of high
Ca2+ in this preparation.
[Ca2+]i dependence of MEPP frequency in ionomycin-treated terminals
The Ca2+ ionophore ionomycin offers an
additional, independent method to determine the
[Ca2+]i dependence of
MEPP frequency. This approach precludes both development of
Ca2+ microdomains selectively around
voltage-gated Ca2+ channels or a substantial
change in membrane potential. The fura-2-estimated [Ca2+]i dependence of
MEPP frequency from terminals treated with 0.1-10 µM ionomycin
(constant extracellular CaCl2 of 1.8 mM) is
reported in Fig. 3A. It is
evident that for values of
[Ca2+]i at least 1 µM
that this relationship is similar to that found when
[Ca2+]i was elevated by
increasing [KCl]o. Even for values of
[Ca2+]i of 1-5 µM (an
inherently less accurate value due to the relatively low
Kd for Ca2+ of
fura-2), the Ca2+-dependence of MEPP frequency is
not significantly different for high K+ or
ionomycin stimulations. These results with ionomycin further validate
the conclusions based on data obtained by altering
[KCl]o.
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Estimation of Kd
While lack of clear saturation of the Ca2+
dependence of MEPP frequency prohibits a reliable determination of the
Kd for Ca2+
required for transmitter release, estimates of the value of
Kd can be made from these data based
on rearrangement of the standard Hill equation and an estimate of the
Hill coefficient (Ravin et al. 1996). The standard Hill
equation
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(1) |
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(2) |
Another method of estimating Kd is by using
Eq. 1 and assumed values of fMEPPmax.
The highest MEPP frequency we measured was 300 s1, which can serve as a
lower limit for fMEPPmax. Quantal release rates
of 1,000's s
1 have been
reported based on noise analysis of end plate potentials of black widow
spider venom-treated frog neuromuscular junctions (Fesce et al.
1986
). While the release events from venom-treated nerve
terminals do not reflect the Ca2+-dependent
transmitter release under investigation here, they do provide for a
reasonable upper limit for estimates of fMEPPmax. Fig. 3C and Table 1
demonstrate that regardless of the fMEPPmax used
in Eq. 1, these data are consistent with a
Kd
5 µM. The analysis described
in Fig. 3C indicates the data are best described with a Hill
coefficient of 1-2, while there is abundant evidence that evoked
release from this preparation is described with a Hill coefficient of
3-4 (see VanderKloot and Molgò 1994
). We repeated
these analyses with Hill coefficients fixed at values of 1, 2, 3, and 4 and with fMEPPmax fixed at values of 350, 500, and 1,000 s
1 (Table 1).
The relative
2 values from these analyses
indicate that the data are best described with a Hill coefficient of
1-2. This finding regarding the value of the Hill coefficient is
consistent with other studies in which the apparent Hill coefficient
for MEPPs could be described by a value less than that of EPPs
(Andreu and Barrett 1980
) but is different from studies
at the crayfish neuromuscular junction that indicated MEPP frequency
followed a fourth power Ca2+ relationship
(Ravin et al. 1997
). Our main conclusion from the analysis of our data is that the Ca2+-dependent
increase in MEPP frequency is consistent with a
Kd for
[Ca2+]i on the order of 1 µM.
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Changes in MEPP frequency due to phorbol esters could be attributed to elevated [Ca2+]i
Compounds that can affect the activity of protein kinases,
including protein kinase C (PKC), have been reported to alter various types of neurotransmitter release at neuromuscular junctions (e.g., Redman et al. 1997; Shapira et al. 1987
;
for review, see VanderKloot and Molgò 1994
).
Potentiating effects of PKC on evoked transmitter release under certain
conditions has been reported to be Ca2+
independent (Redman et al. 1997
). Since we were in
position to directly test for possible Ca2+
independence of one type of transmitter release, we asked the question:
can pharmacological manipulation of PKC activity cause Ca2+-independent increase in MEPP frequency under
our experimental conditions?
We found that treatment of neuromuscular preparations with the protein
kinase activator PMA (400 nM) increased the MEPP frequency observed in
10 mM KClo (Fig.
