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The Journal of Neuroscience, January 1, 2000, 20(1):140-148
Recovery from Open Channel Block by Acetylcholine during
Neuromuscular Transmission in Zebrafish
Pascal
Legendre1,
Declan W.
Ali2, and
Pierre
Drapeau2
1 Institut des Neurosciences, Centre National de la
Recherche Scientifique, Unité Mixte de Recherche 7624, Université Pierre et Marie Curie, 75252 Paris, France, and
2 Center for Research in Neuroscience, Montreal General
Hospital Research Institute and McGill University, Montreal, Quebec,
Canada H3G 1A4
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ABSTRACT |
At larval zebrafish neuromuscular junctions (NMJs), miniature end
plate currents (mEPCs) recorded in vivo have an
unusually fast time course. We used fast-flow application of
acetylcholine (ACh) onto outside-out patches to mimic the effect of
synaptic release onto small numbers of ACh receptor channels (AChRs).
Positively charged ACh acted at hyperpolarized potentials and at
millimolar concentrations as a fast ("flickering") open channel
blocker of AChRs. Because of filtering, the open channel block
resulted in reduced amplitude of single channel currents. Immediately
after brief (1 msec) application (without significant desensitization) of millimolar ACh at hyperpolarized potentials, a slower, transient current appeared because of delayed reversal of the block. This rebound
current depended on the ACh concentration and resembled in time course
the mEPC. A simple kinetic model of the AChR that includes an open
channel-blocking step accounted for our single channel results, as well
as the experimentally observed slowing of the time course of mEPCs
recorded at a hyperpolarized compared with a depolarized potential.
Recovery from AChR block is a novel mechanism of synaptic transmission
that may contribute in part at all NMJs.
Key words:
fast-flow; single channels; nonstationary kinetics; neuromuscular junction; mEPC; locomotion.
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INTRODUCTION |
A fundamental issue in synaptic
physiology is to determine the relationship between postsynaptic
receptor-channel activation kinetics and the time course of the
synaptic response elicited upon neurotransmitter release. These issues
have been explored extensively at the neuromuscular junction
(NMJ) at which quantal release of transmitter was first
demonstrated (Fatt and Katz, 1952 ). In addition, studies of the
stationary kinetic properties of nicotinic acetylcholine receptor
channels (AChRs) upon prolonged exposure to ACh, particularly at low
concentrations, have yielded detailed kinetic models of the channels in
a variety of species and show remarkably consistent features (Colquhoun
and Sakmann, 1985; Auerbach and Lingle, 1987; Colquhoun and Ogden,
1988; Sine et al., 1990; Liu and Dilger, 1991). At high (millimolar)
concentrations, ACh is known to block open AChRs in many (Sine and
Steinbach, 1984; Ogden and Colquhoun, 1985; Sine et al., 1990; Liu and
Dilger, 1991; Maconochie and Knight, 1992a; Maconochie and Steinbach, 1998) if not all preparations. This is generally believed to limit the
effectiveness of AChR activation after vesicular release. Nevertheless,
a critical issue is to understand how AChRs respond during brief
exposure to the high concentration of ACh released into the synaptic
cleft. This can best be performed by nonstationary analysis of AChR
kinetics. For example, this approach has revealed the critical role of
receptor desensitization in determining the time course and other
features of the postsynaptic responses at central synapses (Jones and
Westbrook, 1996).
It is particularly important to understand the response properties of
AChRs at NMJs at which high rates of release evoke rapid contractions,
e.g., during locomotion. In the adult zebrafish, synaptic potentials
have been recorded in muscle cells and occur in bursts that are
consistent with the fast rate of trunk contractions (20-40 Hz) during
swimming (Liu and Westerfield, 1988). This suggests that the speed of
neuromuscular transmission must be tightly regulated. Similar high
rates of contractions have been observed in zebrafish larvae (Eaton et
al., 1977; Saint-Amant and Drapeau, 1998) in which NMJs are formed
early during embryogenesis (day 1), and swimming can be evoked at
mature rates before hatching (on day 2). In addition, the small size of
the muscle cells in zebrafish larvae (up to 5 d) permits high
resolution recording of synaptic currents using the patch-clamp
technique (Nguyen et al., 1999), which has revealed unusually fast
miniature EPCs (mEPCs) with decay time constants of often <1 msec.
To determine the features of AChRs underlying the fast synaptic
currents, we examined their properties in outside-out membrane patches
(Hamill et al., 1981) during application of high concentrations of
ligand (0.1-10 mM ACh) by fast-flow perfusion for periods
as brief as 1 msec, as described previously (Legendre, 1998). This allowed us to approximate the effect of synaptic release onto small
numbers (10 or fewer) of AChRs. From these results, we developed a
simple kinetic model whereby open channel block and recovery from block
are major determinants of the time course and other features of the
mEPC and suggests a role at other NMJs as well.
