1Physiologisches Institut and 2Neurologische Klinik, Technische Universität München, 80802 Munich, Germany; and 3Department of Neurobiology, Hebrew University, Jerusalem 91904, Israel
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ABSTRACT |
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Dudel, J.,
M. Schramm,
C. Franke,
E. Ratner, and
H. Parnas.
of quantal end-plate currents of mouse muscle by physostigmine
and procaine. Quantal endplate currents (qEPCs) were recorded from hemidiaphragms of mice by means of a macro-patch-clamp electrode. Excitation was blocked with tetrodotoxin, and quantal release was
elicited by depolarizing pulses through the electrode. Physostigmine (Phys) or procaine (Proc) was applied to the recording site by perfusion of the electrode tip. Low concentrations of Phys increased the amplitude and prolonged the decay time constants of qEPCs from ~3
to ~10 ms, due to block of acetylcholine-esterase. With 20 µM to 2 mM Phys or Proc, the decay of qEPCs became biphasic, an initial short
time constant s decreasing to <1 ms with 1 mM Phys and
to ~0.3 ms with 1 mM Proc. The long second time constant of the
decay,
l, reached values of
100 ms with these blocker concentrations. The blocking effects of Phys and Proc on the qEPC are
due to binding to the open channel conformation. A method is described
to extract the rate constants of binding (bp)
from the sums 1/
s + 1/
l, and the rates of
unbinding (b
p) from
0 ·
s
1 ·
l
1 (
0 is the decay time
constant of the control EPC). For Phys and Proc
bp of 1.3 and 5 · 106
M
1 s
1 and b
p of
176 and 350 s
1, respectively, were found. Using these
rate constants and a reaction scheme for the nicotinic receptor
together with the respective rate constants determined before, we could
model the experimental results satisfactorily.
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INTRODUCTION |
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In a number of recent studies, we have established
reaction schemes for nicotinic receptors/channels on mouse muscle using mostly patches containing the embryonic type of channels (Franke et al. 1991a,b
, 1992a
,b
, 1993
). The experimental data have been collected mainly by application of acetylcholine (ACh) to outside-out patches, using a liquid-filament switch that generated pulses of ACh at
the patch with rise and decay times of the agonist concentrations of
<0.5 ms (Bufler et al. 1996a
; Franke et al.
1987
).
Using the same techniques, we have extended these studies to the
"open channel block" in embryonic channels by the local
anesthetic procaine (Proc) and by physostigmine (Phys) (Bufler
et al. 1996a). Phys and Proc blocked the open channels with
rate constants of 6 · 106 M
1
s
1 and 2 · 106 M
1
s
1, respectively, and unblocked with rates of 200 s
1. The blocking rate constants were in the same range as
those found by previous investigators (Adams 1977
;
Albuquerque et al. 1986
; Neher and Steinbach
1978
; Ogden et al. 1981
). Ogden et al. (1981)
reported an unblocking rate for benzocaine similar to
that found by us, whereas Neher and Steinbach (1978)
saw
a rate of unblock for lidocaine derivatives of 2,300 s
1,
corresponding to the "flickering block" caused by these
substances. Such flickering block also has been seen with high ACh
concentrations in hyperpolarized patches, with blocking rates in the
range of 5 · 107 M
1 s
1
and unblocking rates of 5 · 104 s
1.
These unblocking rates correspond to "flickering," short closings of the channel with an average duration of 20 µs (McGroddy et al. 1993
; Ogden and Colquhoun 1985
;
Parzefall et al. 1998
; Sine and Steinbach
1984
; Sine et al. 1990
).
Aside from biophysical aspects, the open channel block by local
anesthetics is of interest for its effect on end-plate currents (EPCs).
First Furukawa (1957), then Maeno (1966)
,
Steinbach (1968)
, Kordas (1970)
, and
Katz and Miledi (1975)
applied local anesthetics to frog
muscle and saw biphasic endplate potentials with a short initial and a
long second phase. Similar effects on EPCs from toad muscle were
reported by Gage and Wachtel (1984)
. Shaw et al.
(1985)
applied Phys to mouse muscle and saw an initial more rapid decay of the EPCs.
Already at low Phys concentrations, EPCs were lengthened by its
well-known block of ACh-esterase, which prolonged the presence of ACh
at the receptors. Both these results agree qualitatively with the
respective blocking rates derived for nicotinic channels (Bufler
et al. 1996a). We decided, therefore, to study the effects of
Phys and Proc on quantal EPCs of adult mouse muscle up to higher concentrations than used before. We developed procedures to derive rate
constants of block and unblock from the effects of Proc and Phys on the
EPCs based on a simplified reaction scheme. Finally, we used the full
reaction scheme found for nicotinic channels (Bufler et al.
1996a
) that includes the rapid desensitization from the open
state and that from the blocked state, and the highly ACh-sensitive
desensitized states, to model the effects of the blockers on the EPC.