4A). Identical treatment with
the related phorbol ester 4-PMA, which does not activate protein kinases, did not alter MEPP frequency (Fig. 4A). Incubation
of neuromuscular preparations with 500 nM staurosporine (a nonspecific protein kinase inhibitor at this concentration) reduced the MEPP frequency both at rest and in conditions of elevated
[KCl]o (Fig. 4), consistent with previous
results demonstrating that staurosporine treatment reduced the elevated
MEPP frequency observed following tetanic stimulation (Henkel
and Betz 1995
). The lack of an effect of 4
-PMA and the
opposite effect of staurosporine suggest that the elevation of MEPP
frequency by PMA is due to protein phosphorylation. These results are
consistent with other studies suggesting that active phorbol esters
enhance neurotransmitter release at frog neuromuscular junctions
(Redman et al. 1997
; Shapira et al.
1987
).
|
To test whether the enhancement of neurotransmitter release in PMA is
due to an increase in
[Ca2+]i or to a
Ca2+-independent mechanism, we performed fura-2
measurements before and after PMA treatment. Under conditions identical
to those used to measure effects on MEPP frequency, PMA caused an
increase in [Ca2+]i (Fig.
4B). The relationship between
[Ca2+]i and MEPP
frequency in PMA-treated terminals was similar to that described for
both high [KCl]o and ionomycin experiments (Fig. 3A, ). This result suggests that in frog motor
nerve terminals, PMA increases MEPP frequency by elevating
[Ca2+]i and does not have
to exert a Ca2+-independent action on any
component of release machinery associated with the type of release we
measured. Our results do not rule out a
Ca2+-independent enhancement of evoked release
(Redman et al. 1997
).
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DISCUSSION |
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Low-threshold, high-affinity Ca2+ dependence of transmitter release
We investigated the relationship between intraterminal
[Ca2+]i and the frequency
of MEPPs at the frog neuromuscular junction. Raising
[Ca2+]i from resting
values of ~100 nM to 1 µM markedly increased the frequency of
MEPPs. This is in full agreement with studies at crayfish neuromuscular
junctions in which
[Ca2+]i was raised
uniformly by either photolysis of caged Ca2+
(Mulkey and Zucker 1993) or by application of ionomycin
(Ravin et al. 1997
). Together these results indicate a
low threshold for Ca2+ in the activation of
asynchronous transmitter release at these synapses. This is comparable
to the low-Ca2+ requirement for exocytosis
revealed by patch-clamp capacitance measurements of synaptic vesicle
fusion in rod photoreceptor cells (Rieke and Schwartz
1996
) and for secretory granule fusion in chromaffin cells
(detected at [Ca2+]i
<0.2 µM) (Augustine and Neher 1992
). These apparent
low-threshold Ca2+ requirements are in striking
contrast to the high threshold of [Ca2+]i >20 µM
[Ca2+]i required for
activation of exocytosis found in retinal bipolar nerve terminals
(Heidelberger et al. 1994
; von Gersdorff and
Matthews 1994
).
Direct measurements of the Kd for
Ca2+ required for transmitter release or vesicle
fusion have only been made in a few preparations. Our estimates of a
Kd on the order of 1 µM for the
Ca2+ requirement for MEPP frequency implicate a
high-affinity Ca2+ binding site for the sensor
responsible for this transmitter release. Ravin et al.
(1997) found the Kd for
Ca2+ required for asynchronous transmitter
release following evoked release at crayfish neuromuscular junctions to
be 2-4 µM. How do these studies of asynchronous release relate to
other studies of Ca2+-dependent exocytosis?
Direct measurements of evoked release at a fast central synapse have
demonstrated that an intraterminal [Ca2+] of
just a few micromolar is required to trigger release (Bollmann et al. 2000
; Schneggenburger and Neher 2000
).
Studies using membrane capacitance as a measure of secretory granule or
synaptic vesicle fusion with the plasma membrane while uniformly
raising [Ca2+]i by
photolysis of caged Ca2+ revealed a
Kd in the range of 7-21 µM for
bovine chromaffin cells (Heinemann et al. 1994
), 27 µM
for pituitary melanotrophs (Thomas et al. 1994
), and
~200 µM for retinal bipolar nerve terminals (Heidelberger et
al. 1994
). This wide range in reported
Ca2+ affinities required for activation of
transmitter release likely reflects involvement of distinct
Ca2+ sensors.
