From the * Department of Anesthesiology, Department of Physiology and Biophysics, University at Stony Brook, Stony Brook, New
York 11794-8480; and || Klinik für Anästhesiologie, Universität Bonn, Bonn 53105, Germany
We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps
within bursts is 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces
the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst
but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of
300 µM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit
with a Hill function; for pentobarbital, Ki = 32 µM, n = 1.09; for barbital, Ki = 1900 µM, n = 1.24. Inhibition is
voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. <0.1 ms at the Ki). Pentobarbital binds 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with
both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic
model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through
the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at >100 µM
pentobarbital.
Barbiturates have several therapeutic effects on humans including light sleep (at low doses), deep coma
(at high doses), amnesia, muscle relaxation, protection
against cerebral ischemia, and reversal of seizures
(Fragen, 1994). Thus, these drugs probably have multiple effects on the central and peripheral nervous systems. Ion channels are among the possible targets of
barbiturates. Barbiturates are known to affect many
types of ion channels including GABAA receptor channels (Tanelian et al., 1993
), some (ffrench-Mullen et
al., 1993) but not all (Hall et al., 1994
) calcium channels, sodium channels (Frenkel et al., 1990
; Barann et
al., 1993
), glutamate receptor channels (Marszalec and
Narahashi, 1993
), 5-HT3 receptor channels (Barann et
al., 1993
), and muscle-type nicotinic acetylcholine receptor (AChR)1 channels (deArmendi et al., 1993
).
Inhibition of AChR by barbiturates has been studied
with electrophysiological (Lee-Son et al., 1975; Gage
and McKinnon, 1985
; Jacobson et al., 1991
; Yost and
Dodson, 1993
), flux (Firestone et al., 1986b
; Roth et al.,
1989
; deArmendi et al., 1993
), and binding (Dodson et
al., 1990
) techniques. Several effects have been observed (Firestone et al., 1986b
, 1994
), but the dominant effect is a direct inhibitory action of the drug on the
open state of the channel. The potency of barbiturates
for inhibiting the channel is related to but not completely determined by lipid solubility (deArmendi et
al., 1993
). A study of the single channel kinetics in the
presence of pentobarbital (PB) allowed Gage and McKinnon (1985)
to dismiss a simple, sequential open channel blocking mechanism for PB action. They suggested
that the mechanism might be allosteric in that the
binding of one molecule of PB to the open channel protein induces a conformational change to a new
closed state of the channel. In this scenario, the binding of PB is not concomitant with inhibition.
Here, we use several patch clamp recording protocols to study the effects of PB and barbital (Barb) on nicotinic AChRs in outside-out patches from BC3H-1 cells. We examine single channel kinetics, the equilibrium and kinetic properties of macroscopic currents, and interactions between PB and Barb. We conclude that inhibition of the AChR by barbiturates is temporally coincident with binding of the drug to a site on the channel protein. This can be described by a model in which barbiturates bind to both the open and closed states of the channel. PB shows a strong preference for binding to the open state. The binding sites for PB and Barb do not coincide but are probably close to each other. There is evidence for an additional, lower affinity binding site for PB.
BC3H-1 cells that express the 2
-type nicotinic AChR were
cultured as described previously (Sine and Steinbach, 1984
). To
prepare cells for patch clamp recording, the culture medium was
replaced with an "extracellular" solution (ECS) containing (in
mM): NaCl (150), KCl (5.6), CaCl2 (1.8), MgCl2 (1.0), and HEPES (10), pH 7.3. Patch pipettes were filled with a solution consisting of (in mM) KCl (140), EGTA (5), MgCl2 (5), and
HEPES (10), pH 7.3, and had resistances of 3-6 M
. An outside-out patch (Hamill et al., 1981
) with a seal resistance of 10 G
or
greater was obtained from a cell and moved into position at the
outflow of a perfusion system. The perfusion system consisted of
solution reservoirs, manual switching valves, and a V-shaped
piece of plastic tubing inserted into the culture dish (Liu and
Dilger, 1991
). For single channel current measurements, the
manual valves were used to switch from drug-free to drug-containing solutions containing 0.2 µM ACh. For macroscopic current measurements, the perfusion system also contained a solenoid-driven pinch valve. One arm of the "V" contained ECS without agonist (normal solution); the other arm contained ECS with
300 µM ACh (test solution). In the resting position of the pinch
valve, normal solution perfused the patch. The pinch valve was
then triggered to stop the flow of normal solution and start the
flow of test solution. After the patch was exposed to test solution
for 0.2 s, the pinch valve was returned to its resting position for
several seconds. In this way, we treated the patch to a series of
timed exposures to agonist-containing solution while minimizing
the desensitizing effects of prolonged exposure to high concentrations of ACh. The perfusion system allows for a rapid (0.1-1
ms) exchange of the solution bathing the patch.
On the day of the experiment, we prepared a stock solution of 20 mM Barb or 1 mM PB (Sigma Chemical Co., St. Louis, MO) in ECS. The 20 mM Barb solution had a pH of 8.3; this was titrated to pH 7.3 with concentrated HCl. The stock solution was then diluted with ECS to obtain the desired concentration of barbiturate. The solution was transferred to a perfusion reservoir, a plastic intravenous drip bag.