There was good agreement of model and experimental results, including
the rate constants of block and unblock of the channels and of the EPCs.
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METHODS |
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Adult mice were killed by cervical dislocation, and the
diaphragm was excised rapidly. Hemidiaphragms were pinned down in a
bath chamber through which Bretag's solution saturated with 95%
O2-5% CO2 was superfused. The solution
contained (mM) 136 Na+, 3.5 K+, 117 Cl, 1.5 Ca2+, 0.7 Mg2+, 26 HCO3
, 1.7 H2PO4
, 10 Na-gluconate, 5.5 glucose, and 7.6 saccharose, pH 7.4. The superfusate
flowed through a theromostated heat exchanger before entering the bath
and was held at 10 or 20°C.
EPCs were recorded through a perfused macro-patch-clamp electrode
(Dudel 1989, 1992
). It had an ~10-µm-wide opening
and contained a current-clamp recording input as well as a stimulating
electrode through which negative current pulses could depolarize the
terminal in a graded manner by shifting the extracellular field
potential. The recording systems had upper frequency limits of 8-10
kHz. The data were digitized at 48 kHz and stored on video tape. On read-out a 10-kHz filter was used. The electrode was perfused with a
solution containing (mM) 162 NaCl, 5.3 KCl, 2 CaCl2, 0.67 NaH2PO4, 15 HEPES, and 5.6 glucose; pH 7.4. The
fluid volume in the tip of the electrode was exchanged several hundred
times per second. Drugs were applied by switching the perfusates,
performing for instance 12 exchanges of solutions while recording from
the same site in the experiment of Fig. 2. Tetrodotoxin (TTX, 0.2 µM)
was added to the perfusate of the electrode to prevent triggering of
action potentials in the nerve terminals by depolarizing pulses. As a
precaution against the generation of twitches of the diaphragm, TTX
sometimes had to be added also to the perfusate of the bath. The
cholinesterase blocker physostigmine (eserine) and the local anesthetic
procaine were obtained from Sigma and could be dissolved in the perfusate.
Recordings were stored on video tape. They were evaluated off-line by means of a series 300 Hewlett-Packard computer, a Sun-system, or PCs. To avoid influences of varying delays of quanta in multiquantal EPCs, only single quantum EPCs were evaluated. The depolarizing stimulation pulses through the electrode were arranged to release on average <0.5 quanta/pulse. Consequently more than half of the pulses produced failures of release and <9% multiquantal releases that could be recognized. Quantitative determination of the time constants, including their amplitudes, of the biphasical decay in EPCs is difficult, especially when the amplitudes of the excitatory postsynaptic currents are reduced in high concentrations of anesthetics. Therefore for the derivation of rate constants of the action of Proc in Figs. 3 and 4, two strategies for evaluation were employed, and the results of both were presented. The first used an ISO-Program developed by M. Friedrich, Köln. The recordings were searched for clearcut one quantum releases and for clearcut nonreleases. Then an average of nonrelease traces was subtracted from the one-release traces, generating artifact-free single quantum recordings (qEPCs). From the latter, the time constants of decay were evaluated automatically by a Levenberg-Marquardt routine. The second method of evaluation (implemented by E. Ratner) also used the artifact-free qEPC recordings just described. The maxima of qEPCs were detected, and the times of the maximum were used for starting an average of the decays of hundreds of qEPCs. With the consequent reduction of noise, the time constants of decay can be evaluated unambiguously. Both methods generate similar results (Fig. 4).
Simulations of three-dimensional spatio-temporal distribution of ACh,
its hydrolysis and binding to receptors, R, were done using a
commercial software package, FIDAP (version 7.05) (Engelman 1995) on SGI workstation. FIDAP is a computer program that
employs the finite element method. Accordingly, the synaptic cleft
around the terminal was represented as a space (Fig. 6) divided into variable brick-shaped elements called MESH. Shape functions link the
MESH to the equations describing the processes detailed in the next
paragraph (Aharon et al. 1994
).
The model of the synaptic cleft where calculations took place is
depicted in Fig. 6A. In this model, at time 0,
ACh is being discharged from a presynaptic vesicle during 0.1 ms
(Khanin et al. 1994). The discharge was modeled as a
step function of 0.1-ms duration. ACh then diffuses through the
synaptic cleft (50 nm width) (Parnas et al. 1989
) toward
the postsynaptic membrane and also throughout the active zone. The
diffusion of ACh was calculated using second Fick law (Crank
1975
). Concomitantly with its diffusion, ACh is also
subjected to hydrolysis by ACh-esterase, E, and binds to receptors, R. E is present all over the synaptic cleft including the active zone.
Hydrolysis was modeled according to Parnas et al.
(1989)
. In contrast to E, the receptors are concentrated in the
active zone only. Binding to receptors and the resulting processes were
modeled according to Scheme 2.