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MATERIALS AND METHODS |
Whole-cell recording. All procedures were performed
in compliance with the guidelines stipulated by the Canadian Council
for Animal Care and McGill University. Zebrafish (Danio
rerio) larvae (3- to 5-d-old) of the Longfin and Tubingen lines
were anesthetized in recording solution (see below) containing 0.2%
tricaine (MS-222; Sigma, St. Louis, MO). The trunk was severed and
pinned on its side through the notochord to the bottom of a
Sylgard-coated Petri dish, and the skin was removed using a fine
forceps. The preparations were then continuously superfused (~2
ml/min) with oxygenated recording solution that lacked tricaine. The
extracellular recording solution was modified from Legendre and Korn
(1994) to contain less Ca and more Mg, consisting of (in
mM): NaCl 145, KCl 4, NaHCO3 26, KH2PO4 1.25, CaCl2 0.6, MgCl2 10, glucose 10, and tetrodotoxin 0.001, adjusted to pH 7.2, 330 mOsm, and
bubbled with
95%O2-5%CO2. Superficial
muscle cells were removed to expose the medial surface of deeper muscle
cells, but the small size of the NMJs prevented us from directly
locating them. We used standard whole-cell recording techniques (Hamill
et al., 1981) at room temperature (22°C) from the middle of deep
muscle fibers. Patch-clamp electrodes were pulled from thick-walled
borosilicate glass and were filled with (in mM):
CsCl 130, MgCl2 2, HEPES 10, EGTA 10, and
Na4ATP 4, pH 7.2, 290 mOsm. The electrode
resistance was 1-3 M , and miniature end plate currents (mEPCs) were
recorded with an Axopatch 1D or 200B patch clamp (Axon Instruments,
Foster City, CA), filtered at 5 kHz ( 3 dB), and stored either on the
computer or on a digital tape recorder. Data were acquired with pClamp
6.0 software (Axon Instruments) by digitizing at 50 kHz and were
analyzed off-line with Axograph 4.0 software (Axon Instruments). The
mEPCs were detected as events rising five times above the SD of
the noise with a time course resembling that of a well resolved mEPC
used as a template. The events were aligned at the corresponding time point of detection. We excluded from analysis a small fraction (<25%)
of low-amplitude (less than 50 pA at 50 mV) slow events (rise times
of >0.5 msec and decay time constant of >2 msec) thought to result
from electrical coupling with neighboring muscle cells (Nguyen et al.,
1999). The series resistance (Rs) was 3-10 M, was compensated by
60-95%, and was verified throughout the recordings. Typically, mEPCs
were recorded for 5 min at 50 mV, the potential was changed to +50
mV, Rs was verified, and a 5 min recording was then obtained at +50 mV,
after which Rs was verified again. In some experiments, this procedure
was repeated for further recording sessions at either potential, and Rs
was checked between each session. Recordings were not analyzed if Rs
increased more than twofold or reached 10 M. Because the membrane
capacitance was ~50 pF, this limited the recording bandwidth to 5 kHz, the filter cutoff frequency chosen during data acquisition.
Fast-flow recordings. Electrodes of 10-15 M resistance
were used to obtain a whole-cell configuration near the middle of deep
muscle fibers; outside-out membrane patches were then isolated by
slowly pulling the pipette off the cell. Fast-flow applications were
performed as described previously (Legendre, 1998). The outside-out patch was positioned obliquely approximately 100 µm away from the
stream of a twin-barreled application pipette, close to the interface
formed between the solutions. One barrel contained control solution
consisting of (in mM): NaCl 145, KCl 1.5, CaCl2 0.6, MgCl2 10, glucose 10, and HEPES 10, adjusted to pH 7.2, 330 mOsm. The other
barrel contained ACh (0.1, 1, or 10 mM) dissolved
in control solution. The solution exchange was performed by rapidly moving the solution interface across the tip of the patch pipette, using a piezo-electric translator (model P245.30, Physic Instruments). Current was recorded with an Axopatch-1D amplifier (Axon Instruments), filtered at 10 kHz (3 dB), and stored using a digital tape recorder. Data were acquired with pClamp 6.0 software (Axon Instruments) by
digitizing at 50 kHz and were analyzed off-line with Axograph 3.5 software (Axon Instruments). In control experiments, the rising phase
of the pipette junctional current had a time to peak (20-80%) of 0.08 msec. However, the real exchange time results partially from an
unstirred layer around the patch. The theoretical limit to the full
exchange time was therefore estimated (Legendre, 1998) by assuming a
patch diameter of 0.4-0.5 µm and found to be closer to 0.11 msec.
Because the activation kinetics of AChRs can be as fast as our
application technique, we obtained an estimate of the maximum
activation time by applying a saturating concentration of agonist (10 mM ACh). The activation phase of the current
evoked by 10 mM ACh application (20-80% rise
time, 0.04 ± 0.01 msec; mean ± SD; n = 10;
patch potential Vh, +50 mV) was faster
than the junctional current and is likely to reflect the real kinetic values. To the contrary, the time course of the current evoked by 1 mM ACh was close to the solution exchange time,
which therefore appears to limit the onset of the response.
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RESULTS |
AChR properties
When 10 mM ACh was applied at a positive potential
(+50 mV), a step-like outward (upward) current appeared during the
period of application (Fig.