Diffusion of Ca2+ from Ca2+ channels to transmitter release sites
The short delay between a presynaptic action potential and a
postsynaptic response at the neuromuscular junction suggests a close
spatial relationship between the site of Ca2+
influx and the Ca2+ binding site that triggers
evoked transmitter release (reviewed in Vander Kloot and
Molgò 1994). Theoretical considerations predict a local
domain of high Ca2+ concentration around an open
Ca2+ channel (e.g., Fogelson and Zucker
1985
; Simon and Llinás 1985
; Yamada
and Zucker 1992
). Such models predict short-lasting
(~100 µs) domains of Ca2+ that reach values
as high as a few hundred micromolar in a region
20 nm from the mouth
of a Ca2+ channel. The existence of such domains
of high Ca2+ during an action potential have been
demonstrated in the presynaptic terminal of the squid giant synapse
(Llinás et al. 1992
) although the limits placed by
the resolution of light do not allow for the measured domains to be
assigned to a single channel. The location of the
Ca2+ sensor for transmitter release relative to
domains of high [Ca2+] around
Ca2+ channels has important implications for the
Ca2+ dependence of release. If the sensor is <20
nm from the mouth of a Ca2+ channel, it would be
exposed to very high [Ca2+] (~100 µM) for
tens of microseconds and would be dominated by Ca2+ from the Ca2+ channel,
whereas at distances of 100-200 nm from a channel the sensor would be
exposed to [Ca2+] of <10 µM for up to 10 ms
and would sense Ca2+ overlapping from several
channels (reviewed in Neher 1998
; Stanley 1997
).
There is good evidence for clustering of Ca2+
channels at release sites of the neuromuscular junction. It has been
suggested that large intramembranous particles evident at active zones
in freeze fracture electronmicroscopy are Ca2+
channels (e.g., Heuser and Reese 1981) and fluorescently
labeled toxins that specifically bind to Ca2+
channels intensely stained active zones of the neuromuscular junction
(Robitaille et al. 1990
). However, these approaches do not allow for the functional relationship between
Ca2+ domains and transmitter release to be addressed.
The "single-channel-domain" and "overlapping-domain" hypotheses
can be experimentally distinguished by determining the
Ca2+ dependence of transmitter release and by
addition of exogenous Ca2+ chelators
(Neher 1998). Our finding that EGTA and BAPTA were equally effective at competing with the release sensor for binding Ca2+ when
[Ca2+]i was elevated by
mild depolarizations (Fig. 2) suggests that a single-channel domain of
Ca2+ was not responsible for increases in
spontaneous release at least when low-threshold
Ca2+ channels were responsible for
Ca2+ influx. This conclusion is further supported
by our finding that the
[Ca2+]i dependence of
release was similar when Ca2+ was elevated by
either activation of voltage-activated Ca2+
channels or application of ionomycin, which will not form
Ca2+ domains related to
Ca2+ channels. Under our depolarizing conditions
([KCl]o of 10-20 mM), we may have activated
Ca2+-induced Ca2+ release as has been
reported to occur during trains of action potentials and found to
enhance asynchronous release in this preparation (Narita et al.
1998
). Regardless of the source of
[Ca2+]i during our high
K+ experiments, it is clear that
microdomains of Ca2+ are not necessary to enhance
MEPP frequency.
The use of exogenous Ca2+ chelators to
investigate the spatial relationship between Ca2+
domains and transmitter release was first employed by Adler et al. (1991) at the giant synaptic terminal of the squid. They
found that presynaptic injection of BAPTA could inhibit transmitter release, but high concentrations of EGTA were completely ineffective at
reducing transmitter release. This was taken as evidence of local
domains of high Ca2+ being necessary for
transmitter release. Further support for the single-domain hypothesis
has been reported at a chick calyx synapse (Stanley
1993
). In contrast, simultaneous pre- and postsynaptic recordings at a central calyx-type synapse in the rat auditory system
revealed that EGTA and BAPTA were equally effective even at relatively
low concentrations in reducing transmitter release, arguing for
overlapping domains of Ca2+ from neighboring
Ca2+ channels being required for triggering
transmitter release (Borst and Sakmann 1996
;
Borst et al. 1995
). Evidence for the presence of both
mechanisms in the same cell has been provided by capacitance measurements from mouse adrenal medulla slices (Moser and Neher 1997
) and retinal bipolar terminals (Mennerick and
Matthews 1996
) where ultrafast components of exocytosis were
more sensitive to BAPTA than EGTA, whereas slower components of
exocytosis were equally sensitive the two chelators.