The size of the domain (its limit is denoted by Rb in Fig. 6A) wherein calculations of ACh distributions take place affects strongly the results. We took Rb to be 3,000 nm, that is, ~10 times larger than the active zone. This distance was found to be the minimal distance needed in order not to affect the results.
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RESULTS |
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Blocking of qEPCs by Phys and Proc
To record EPCs, the electrode was shifted slowly in contact with
the surface of the diaphragm in the region of the end plates while
depolarizing current pulses were applied at a rate of five per second.
When touching on a superficially located end plate, EPCs were elicited.
The recording position then was optimized to give a maximal amplitude
and a minimal rise time of the currents. It should be understood that
these recordings with a 10-µm electrode opening are only from part of
an end plate; they elicit ~10 quanta per pulse for saturating
depolarization amplitudes, and ~2 quanta per pulse in case of
triggering action potentials in the nerve terminal in the absence of
TTX. The total EPC elicited by excitation of the motor axon contains
~500 quanta (see van der Kloot and Molgó 1994).
Recordings were stable for hours in successful experiments, with
amplitudes and time courses of qEPCs remaining constant in the controls
after the drug applications. When a control declined, the experiment
was broken off. The stable recording conditions necessitate that the
resting potential of the muscle fibers was at control values of 80 to
90 mV.
To study the effects of Phys and Proc, the strength of the
depolarization was arranged to result in the release of on average 0.1-0.5 quanta per pulse. At this release rate, almost all endplate currents were single quanta (qEPCs), the amplitude and decay of which
could be evaluated readily. In an initial set of experiments, we worked
at 10°C to prolong the presence of ACh at the receptors and possibly
to increase the effectivity of the blockers. An example is shown in
Fig. 1. In average EPCs (Fig.
1A), on application of 30 µM Phys, the amplitude of the
qEPCs increased and the time constant of their decay was lengthened to
15 ms. This potentiation and lengthening of the qEPC is interpreted to
be due to the cholinesterase blocking activity of Phys (Katz and
Miledi 1975). Higher concentrations of Phys decreased the
average amplitude of the qEPCs progressively. Simultaneously, the decay
phase of the qEPCs developed a rapid and a slow phase. The time
constants
l of the slow phase increased from 30 to
~100 ms from 30 to 1,000 µM Phys, whereas the amplitude of this
component decreased (Fig. 1A). Through the respective range
of Phys concentrations, the relative amplitude of the short component,
s, grew with rising concentrations, whereas the time constant of this component decreased from 3.8 ms with 100 µM Phys to
1.4 ms with 1 mM Phys.
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Single recordings (Fig. 1B) on which the averages in Fig.
1A were based are generally shorter than the respective
averages. This difference was small in the long EPCs with 30 µM Phys;
here the mean decay time constant of the single qEPCs was 13.6 ms
compared with 14.5 ms in the averaged recordings. With 1 mM Phys, the
average short decay time constant s was 0.86 ms, clearly
less than
s = 1.4 ms in the average current (Fig. 1,
A and B). The lengthening of the decays in
averaged recordings is due to the temporal spread of the delays of
release of quanta, which amounts to several milliseconds at 10°C
(Dudel 1984
). This temporal dispersion of the quanta
lengthens the averaged recordings, and this distortion is relatively
more effective for short qEPCs. The temporal dispersion of the short spikes also depresses the amplitude of the average EPC, especially when
the qEPCs are short. We stress these points because the total EPC
produced by an action potential in the motor axon is the sum of several
hundred qEPCs, and short components of the decay will reduce the
amplitude but not necessarily the duration of such endplate currents.
In recordings at 20°C, the temporal dispersion of the quantal releases is smaller and the respective distortion of the average EPCs is less developed than at 10°C. In the experiment of Fig. 2, the effects of Proc and of Phys were compared at 20°C. On application of 10 µM Phys, the cholinesterase was blocked to a large extent and the decay time constant of the EPCs increased from 3 to ~8.5 ms. Because the open channel block effect is very small at this low concentration of Phys, these recordings with 10 µM Phys represent the "control" condition for the further effects of Proc and Phys. In averages of the recordings (not illustrated), the amplitude clearly was reduced only with 500 µM Proc or Phys and more so, to a quarter of the control amplitude, with 1,000 µM Proc or Phys.
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The evaluations in Fig. 2 are from single traces like in Fig.
1B and show s and
l as well as
their relative amplitude As (As + Al)
1
versus the blocker concentration. Control qEPCs have average time
constants of decay of 2-3 ms at 20°C (see also Figs.
3 and 4).