1A). In contrast, when
the same patch was held at a negative potential (50 mV), a clear
plateau of inward (downward) current (reversing near 0 mV) was evident
during ACh application (labeled "1"). Upon removal of ACh, a large
transient rebound current appeared (labeled "2"). At a lower ACh
concentration of 1 mM (Fig.
1B), a smaller current was evoked at 50 mV compared with at +50 mV during ACh application, and a small rebound current appeared immediately after ACh removal and then decayed to baseline. When only 0.1 mM ACh was applied, similar current
steps were observed at +50 and 50 mV (Fig. 1C), presumably
attributable to a lack of block at this concentration. The extent of
current block was estimated from the difference between the peak
rebound current after removal of ACh and the early plateau current
level during ACh application. The block (summarized in Fig.
1D) was negligible (<5%) at 0.1 mM and at all ACh concentrations tested at +50 mV (black bars) but was increasingly obvious at higher ACh
concentrations when the patches were held at 50 mV (stippled
bars). For responses evoked by the application of 10 mM ACh, the block reached 79 ± 10%
(n = 10), which presumably is limited by equilibration
between the open and blocked states of the AChR (see below).
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Figure 1.
Currents evoked in outside-out patches
by fast-flow application of ACh. A-C, A series of
fast-flow perfusion trials (10-15) separated by 2 sec or more were
used to generate an average trace under each condition for the same
patch. The top traces are recordings of the junctional
current recorded from the open pipette upon rupturing the patches at
the end of the experiment and indicate the period of ACh application.
Current responses during application of 10 (A), 1 (B), and 0.1 (C)
mM ACh to the same patch held at +50 (top
traces) or 50 (bottom traces) mV. The
bottom trace in A (at 10 mM
ACh) demonstrates an early plateau (1), followed
by a rebound phase (2). D, Summary
of the extent of current blocked (n = 5) at 50
(black bars) and +50 (stippled bars) mV
for patches exposed to 0.1 (left pair of bars), 1 (middle pair of bars), and 10 (right pair of
bars) mM ACh. E, Summary of the rise
times (20-80%) for currents evoked at +50 and 50 mV at either 0.1 (left pair of bars) or 10 (right pair of
bars) mM ACh, including the onset of the plateau
phase (1) and rebound phase
(2) observed at 50 mV. F,
Summary of the exponential time constants ( off)
for deactivation of the currents after termination of the ACh
application at +50 and 50 mV at 0.1 (left pair of
bars) and 10 (right pair of bars) mM
ACh.
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Other features of the currents were examined by comparing the
activation and deactivation time courses observed under different conditions. At 10 mM ACh, the 20-80% rise time at 50 mV
(Fig. 1E, stippled bars) was 0.04 ± 0.01 msec (n = 10) for the plateau current (1) and
0.05 ± 0.03 msec (n = 10) for the rebound current (2). These values were not significantly different from that measured at +50 mV (black bar; 0.04 ± 0.01 msec;
n = 10; paired t test; p > 0.05). The rise times of the responses evoked by 1 mM ACh application and for both components of the
responses evoked by 10 mM ACh were limited by the
solution exchange time and recording bandwidth (see Materials and
Methods). Application of 0.1 mM ACh (for 1 msec)
evoked responses with a longer 20-80% rise time at both potentials
(Fig. 1E), consistent with the slower activation of
AChRs expected at this lower agonist concentration. The rise time was
0.17 ± 0.05 msec (n = 5) at
Vh of 50 mV and 0.17 ± 0.07 msec (n = 5) at Vh of
+50 mV. The deactivation time constant (off) of responses evoked at this concentration had similar values at both
negative (0.18 ± 0.03 msec; n = 5) and positive
(0.17 ± 0.05 msec; n = 5) holding potentials
(Fig. 1F), suggesting that, at this agonist
concentration, zebrafish AChR closure after removal of ACh was
voltage-independent. When Vh was +50
mV, off of the responses evoked by the
application of 10 mM ACh (0.20 ± 0.11 msec;
n = 10) had similar values to those obtained when 0.1 mM ACh was applied. But at
Vh of 50 mV,
off was significantly longer (0.30 ± 0.10 msec; n = 10) at the higher concentration (paired t test; p < 0.05) (Fig.
1F). This slower decay time of the rebound current
obtained at negative potentials appears to be related to an additional
delay in unbinding of ACh from open but blocked channels under these
conditions (see below). A similar rebound of the ACh-evoked current
during recovery from open channel block was also reported for AChRs
from chromaffin cells (Maconochie and Knight, 1992a) and mouse muscle
(Maconochie and Steinbach, 1998).