Conflicting results from different preparations suggest that the
spatial relationship between Ca2+ channels and
the Ca2+ sensor for release as well as the
apparent affinity of the Ca2+ sensor may be
different in different secretory cells. Moreover, it seems that in at
least some cell types both the single Ca2+ domain
and overlapping Ca2+ domain models may apply
under different conditions. Freeze-fracture electron microscopy of frog
neuromuscular junctions stimulated with 20 mM KCl demonstrated that
most fusion events observed in the first minute of KCl treatment
occurred near the active zone, whereas fusion events after 5 min of
stimulation occurred uniformly over the presynaptic membrane
(Ceccarelli et al. 1988), consistent with distinct
populations of vesicles based on cellular localization. This also
suggests that fusion events measured in our KCl experiments occurred
primarily near the active zone, although the channels opened by
elevated [KCl]o are not necessarily the same
Ca2+ channels as those used for evoked release
(Katz et al. 1995
; Robitaille et al.
1993
), as evident from a report that distinct channel types are
coupled to release from sympathetic ganglia depending on whether
stimulation was by a brief pulse or by sustained depolarization
(Gonzalez Burgos et al. 1995
).
Are the vesicles involved in synchronous and asynchronous release
distinct? Several studies are suggestive of distinct mechanisms for
spontaneous and evoked release. For example the finding that EPPs and
MEPPs have different temperature dependencies at the frog neuromuscular
junction (Barrett et al. 1978) is suggestive of
different exocytic mechanisms. Another experimental manipulation that
points to distinct triggers for exocytosis is the ability of
Sr2+ or Ba2+ to trigger
exocytosis to varying extents, a phenomena that may be due to the
presence of different synaptotagmin isoforms (Li et al.
1995
). The different divalent cation requirements for
synchronous and asynchronous release at frog neuromuscular junctions
(Zengel and Magleby 1981
) and at hippocampal synapses
(Goda and Stevens 1994
) as well as the reduction in
synchronous but not asynchronous release in synaptotagmin I knockout
mice (Geppert et al. 1994
) are suggestive of two
populations of vesicles with different Ca2+
sensors. Other studies using either mutations or toxins to target SNARE
complexes are also suggestive of biochemically distinct fusion events.
Synaptobrevin mutant Drosophila show complete loss of evoked
release but retain 25% of MEPPs (Deitcher et al. 1998
). These residual MEPPs, which were still sensitive to
[Ca2+], may have been due to other
synaptobrevin isoforms. More clear evidence for different SNARE complex
involvement in evoked and asynchronous release comes from studies of
the crayfish neuromuscular junction in which tetanus toxin blocked
evoked but not spontaneous release whereas botulinum toxin D blocked
both forms of release (Hua et al. 1998
).
How does the Ca2+ dependence of spontaneous or
asynchronous release relate to the Ca2+
dependence of evoked release at the frog neuromuscular junction? We do
not assume that the
[Ca2+]i dependence of
MEPP frequency that we measured has a direct relationship to the
Ca2+ dependence of synchronous release at frog
motor nerve terminals. Incorporation of EGTA into motor nerve terminals
caused a reduction in both spontaneous release (this study, Fig. 2) and
evoked release (Robitaille et al. 1993). In the study of
evoked release by Robitaille et al. (1993)
BAPTA was
more effective than EGTA at reducing evoked release, whereas in our
study both chelators [loaded in an identical manner to that of
Robitaille et al. (1993)
] had the same inhibitory effect on MEPP frequency. This strongly suggests the
Ca2+ dependence
(Kd and/or microdomains) differs for
evoked and synchronous release at the frog neuromuscular junction.
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ACKNOWLEDGMENTS |
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We thank Dr. Ling-Gang Wu (Washington University, St. Louis, MO) for advice on fura-2 measurements. S. Fadul provided expert technical assistance on all aspects of this work.
This work was supported by National Institutes of Health grants to J. K. Angleson and W. J. Betz and a grant from the Muscular Dystrophy Association to W. J. Betz.
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FOOTNOTES |
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Address for reprint requests: J. K. Angleson (E-mail: jangleso{at}du.edu).
Received 24 January 2000; accepted in final form 15 September 2000.
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REFERENCES |
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