On application of
10 µM Phys, the qEPCs decayed with one time
constant that was higher than in the controls due to block of
cholinesterase. With 10 µM Phys in Fig. 2, this time constant became
8.0 and 8.9 ms in different controls at the beginning, in the middle,
and at the end of the experiment. On further increasing the Phys
concentration, the decay of qEPCs became obviously biexponential (see
also Fig. 1A). An initial spike was shortened from
s = 4 ms with 20 µM Phys to 0.7 ms with 1,000 µM
Phys. Concurrently, a long decay component developed that reached 90 ms
with 1,000 µM Phys. With regard to the amplitude of the qEPCs, the
proportion of the initial spike [As
(As + Al)
1
in Fig. 2] increased with rising Phys concentration, amounting to 94%
of the qEPCs with 1,000 µM Phys, the long component forming a 6%,
90-ms time constant tail. Proc was almost as effective in changing the
time course of the qEPCs as Phys, becoming somewhat more efficient at
high concentrations. For both blockers, the reduction of average EPCs
seems to be due completely to the reduction of the amplitude of qEPCs.
There is no evidence that the quantal content of EPCs was reduced by
Phys or Proc
their effect seems to be completely postsynaptic.
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In the experiment of Fig. 2, the effect of Proc was shown on the background of a constant low Phys concentration, which largely blocked the ACh-esterase and thus prolonged the presence of ACh at the receptors (see Fig. 1). The blocking of the cholinesterase, by lengthening the presence of ACh, increases the effectivity of the local anesthetics but, on the other hand, adds a second drug effect. When applying another blocker of acetylcholinesterase, diisopropylfluorphosphate (DFP), the qEPCs were prolonged much more than with 10 µM Phys, and we are not sure whether 10 µM Phys totally blocks the esterase or DFP has additional effects on the decay of the qEPC. We plan to pursue this matter further elsewhere. To extract the rate constants of the blocking effect of Proc, we avoided block of the acetylcholinesterase and applied only rising concentrations of Proc.
For the evaluation of rate constants of block by Proc, we used optimal
recordings of qEPCs as shown in Fig. 3. The controls had rise times of
on average 0.33 ms and decayed with the time constant of 1.65 ms (Fig.
3 and Table 1, preparation A).
The s were distributed with a standard deviation of 0.12 ms. On application of 300 µM Proc, the rise time shortened to 0.25 ms
and the decay of the EPCs split into short and long components,
s and
l of on average 0.33 ± 0.075 (mean ± SD) and 12 ± 3.8 ms, respectively (Fig. 3 and also
Table 1, preparation A).
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Figure 3 also presents average decays of qEPCs from the same experiment as the single traces and for more Proc concentrations. These averages used single traces like shown to the left, starting the averaging in each trace from the peak of the qEPC and thus eliminating the averaging error caused by varying delays of qEPCs from the depolarizing pulse (see METHODS). Also these averages demonstrate the development of short and long decay phases of the qEPC with rising Proc concentrations.
Average values describing the time course of the EPC are listed
in Table 1 and Fig. 5 for a wider range
of Proc concentrations for two experiments. The average delays of the
qEPCs from the beginning of the depolarizing pulse are not affected by
Proc. The rise times of the qEPCs are reduced clearly by >100 µM
Proc, and with 1 mM Proc this reduction amounts to about one-third of the control value (Table 1). Obviously at high Proc concentrations, the
rapid block open channel shortens the rise of the EPC (see also the
simulation in Fig. 8). The decay time constants and their amplitudes in
the graphs of Fig. 4 were determined either as averages of single trace
evaluations (Figs. 3, left, and 4, ) or from average
decays (Figs. 3, right, and 4,
). The results of both evaluations agree well. The dotted line approximately fitting the
results was generated by simulations and will be discussed later. The
general shape of the graphs in Fig. 4 is quite similar to that in Fig.
2; in the latter the
values are naturally higher due to the block
of ACh-esterase.
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The effects of Phys on the EPC can be evaluated more easily from single
quantal currents than those of Proc because a very low concentration of
Phys blocks the acetylcholinesterase and prolongs the EPC. Even with 1 mM Phys, the open channel block shortens the initial decay phase only
to a time constant s of 0.6-0.7 ms (Fig. 5), which can
be evaluated easily.
Figure 5 presents evaluations from two experiments, which have
almost identical results. Single quantal currents were evaluated, analogous to the evaluations of Fig. 4 (). With 10 µM Phys in Fig.
6, the EPCs decayed with a single time
constant of 8.5 ms. This corresponds to the EPCs in Fig. 1 with 30 µM
Phys, which decayed with one time constant of ~14 ms at 10°C lower
temperatures. With higher Phys concentrations, the decays of the EPCs
became biexponential with a short initial decay
s and
long later decay
l (Fig. 5). As in case of Proc (Fig.
4),
s decreased and
l increased with
rising concentrations of Phys. Very similar to the situation with Proc,
both the shortening of
s and the lengthening of
l amounted to about a factor of 10 in the concentration
range from 10 µM to 1 mM Phys. The proportion of the amplitude of the short decay component, As in relation to the
total amplitude of the EPC (As + Al) rose from 0.4 with 20 µM Phys to 0.9 with
1 mM Phys (Fig. 5).