Open channel block
We examined the voltage dependence of the effect of 10 mM ACh in greater detail, as shown in Figure
2. ACh was applied for 1 msec at holding
potentials ranging between +40 and 70 mV in this example. As the
potential was held more negative, the early plateau current reached a
limiting amplitude, whereas the rebound current became progressively
larger (Fig. 2A). As shown in Figure 2B,
the peak of the rebound current varied linearly over this potential
range (open circles), whereas the steady current increased similarly at positive potentials but was reduced (rectified) at voltages of 20 mV or less (filled circles). The
voltage dependence of the block was estimated as shown in Figure
2C in which the difference between the amplitudes of the
peak of the rebound current and the early plateau current, normalized
as a percentage of the peak current, is plotted at different holding
potentials. The proportion of blocked current can be seen to increase
as the voltage was decreased and reached a maximum of ~80% block at
70 mV. In Figure 2D, these results are plotted on
semilogarithmic coordinates. The relationship between the extent of
block (on a logarithmic scale) and the holding potential could be well
fitted by a single exponential function (straight line) between 40
and +50 mV. Accordingly, the block increased with a limiting slope of
e-fold/32 mV of hyperpolarization. A similar steady-state voltage
dependence has been reported for block of BC3H1 clonal muscle cell
AChRs by millimolar ACh (Liu and Dilger, 1991) and for local anesthetic
block of AChRs in denervated frog muscle (Neher and Steinbach, 1978),
and slightly lower voltage dependencies have been reported in other
preparations (Ogden and Colquhoun, 1985; Sine et al., 1990). Therefore,
ACh appears to bind to a site well within the membrane field in a
variety of AChRs.
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Figure 2.
Voltage dependence of the effect of 10 mM ACh. A, Series of current responses
evoked in the same patch upon exposure to 10 mM ACh at
potentials of +40, +20, 10, 20, 30, 40, 50, 60, and 70 mV
(from top to bottom traces; averages of 8 trials). The filled circle denotes the early plateau
phase, and the open circle denotes the peak of the
rebound phase. B, Plot of the current during the early
plateau phase (filled circles) and peak of the
rebound phase (open circles) at each potential examined
for the traces shown in A. C,
Summary of the difference between the peak rebound current and early
plateau current (Blocked current), plotted as a
percentage of the total current observed at each potential. SDs
(n = 5) are indicated by the error bars.
D, Same results as in C normalized for
the maximal block (at 70 mV) on a logarithmic scale and plotted as a
function of voltage. The solid line is the best linear
fit of the foot of the curve and indicates an e-fold increase in block
for a 32 mV hyperpolarization.
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Because ACh is positively charged, it may enter and permeate the AChR
as the pore discriminates only poorly between cations (Dwyer et al.,
1980). At high (millimolar) concentrations of ACh, such as that reached
in the synaptic cleft during neuromuscular transmission (Clements,
1996), a reversible block of the channels has been described previously
(Sine and Steinbach, 1984; Ogden and Colquhoun, 1985; Sine et al.,
1990; Liu and Dilger, 1991; Maconochie and Knight, 1992a; Maconochie
and Steinbach, 1998). Similarly, open channel block has been observed
in some AChRs by 5-hydroxytryptamine (Grassi et al., 1993),
extracellular (Marty, 1980) and intracellular
Mg2+ ions (Sands and Barish, 1992; Ifune
and Steinbach, 1991), and intracellular spermidine (Haghighi and
Cooper, 1998). An open channel block of AChRs by ACh at negative
voltages causes fast closures of the channel (Sine and Steinbach, 1984;
Ogden and Colquhoun, 1985; Sine et al., 1990; Liu and Dilger, 1991;
Maconochie and Knight, 1992a; Maconochie and Steinbach, 1998) with
durations depending on the association and dissociation rates of the
blocker within the pore. The presence of a rebound current for
zebrafish AChRs, with an onset in the microsecond domain after agonist
removal, predicts fast association and dissociation rates. If these
kinetics exceed the bandwidth of the recordings, the block will be
filtered and consequently reduce the single channel current, resulting in a flickering block as first described for open AChR block by local
anesthetics (Neher and Steinbach, 1978). This was effectively the case
as shown in Figure 3A for a
patch with at least three channels. In this example, a 100 msec
application of 10 mM ACh evoked single channel
activity with an apparent approximate fourfold lower single channel
current amplitude at negative potentials because of the flickering
block. At a positive potential (top trace), we observed
occasional long-lasting closures, which eventually terminated the
current as a result of slow desensitization (see below). When ACh was
applied to the same patch at a lower concentration of 0.1 mM (Fig. 3B), we observed better
resolved current transitions because of slower, less filtered channel
openings and closures at this lower concentration, and the amplitudes
were similar at both potentials. These observations confirm that an
open channel block occurred at high ACh concentrations and at negative
potentials.
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Figure 3.
Open channel block by ACh. A, The
top line indicates the 100 msec period of application of
10 mM ACh to a patch containing three AChRs. The top
trace shows the current (filtered at 2 kHz) elicited during a
single application at +50 mV. The baseline is indicated by
c on the left; each of three consecutive
open channel current levels are indicated by o. The
bottom trace shows a response by the same patch during
ACh application at 50 mV. Note the much smaller current amplitude.
B, In contrast, the single channel current was
insensitive to voltage when 0.1 mM ACh was applied to the
same patch. C, Long pulses (100 msec) of 0.1 mM ACh also evoked a voltage-independent desensitization in
this patch. In this example, 45 traces were averaged. At both voltages
(+50 and 50 mV), the averaged trace decayed with a time course, which
was well fitted by a single exponential curve with time constants
( d) of 43 msec at +50 mV and 64 msec at 50
mV.