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Calculation of the spatio-temporal concentration profiles of ACh at the receptors
The first step on modeling the EPC is to calculate the time course
of the ACh concentration in the synaptic gap after release of a quantum
of ACh from a terminal (Fig.
7A). Calculations were done as
described in METHODS. The dimensions and concentrations used are given in Table 2 with the rates
e+1 and e1. ACh is
bound to cholinesterase (E) and then is split with a rate e+2 into acetyl and choline (Scheme
1), even before reaching the
receptors R.
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When the rate constants in the model will have been determined completely, the complex spatiotemporal concentration profiles of ACh for the different conditions of block of ACh-esterase, and channels will be entered into the reaction scheme of the channels to describe the generation of the EPC.
Adjustment of rate constants to fit the time course of control EPCs
When control EPCs (no ACh-esterase or channel blockers) were
simulated using reaction Schemes 1 and 2 and the
rate constants estimated by Franke et al. (1991b) for
the adult type of nicotinic channel, the EPCs decayed with a time
constant of 2.2 ms (not illustrated). Experimentally, we found average
decay time constants of control EPCs of 1.6-2.4 ms at 20°C (Table 1,
0 Proc). As seen in Fig. 6D, the ACh concentration has
declined to insignificant levels under these conditions after <0.5 ms,
allowing most of the ACh liganded receptors to reach the states
A2R and A2O within 0.2 ms. In Scheme
2, once A2R is reached, the receptor oscillates rapidly between A2R and A2O, forming a
"burst of openings" independent of the fact that the
concentration of A might be high or zero. The short spike of high ACh
concentration in the control EPCs thus will generate a burst of
openings, which will be terminated by the unbinding of A from
A2R with the rate 2k
1. The burst duration according to Scheme 2 is given by (Colquhoun
and Sakmann 1985
)
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When the ACh-esterase is blocked by low Phys concentrations, the EPCs
are lengthened by the prolonged presence of ACh (Fig. 6), which is
determined largely by diffusion of ACh from the active zone. The
diffusion constant of ACh given in the literature is DACh = 4 · 106
cm2 s1 (Anglister et al.
1994
), and qEPCs simulated with this value decay with a
time constant of 6.1 ms (not illustrated). With
10
5 M Phys, the decay time constant of the EPCs was 8.6 ms (Figs. 2 and 5), but when a value of
DACh = 2 · 106
cm2 s
1 is adopted (Table 2), a satisfactory
decay time constant of 8.7 ms results.
Extraction of the blocking rate constants of Phys and Proc from the time courses of EPCs
Scheme 2 is inconveniently complicated for extracting
the blocking rates bp and
bp from the decay phase of the EPC. Because
the decay phase of the control can be fitted adequately by a single
exponent even in case of block of the cholinesterase, we lumped all the
relevant steps in Scheme 2 into one step with a rate
constant of 1/
0. This treatment disregards
desensitization from A2BP, but this is a slow process in
comparison to the time course of the EPC. With these simplifications
the open channel block is formulated by Scheme
3.
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1/s + 1/
l are plotted against the
concentration of Proc or Phys in Fig. 7, using the values from the
experiment of Figs. 4 (
) and 5, respectively. The sums of reciprocal
values rise with a greater slope at low Proc concentrations than at
high concentrations (Fig. 7A). The values plotted for 1 mM
Proc especially deviate. Regression lines for the plot in Fig.
7A result in slope bp = 2.8 · 106 M
1 s
1 (Eq. 10).
If the value at 1 mM Proc is excluded, a higher
bp of 6.1 · 106
M
1 s
1 results. The same trend is seen in
all six experiments of this type.
Evaluated from single traces (Fig. 3, left),
bp for the whole range of Proc concentrations
(2 mM Proc) was on average 3.1 · 106
M
1 s
1, and, excluding measurements with
Proc concentration >0.5 mM, bp = 4.6 · 106 M
1 s
1 resulted. In contrast
to the deviation of bp, the
b
p calculated from Eq. 10 was
little influenced by including or excluding data at Proc >0.5 mM. The
respective b
p for the experiment of Fig. 4 was
357 s
1, and with 1 mM Proc
values excluded it was 323 s
1. For all experiments, the average
b
p was 349 s
1, and, excluding
measurements for Proc >0.5 mM, it was 336 s
1.
Plots analogous to Fig. 7A were made for the Phys
experiments of Fig. 5 (Fig. 7B), and the average slope
representing bp was 1.3 · 106
M1 s
1. In this case, values at 1 mM Phys
did not much deviate from the trend of the other values and were
included into the evaluation. The value of b
p
derived from the products
s ·
l
was 176 s
1.
Finally we calculated time courses of EPCs (Figs. 8 and
9) using the time courses and spatial
distributions of ACh-concentration at the receptors (Fig. 6) and
reaction Scheme 2 for the reaction of the receptors with ACh
and Proc or Phys. In the case of Proc, from the simulated EPCs, the
decay time constants and their amplitudes were evaluated and compared
with the experimental results. To specifically fit the results in Figs.
4 and 5, we first had to adjust to obtain the measured decay time
constant of the control. For the experiment of Fig. 4A,
= 1,600 s
1 and for those of Fig. 5
= 750 s
1 had to be assumed (see preceding text). When using the
bp and b
p derived from
s and
l by means of Eq. 10
including measurements also at high Proc, the fit was good for 1 mM
Proc but not for lower Proc concentrations. Much better fits for
s and
l in Fig. 4(· · ·) were obtained when
s and
l were
included only from Proc concentrations <0.5 mM, i.e.,
bp = 6 · 106 M
1
s
1 and b
p = 360 s
1. The resulting fits reached r = 0.98 and deviated only at 1 mM Proc. As an average estimate, including also
the four other experiments discussed earlier, we suggest
bp = 5 · 106 M
1
s
1 and b
p = 350 s
1. Another shortcoming of the fits in Fig. 4 are the
relative amplitudes of the
s component: they tend to be
higher in the simulations than in the measurements. This divergence is
not reduced when decreasing bp to half.
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The deviation of the measured s from the fitting
simulations in Fig. 4 may indicate an error of measurement. In fact,
EPCs with 1 mM Proc are short and small (Fig. 3), and their time course is difficult to evaluate. However with bp = 5 · 106 M
1 s
1 we have
found consistently
s of ~0.3 ms with 1 mM Proc when the blocking rate should be 5,000 s
1 at 1 mM Proc. When
the ACh-esterase was blocked and ACh present for a longer period of
time,
s was even 0.54 ms at 1 mM Proc (Fig. 2). One
possibility for obtaining too-large values of short
s is
filtering by the recording system. We have simulated such a filtering
function and found a 2-kHz low-pass filter to produce approximately the
measured 0.3-ms values of
s with
bp = 5 · 106 M
1
s
1. However, the macropatch systems including the
electrodes used in our experiments were checked to present 8- or 10-kHz
low-pass filters. Another possible error could arise by misinterpreting double releases as single ones in the short, low-amplitude recordings at 1 mM Proc. Two qEPCs with a 0.2-ms decay time constant, starting with a 0.1-ms interval, may look like one qEPC and would present an
average decay lengthened to 0.3 ms. However, with a mean quantum content of the release in the experiments in Fig. 5 of 0.5, there would
not be enough double releases to affect the average decays sufficiently. Further, if double releases were responsible for apparently lengthening the decay of the EPC, the
s
should be correlated positively with the amplitudes of EPCs. However,
there was absolutely no such correlation. We thus do not see an obvious error of measurement responsible for the
s at 1 mM Proc
to be longer than predicted by the theory.
It should be noted that also in the patch-clamp measurements of the
reaction of nicotinic channels to ACh and Phys or Proc (Bufler
et al. 1996a) (Figs. 3 and 5), the decay time constants for 1 mM blocker concentration are lower than predicted by the reaction
Scheme 2. Like in our experiments with Phys, the
bp value fitting the experimental results at 1 mM Proc or Phys would have to be about half that producing a good fit
for lower blocker concentrations.
In case of the results with Phys, the values of
bp = 1.3 · 106
M1 s
1 and b
p = 176 s
1 derived by means of Eqs. 9 and 10 produce good fits of the results in simulated EPCs (Fig.
5). Even the proportions of the short and long decay components of the
EPC [As (As + Al)
1] in Fig. 5 are fitted
impressively well. Also the measured
s and
l at 1 mM could be included, deriving the blocking
and unblocking rate constants. The absence of a deviation of the
values with 1 mM Phys from the regression lines fitted for low
concentrations may reflect the 4.5-times lower blocking rate constant
of Phys in comparison with Proc.
Time courses of occupancies of receptor states in simulated EPCs
As discussed earlier, EPCs under different conditions were
simulated with the parameters in Table 2, and their time courses were
evaluated. Such simulations generate not only the time course of the
open state A2O equivalent to that of the EPC but also of other states of the receptor. The most interesting ones are plotted in
Fig. 8 for a control and for 1 mM Proc. Figure 8, left, has graphs with a linear time scale, those in Fig. 8, right,
have a logarithmic one to show the beginning of the EPC in greater detail. The open state, A2O, reaches its peak after 0.33 ms
in the control and much earlier, after 0.18 ms, in presence of 1 mM
Proc. This shortening of the rise time is due to the rapid filling of
the blocked state, A2BP, which attains a maximum occupation of 0.55 after 1 ms. Shortened rise times with high Proc concentrations were found also experimentally (Table 1). The block by Proc reduces the
amplitude of the peak of A2O from 0.58 in the control to
0.35 in 1 mM Proc. The decay of the A2O shortens
dramatically, from 2.4 ms in the control to 0.3 ms in 1 mM Proc. In
Proc, a slow further decay follows described by l.