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A 100 msec application of a low or high concentration of agonist also
revealed a desensitization process characterized by a progressive
decrease in single channel activity with time. Desensitization was
nearly complete by 100 msec and is the reason a rebound current is not
observed in Figure 3. The time course of desensitization is shown more
clearly in Figure 3C for averaged currents evoked in the
same patch with 0.1 mM ACh. The desensitization
time constant (d) appeared to be independent
of the voltage and ACh concentration at the levels tested. The values
of d at 0.1 mM ACh were
53 ± 10 msec (n = 7) at +50 mV and 51 ± 5 msec (n = 7) at 50 mV; at 10 mM
ACh, they were 43 ± 14 msec (n = 7) at +50 mV and
45 ± 15 msec (n = 10) at 50 mV. These values
did not differ significantly (paired t test;
p > 0.05). The desensitization process was therefore too slow to participate significantly in the phenomena occurring during
brief (1 msec) applications, and paired-pulse experiments confirmed
this point (data not shown).
Kinetic model of the AChR and mEPC time course
A simple kinetic model of the AChR with a blocked "open"
channel state can well describe our main results of
concentration-dependent (Fig.
4A) and
voltage-dependent (Fig. 4B) block by ACh. This model was originally proposed for chromaffin AChRs (Maconochie and Knight, 1992b). The rate constants were chosen based on our experimental data
and on detailed kinetic studies of AChRs performed in other preparations. We assumed two equivalent closed states that each bind
ACh and rapidly interconvert, as described for muscle AChRs in other
species (Colquhoun and Sakmann, 1985; Auerbach and Lingle, 1987;
Colquhoun and Ogden, 1988; Sine et al., 1990; Liu and Dilger, 1991),
leading to opening of the doubly occupied receptor and subsequent
concentration- and voltage-dependent block by ACh.
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Figure 4.
Model of AChRs and predicted properties of mEPCs
during open channel block. A, Model predictions of the
effects of a 1 msec application of 0.1, 1, and 10 mM ACh
(as indicated). The solid curves are exponential fits of
the current decay after application of 0.1 and 10 mM with
the indicated time constants. B, Model prediction of the
voltage dependency of open channel block evoked by ACh (10 mM applied for 1 msec) at +50 and 50 mV. The solid
curves are exponential fits of the current decay at either
potential with the indicated time constants. Model prediction of the
evolution of the kinetic states during a 1 msec application of 10 mM ACh at +50 (C) and 50
(D) mV. Each trace represents a
different state: monoliganded (AC) and diliganded closed
states (A2C), the open channel
(A2O), and the blocked channel
(A3B). E,
Prediction of the time course of the mEPC at 50 and +50 mV elicited
by a 0.125 msec duration pulse of 10 mM ACh. The mEPCs
are scaled to a similar peak. The solid curves are
exponential fits of the mEPC decay at either potential with the
indicated time constants. The rise times were 0.08 msec at +50 mV and
0.12 msec at 50 mV.
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Explicitly, the mechanism of block of zebrafish AChRs can be well
described by the following linear kinetic model in which A denotes a molecule of ACh:
For simplicity, we assumed two identical, interconnected binding
states (C and AC) with an optimum forward rate
constant (kon) of 1.6 × 108
M 1
sec1 and an optimum dissociation
rate constant (koff) of 9 × 103 sec1.
This is close to that previously proposed for muscle AChRs (Colquhoun and Sakmann, 1985; Auerbach and Lingle, 1987; Colquhoun and Ogden, 1988; Sine et al., 1990; Liu and Dilger, 1991). The value of
koff was adjusted depending on
kon to limit reopening of the channel as suggested by the fast deactivation phase of the outside-out currents
we observed (Fig. 1F), but the exact values chosen
were not as critical as the values of the other rate constants in the model. The doubly bound closed state
(A2C) led to an open state (A2O) with an open rate constant
( ) of 3.5 × 103
sec1, similar to that proposed for the
AChR of the frog (Colquhoun and Sakmann, 1985). The estimated closing
rate constant ( ) needed to be faster (9.5 × 103 sec1)
to obtain a fast rise time and decay time constant. A fast rate for has also been observed in high-resolution stationary kinetic analyses
of AChRs at high (>0.1 mM) ACh concentrations
(Sine et al., 1990). As for other muscle AChRs (Sine and Steinbach,
1984; Ogden and Colquhoun, 1985; Sine et al., 1990; Liu and Dilger, 1991; Maconochie and Knight, 1992a; Maconochie and Steinbach, 1998), we
propose that the open state was blocked
(A3B) by ACh with an essentially
diffusion-limited rate constant (b) estimated as 7 × 106
M1
sec1. To predict the voltage dependence
of the mechanism of block, b was set using the relationship
b × exp [(Vh + 50 mV)/30 mV] so that the block decreased e-fold/30 mV
depolarization, which gave a somewhat better fit than the value of
e-fold/32 mV observed experimentally (Fig. 2D). The
recovery rate constant (r) was estimated as 3.5 × 103 sec1 at
50 mV, which is an order of magnitude slower than the recovery rate
estimated for other AChRs. This indicates a more stable blocked state
that results in a prolonged recovery (rebound) phase.