During this long decay, A2O is refilled continuously from
the blocked state, A2BP, when P unbinds. Occupation of
A2BP declines slowly, from 0.55 at 1 ms to 0.08 at 50 ms. P
unbinds from A2BP with the rate b
p = 350 s
1, i.e., with a time constant of ~3 ms. However,
during the average life time of A2O before dissociation to
A2R
AR + A, which is equivalent to the burst duration
of 2.4 ms, the binding of 1 mM Proc with the rate of 5,000 s
1 will return most of the open channels back to the
blocked A2BP state. The slow decay of the
l
of the EPC in presence of 1 mM Proc thus is generated by multiple
cycles of unbinding and binding of P that are terminated only when
A2R can dissociate to AR + A in one of the short time
intervals at A2R. In control EPCs, the desensitized state
A2D reaches an insignificant occupation of 0.03, which
lasts for >50 ms. In presence of 1 mM Proc, the filling of
A2D is delayed and reduced because after 1 ms, most of the
channels are in the blocked state and desensitization from this state
is slower than that from A2O and goes first to the A2DP state, which is not plotted here.
Figure 9, top, in comparison with the control in Fig. 8, demonstrates the effect of block of the acetylcholine-esterase on the EPC. This block by 10 µM Phys (Fig. 9, top) increases the amplitude of the EPC relative to the control in Fig. 8, from 0.58 to 0.82, and prolongs its decay time constant to 8.1 ms (see Fig. 2). This slow decay leads to relatively much desensitizitation to A2D in Fig. 9. The maximal occupation of the open channel block state A2BP is 0.025 and does not affect the time course of the EPC appreciably. With 1 mM Phys (bottom), the peak of the EPC is relatively less depressed than with 1 mM Proc in Fig. 8, and the decay time constant is reduced to 0.8 ms with 1 mM Phys. In Fig. 2, the respective value is 0.7 ms. These weaker effects of Phys in comparison to Proc reflect the 4.5-times lower blocking rate constant bp. Other characteristics of the block by 1 mM Phys in Fig. 9 are qualitatively the same as with 1 mM Proc; with 1 mM Phys the rise time of the EPC decreases, the blocked state A2BP rises to a level higher than the initial open state, A2O, and the desensitized state remains almost empty.
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DISCUSSION |
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The paper compares experimental results on the shape of EPCs with
predictions by models. These contain the reactions of the receptor with
the transmitter, but also the time course of the presence of the
transmitter at the receptors. The latter implies modeling of the
release, diffusion, hydrolysis, and the binding to receptors of the
transmitter. The model we employ differs from that of Wathey et
al. (1979). They assumed instantaneous release (as also
Parnas et al. 1989
) and diffusion in a plane disk. The model of transmitter action did not contain the A2R and the
desensitized states. Bartol et al. (1991)
used
instantaneous release, another geometry with membrane folding, a high
value of the ACh diffusion constant and the Monte-Carlo method of
calculation, and they did not include receptor desensitization. We
think that the model we used (see METHODS) describes the
relevant processes sufficiently well. In the outcome, the differences
to the model of Anglister et al. (1994)
and
Bartol et al. (1994)
are small.
The reaction of a local anesthetic P with a nicotinic receptor/channel
in the open state A2O according to our Scheme 2 is an expansion of the sequential open-channel block scheme suggested first by Adams (1976) that is equivalent to our
simplified Scheme 3. This scheme explains the steep and the
following slow phases of the decay of the EPC in presence of the
blockers by the rapid binding of the blocker, which closes the channel,
followed by a relatively slow return from the blocked state by
dissociation of the blocker, which leads to reopenings generating the
slow phase of the decay. In this reaction scheme, there is only one open state, A2O. Adams (1977)
supported his
scheme by ingenious voltage-jump experiments and Katz and Miledi
(1975)
by measurements of membrane noise spectra.
Neher and Steinbach (1978) first measured single channel
currents in presence of ACh and lidocaine derivatives; they saw the one
open state and the openings elicited by ACh alone to break up into
repeated short openings in presence of the blocker as predicted by the
sequential open-channel block scheme. In a continuation of this work,
Neher (1983)
found quantitative discrepancies between theory and experimental results for relatively high blocker
concentrations in which the number of openings due to blocker
dissociation and rebinding was smaller than predicted. In our
experiments, this deviation would be equivalent to shorter than
predicted slow decays of the EPCs, which was not observed. Gage
and Wachtel (1984)
applied Proc to toad end plates, and their
results also could be described by the sequential open channel block
scheme. But they further reported quantitative deviations from the
theoretical predictions. In their experiments, the proportion of the
long phase of the EPC,
Al(As + Al)
1, was larger than calculated
from the theory; in our results, there was the same tendency at the
Proc concentration they used (100 µM), but the fit improved at higher
Proc concentrations (Fig. 4). For Phys the fit was excellent.