It was critical that the value of r should be close to that
of to yield a flat plateau phase during the block, followed by a
more slowly decaying and voltage-dependent current during the recovery
phase. However, the rise time calculated for application of 10 mM ACh was approximately twice that observed with
outside-out patches (Fig.
1E,F). Increasing the values
of and r yielded more appropriate rise times, but the
decay rate became voltage- and concentration-independent. In the
absence of more direct information on activation kinetics in the
microsecond domain, we compromised the model by allowing for slightly
slower rise times while retaining the essential biophysical and
physiological features (see below) of the block. These features include
a slowing of the deactivation phase at higher ACh concentrations (Fig.
4A) and at hyperpolarized compared with depolarized
potentials (Fig. 4B), which resemble the rebound
current observed experimentally (Figs.
1A,F, 2A). Desensitization was not taken into account because it was too slow
(Fig. 3C) to shape the time course of the current evoked by
a short pulse of ACh. We rejected other models, such as multiple blocking sites (Maconochie and Steinbach, 1998), interconversion between blocked and closed states, or a second open state reached from
the monoliganded state, because these failed to account for the slowing
of the decay time constant.
The role of open channel block in the single channel current behavior
is indicated more clearly in Figure 4, C and D.
Here, we have plotted the evolution predicted for different states of the channel during a 1 msec application of 10 mM
ACh with minimal block at +50 mV (Fig. 4C) and with a strong
block at 50 mV (Fig. 4D). During ACh application at
+50 mV (Fig. 4C), the monoliganded state (AC) is
almost entirely dissipated as the diliganded state (A2C) is rapidly reached at the
onset of the application, resulting in ~25% of channels opening
(A2O) after a similar, rapid
time course. In addition, a somewhat smaller fraction (~15%) of the channels become blocked (A3B)
after a slower time course. Removal of the ACh results in dissipation
of the diliganded closed and open states and a delayed, transient
appearance of the monoliganded closed. Reversal of the block
contributes only a small peak (<5%) of open channels at the onset, as
observed experimentally (Fig. 1A,D), and the blocked channels
recover at a similar rate as the closing of the open channels (Fig.
1F).
During application of ACh at 50 mV resulting in strong open channel
block (Fig. 4D), the monoliganded closed state is
again essentially entirely dissipated as the diliganded closed state is
rapidly reached at the onset of the application. However, the diliganded closed state declines to a much greater extent and results
in a smaller fraction (<5%) of openings because of a large accumulation of blocked channels. Removal of the ACh results in immediate reversal of channel block and a delayed opening of the channels, which consequently close at a slower rate than observed in
the absence of block. Closing of the channels sustains the (initially
more rapid) decline of the diliganded closed state and results in the
further delayed and transient appearance of the monoliganded closed
state. It can be seen that at this potential that the openings are
generated mostly from blocked channels.
Properties of mEPCs
Based on this model, we predicted the time course of mEPCs
expected at +50 and 50 mV (Fig. 4E) i.e., with weak
and strong open channel block. We assumed for simplicity that ACh is
released instantaneously into the synaptic cleft as a brief, square
pulse (duration of 0.125 msec) at a concentration of 10 mM. This resulted in mEPCs with rise times
comparable with those observed experimentally (data for unfiltered
events taken from Nguyen et al., 1999, and see below). The mEPCs
predicted at Vh of 50 mV were
somewhat lower in amplitude (by ~25%) than those predicted at +50 mV
(see Fig. 6C; scaled to the same amplitude in Fig.
4E), and both their rise time and decay time courses
were slower. These differences in the mEPCs predicted at the two
potentials result from the reduction in channel current amplitude and
slowing of the rebound current described above for the single channel recordings.
We then examined the properties of mEPCs recorded in vivo at
these two potentials to see whether similar differences occur. In the
presence of TTX and a low Ca-high Mg solution, quantal mEPCs at either
potential fluctuated highly in amplitude, as we reported previously in
recordings at physiological potentials (Nguyen et al., 1999). We
suggested that the lack of a simple, gaussian distribution of mEPC
amplitudes was caused by the polyinnervation of zebrafish muscle cells
by both primary and secondary motoneurons (Myers, 1985; Westerfield et
al., 1986). Because our model predicted a limited reduction in
amplitude at 50 mV compared with +50 mV, we were unable to detect
this effect given the larger experimental variability. Individual
events at a given potential had a relatively homogenous time course
(Fig. 5A). Cumulative
histograms of individual mEPC rise times (Fig. 5B) and decay
time constants (Fig. 5C) from a representative experiment
clearly show that both values were larger at the hyperpolarized,
blocking potential. These observations were significant for all
(n = 15) experiments (Fig. 5D) and suggest that a strong open channel block occurs during ACh release under physiological conditions and that its reversal contributes most of the
mEPC.
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Figure 5.
Properties of mEPCs recorded in
vivo. A, Overlap of 25 mEPCs recorded from the
same muscle fiber at +50 (top traces) and 50
(bottom traces) mV. Cumulative histograms of the
20-80% rise times (B) and monoexponential decay
time constants (C) for all events recorded at +50
(n = 99) and 50 (n = 85) mV.
D, Summary histogram of the values obtained in all
(n = 15) recordings. The mean ± SD for the
rise times (left) and decay time constants
(right) at 50 (black bars) and +50
(gray bars) mV. The asterisks
indicate significance at p < 0.01.
|
|
To examine the physiological implications of the block in greater
detail, we predicted the effects of gradually varying several parameters of the kinetic model on the properties of the mEPCs calculated at +50 and 50 mV. Increasing the duration (from 0.05 to
0.50 msec) of the ACh pulse eliciting the mEPC resulted in a small but
progressive increase in the decay time constant (Fig. 6A), but this remained
slower at 50 mV compared with +50 mV. At an ACh concentration of 0.1 mM, the mEPC decay time constant was similar at
the two potentials but became slower at 50 mV as the concentration
increased (Fig. 6B); above 2 mM, the relative difference in the time constants
became more constant, reaching a twofold difference at 10 mM. This difference is comparable with that
observed in vivo (Fig. 5D), indicating that
millimolar concentrations of ACh are present in the cleft during the
mEPC, as observed at other NMJs and for other transmitters (Clements,
1996). At submillimolar concentrations of ACh the peak amplitude of the
mEPC rose rapidly at both potentials, but at millimolar concentrations
it became consistently smaller at 50 mV because of the block (Fig.
6C). At millimolar ACh concentrations, the reduction of the
mEPC amplitude at 50 mV thus seemed to be offset by the prolongation
of the decay time constant described above. To further examine this
balance of mEPC properties, we estimated the charge contributed by the mEPC at each potential as the product of the amplitude and time constant predicted at each ACh concentration as shown in Figure 6,
B and C, respectively. It can be seen in Figure
6D that the estimated charge rose rapidly with the
ACh concentration at both potentials but was consistently greater at
millimolar concentrations at 50 mV. This indicates that, despite the
reduction in current amplitude because of the block, the delayed
reversal of the block results in a prolonged current that allows more
charge to enter the muscle cell at physiological potentials.
View larger version (22K):
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|
Figure 6.
Effects of model parameters on predicted
properties of the mEPC. A, Decay time constants are
plotted as a function of the duration of a square pulse of 10 mM ACh. The values for the durations are incremented by
0.025 msec from 0.050 to 0.525 msec at 50 (filled
symbols) and +50 (open symbols) mV. Effects of
the ACh concentration on decay time constants
(B), peak amplitudes
(C), and charge (D) during
a 0.125 msec pulse. The concentrations are 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, and 10 mM.
|
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| |
DISCUSSION |
AChR properties
We observed a voltage-dependent block of AChRs during application
of millimolar concentrations of ACh, followed by a rapid, rebound
current upon removal of ACh. Open channel block of muscle AChRs by ACh
has been observed in a variety of preparations in which the block was
supposed to reduce the synaptic current as estimated from the results
obtained during stationary kinetic measurements (Ogden and Colquhoun,
1985). In our nonstationary analysis, we observed a rebound current
after removal of ACh, suggesting that, although a block occurs
initially, a current can nevertheless be generated during recovery from
the block. In fact, the same maximal conductance was observed at
positive potentials in which a sustained current (with minimal block)
was generated during brief application and at negative potentials at
the peak of the rebound (linear I-V relationship of Fig.
2B, open circles). This demonstrates that
all of the activated channels can eventually generate a maximal
current, even if they are initially blocked. Although the block does
not modify the effectiveness of the channels, its reversal determines
the time course of the response to ACh.
A rebound current was observed during nonstationary analysis of
chromaffin cell AChRs (Maconochie and Knight, 1992a) in which the
physiological significance remains unclear and for cloned mouse muscle
AChRs (Maconochie and Steinbach, 1998). For zebrafish AChRs, we
estimated similar association, dissociation, opening, and block onset
rate constants as for most other muscle AChRs (Sine and Steinbach,
1984; Ogden and Colquhoun, 1985; Colquhoun and Sakmann, 1985; Auerbach
and Lingle, 1987; Colquhoun and Ogden, 1988; Sine et al., 1990; Liu and
Dilger, 1991). Our value for the closing rate ( = 9500 sec1) is among the highest reported,
e.g., as for torpedo AChRs (8000 sec1)
(Sine et al., 1990). However, the unblocking rate (r) was
estimated to be an order of magnitude slower than for other AChRs,
accounting for the prolonged effect observed with the otherwise fast
AChR kinetics described here.