There were some early alternatives to the open channel block scheme,
assuming two different open states produced by the binding of the
blockers (Ruff 1976, 1977
; Steinbach
1968
). Such schemes were ruled out mainly by the single channel
recordings, which consistently showed only one species of open channels.
Some of the studies estimated blocking rate constants of the local
anesthetics and found 106-107 M1
s
1 in the different preparations and with different drugs
(Adams 1976
; Gage and Wachtel 1984
;
Neher and Steinbach 1978
; Shaw et al.
1985
). The blocking rates in our studies also were in this range (Table 2). For the lidocaine derivates, Neher and
Steinbach (1978)
reported a high unbinding rate of 2,200 s
1, producing rapid "flickering" of the channel.
For other local anesthetics, lower unbinding rates of 200-400
s
1 were seen, in agreement with our results (Table 2).
In the present study, the experimental conditions were varied by
blocking the ACh-esterase with 10 µM Phys. This prolonged the
presence of ACh at the receptors considerably; although 0.5 ms after
opening of the vesicle the ACh concentration in presence of the
esterase is <105 M and causes negligible further channel
opening, while with the esterase blocked the ACh concentration is eight
times higher and contributes to channel opening. Even at 5 ms, the ACh
concentration with blocked esterase is still 17 µM, which could
elicit about one-tenth maximal channel opening (Fig. 6) (see
Franke et al. 1991b
). In the simulations, the slower
decay of the ACh concentration with blocked esterase prolonged the time
constant of decay of the qEPC to 8.1 ms (Figs. 5 and 9). Although thus
the duration of presence of ACh was greatly altered, the simulations
with the open channel block model fitted the effects of high Phys
concentrations on the pEPCs very well, even better than in case of the
unblocked esterase (Figs. 4 and 5).
Although the model fitted the results with Phys very well, there
was one significant discrepancy between the predictions of the model
and the experimental results with Proc. At high Proc concentrations,
the EPCs did not decay as fast as predicted. We have discussed possible
errors of measurement and think that we can exclude them. Similar
defects in the effectivity of the block at high drug concentrations
were reported also by Gage and Wachtel (1984) and also
in our channel measurements (Bufler et al. 1996a
). It
may be concluded that the reduced blocking effectivity at high drug
concentrations in the experiments in comparison with the predictions
reflects a defect of the reaction scheme. Already Neher and
Steinbach (1978)
pointed out that reduced blocking efficiency at high blocker concentrations is in conflict with a pure open channel-block mechanism. It would be necessary to add at least another
binding step of the blocker to accommodate the experimental results
more completely, and tentative simulations show that additional competitive block at R with an equilibrium near 1 mM Proc and a rate of
unbinding of ~10,000 s
1 would do this.
A combination of open channel block with competitive block was found
also for tubocurarine. In the competitive block component, the main
difference between Proc and curare would be a 105-times
lower rate constant of unbinding from R for the latter (Bufler
et al. 1996b).
In the single-channel measurements, we have derived blocking rate
constants for Phys and Proc of 6 · 106 and 2 · 106 M1 s
1 and unblocking
rates of 200 s
1 for both. In the present study, analysis
of the changes in shape of the EPC rendered bp = 1.3 · 106 M
1 s
1 for Phys
and 5 · 106 M
1 s
1 for
Proc, with the b
p = 176 s
1 and
350 s
1, respectively. These values are not far apart,
Phys being more potent in the channels and less potent than Proc in the
EPCs. It should be noted that the investigated channels were of the embryonic type and that the "adult" channels of the EPCs had one different subunit of the receptor. This may explain the small quantitative differences in blocking efficacy.
Thus we have shown that the reaction schemes developed for nicotinic
channels (Bufler et al. 1996b) serve also to predict the
effects of Proc and Phys on EPCs. They do this for a much larger
concentration range than used so far, relying also on evaluations of
serverely blocked EPCs. For the first time in this type of studies, the
evaluations and models extend also to EPCs in which cholinesterase was
blocked, and the same models and rate constants cover these
quantitatively quite different situations. Last, the derivation of the
Eq. 10 allows a quantitative evaluation of the blocking rate
constants in EPCs.
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ACKNOWLEDGMENTS |
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The authors thank I. Horstmann for technical assistance and M. Griessl and W. Reinhardt for secretarial help.
This work was supported by Deutsche Forschungsbemeinschaft Grant SFB 391.
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FOOTNOTES |
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Address for reprint requests: J. Dudel, Physiologisches Institut der Technischen Universität München, Biedersteiner Str. 29, 80802 Munich, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 April 1998; accepted in final form 8 January 1999.
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REFERENCES |
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