End plate currents
We were able to simulate (data not shown) a synaptic-like rebound
current using the rate constants for previously published studies of
muscle AChR (Sine and Steinbach, 1984; Ogden and Colquhoun, 1985; Liu
and Dilger, 1991; Maconochie and Knight, 1992a; Maconochie and
Steinbach, 1998), including the Torpedo electroplax (Sine et
al., 1990). This suggests that recovery from open channel block is a
ubiquitous component of synaptic transmission at the NMJ that plays a
more important role at synapses in which the unblocking rate is closer
to the channel activation rate. The slow unblocking rate estimated for
the zebrafish AChR together with the lack of channel reopenings result
in the reversal of block being the major determinant of the decay time
course of the mEPC. At other NMJs with faster unblocking rates and
longer mean open times, consisting of bursts with multiple reopenings,
the block may slow the first opening in a burst (when the blocking ACh
molecule rapidly dissociates) but not later openings. Under these
conditions, the block would have a minimal effect on the mean channel
open time and consequently the mEPC duration. An additional factor in
determining the end plate current time course is ACh diffusion (Bartol
et al., 1991), particularly when multiple quanta are secreted during
evoked release at NMJs with extended postsynaptic receptor matrices.
We conclude that, at larval zebrafish end plates, ACh is rapidly
released at millimolar concentrations and blocks the AChRs as soon as
they open. It is important to stress that the AChRs are rapidly blocked
by millimolar concentrations of ACh because this prevents the membrane
from depolarizing during vesicle release under physiological
conditions, which otherwise would reduce the effect of the block. Once
ACh is removed from the cleft, bound ACh slowly leaves the pore of the
blocked channels, and a transient, synaptic current is generated by
reversal of open channel block. Millimolar concentrations of
transmitter have been estimated at a number of synapses, including NMJs
and central synapses (Clements, 1996). A variety of kinetic mechanisms
are thought to define the decay time course of synaptic events, ranging
from fast to slow desensitization and simple closure of the channels
after clearance of transmitter (Jones and Westbrook, 1996). The
reversal of open channel block that we observed is a novel mechanism of
synaptic transmission. The delayed appearance of the ACh-evoked current (because of recovery from block) is reminiscent of the presynaptic calcium tail current (because of calcium channel closure) that is
delayed until the repolarization phase of the action potential (Llinas
et al., 1982). It is thus paradoxical that, at the zebrafish NMJ, much
of the action appears to take place after rather than during the stimuli.
Physiological significance of open channel block
According to our model simulations, the duration of the synaptic
current is not particularly sensitive to variations in the duration of
ACh exposure (e.g., 0.05-0.50 msec) whether or not a block occurs
(Fig. 6A). In contrast, the duration of the rebound current is far more sensitive to variations in the peak concentration of ACh. At lower ACh concentrations the time course of the synaptic current is limited mainly by the rate of activation and deactivation of
the AChRs, whereas at higher concentrations the rebound current is
determinant. At the zebrafish NMJ, the time course of the postsynaptic response should therefore be potentiated when higher concentrations of
ACh are released into the synaptic cleft. This potentiation could be
significant during development because of variations in the extent of
synaptic maturation and at mature synapses after multivesicular release
evoked by a presynaptic action potential. Reversal of open channel
block could thus serve as a simple and reliable biophysical mechanism
to prolong the time course of synaptic transmission and thus enhance
the postsynaptic action of ACh. We speculate that this may also be the
case at the Torpedo electroplax and at other NMJs and may be
especially important at those in which the rate of decay of mEPCs is
related to the rate of muscle contractions (Dionne and Parsons, 1978,
1981; Miledi and Uchitel, 1981).
Although the block slows the time course of the mEPC, the amplitude is
also predicted to be reduced. However, our estimates of the synaptic
charge suggest that the former outweighs the latter. The efficacy of
the NMJ is usually thought to be a direct consequence of the
depolarization produced at the single, discrete end plate. However,
zebrafish muscle cells are polyinnervated, with tiny synapses
contributed by each motoneuron (Liu and Westerfield, 1992) and
thus more closely resembling immature NMJs in other species. Because
the small synapses are spread over the surface of each muscle fiber, we
suggest that the greater charge generated by reversal of open channel
block allows for a better distribution of the depolarization along the
equivalent cable of the muscle cell. This mechanism would be thus be
more akin to synaptic activation of neurons in which distant synapses
are thought to be integrated by dendritic cable properties (Rall,
1969).
| |
FOOTNOTES |
Received Aug. 19, 1999; revised Oct. 13, 1999; accepted Oct. 18, 1999.
This work was supported by grants from Institut National de la
Santé et de la Recherche Médicale (INSERM) and Association contre les Miopathies of France (P.L.), the Natural Sciences and Engineering Research Council of Canada (NSERC) (P.D.), an NSERC-INSERM Collaborative Exchange (P.L. and P.D., for support of D.W.A.), an NSERC
Fellowship (D.W.A.), and a Medical Research Council of Canada-INSERM
Visiting Scientist award (P.D.). We thank Dr. N. Leurèche for
access to equipment used for some of the whole-cell recordings, Dr. E. Cooper for discussions and comments on this manuscript, and S. Girls
for inspiration.
Correspondence should be addressed to Dr. Pierre Drapeau, Department of
Neurology, Montreal General Hospital, 1650 Cedar Avenue, Montreal,
Quebec, Canada H3G 1A4. E-mail: mcpd{at}musica.mcgill.ca.
Dr. Ali's present address: Programme in Brain and Behavior, Hospital
for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
| |
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TOP
ABSTRACT
INTRODUCTION