The currents flowing during exposure of the patch to ACh
were measured with a patch clamp amplifier (EPC-7; List Electronic, Darmstadt, Germany), filtered at 3 kHz (3 db frequency,
8-pole Bessel filter, 902LPF; Frequency Devices, Haverhill, MA),
digitized and stored on the hard disk of a laboratory computer
(PDP-11-73; Digital Equipment Corp., Maynard, MA). Data analysis was performed off-line with the aid of our own computer programs. Experiments were performed at room temperature (20-
23°C). Results are expressed as means ± SD.
For macroscopic currents, the first step was to record current
responses (at 50 mV) during 200-ms applications (at 5-s intervals and sampled at 100-200 µs per point) of ECS containing 300 µM ACh, a concentration that opens ~95% of the AChR channels from BC3H-1 cells (Dilger and Brett, 1990
). This current
served as a reference point for estimating the number of channels in the patch. We returned to this test solution frequently
during the life of the patch to quantify any loss of channel activity. Both the normal and test perfusion solutions were then
switched to barbiturate-containing solutions by means of manual
valves. Responses of the patch to applications of 300 µM ACh
during continuous exposure to barbiturate were recorded. The
drug-free solutions were then re-introduced, and recovery currents were measured. This protocol was continued with other
barbiturate concentrations until the demise of the seal or a large
loss of channel activity (anywhere from 10 min to 3 h). Data were
accepted when the drug-free currents obtained before and after
exposure to drug had not changed by >10%. For some experiments (see Figs. 10 and 11), only the test solution contained barbiturate. Using this protocol, a simultaneous jump in the concentration of both ACh and barbiturate was made. The resulting current provides information about the kinetics of drug inhibition.
The ensemble mean current was calculated from between 10 and 60 individual current traces. Mean currents were fit to a 1- or 2-exponential function.
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The time constant of the 1-exponential fit and the slower time
constant of the 2-exponential fit, 1, measures the current decay
due to desensitization (Dilger and Liu, 1992
). In the presence of
PB, the current contained an additional fast component,
2. This
represents the time course of inhibition by PB (see RESULTS). Fractional inhibition of the peak mean current, Ip, the maximum inward current obtained after perfusing ACh, was calculated as the ratio of the current in the presence of drug, Ip
to the current in the absence of drug, Ip. For PB experiments, Ip
was obtained from the amplitude of the slow component of the decay, Ip
= I
+ I2 (see RESULTS).
Single channel recordings were made while the patch was exposed to ECS + 0.2 µM ACh at a patch potential of 100 mV.
Data was digitized in 5-s segments at a rate of 50 µs per point. 3-10 data segments were collected (enough to obtain 200-1,000 single channel events, depending on the channel activity in the patch). Data collection was repeated with ECS + ACh + barbiturate and then again with ECS + ACh (recovery). Data were accepted if, after analysis, we found that the channel kinetics during recovery
were within 20% of the values obtained during the initial data
collection segments.
Single channel analysis consisted of identifying opening and closing transitions, obtaining the distribution of open, closed, and
burst durations, and fitting the distributions (expressed as the
number of events vs. log-binned duration, 10 bins per decade) to
1- and 2-exponential probability distribution functions by finding
the maximum log-likelihood using a simplex algorithm. The single-exponential fit was considered adequate when the fractional amplitude of one of the components of the 2-exponential fit was <0.1. The definition of bursts was based on the distribution of short (gap) and long closures (Colquhoun and Sakmann, 1985).
Mean gap and open durations and the number of openings per
burst were corrected for undetected events using equations derived for a two-state mechanism (Colquhoun and Hawkes, 1995a
).
This approximation is probably adequate because of the large
time separation between brief and long closed durations and our
observation that the open time histogram in the presence of barbiturate usually has only one component. Mean single channel
amplitudes were calculated by taking the average of the amplitudes of those openings lasting >0.25 ms; these are not attenuated by the 3 kHz low-pass filter.
The Effects of Barbital and Pentobarbital on AChR Single Channel Currents
The effects of Barb and PB on single AChR channels
are illustrated in Fig. 1. Inward single channel currents
at 100 mV were activated by a low concentration of
ACh (0.2 µM). Under control conditions (Fig. 1, left),
channel activity consists of
4 pA openings lasting an
average of 4 ms and separated by tens of milliseconds. Occasionally, a brief closing transition is seen. Both 100 µM PB and 500 µM Barb (Fig. 1, right) induce a bursting pattern of channel activity. The closures within a
burst are much longer for PB than for Barb. The Barb-induced closures are so brief that the single channel
amplitude appears to be attenuated by ~8%. The attenuation is more pronounced at 1,000 µM Barb (see
Fig. 5 D).
Table I. |
The closed duration histograms constructed from
single channel data have two components (Fig. 2). The
dominant component of the control histogram occurs
near 60 ms and corresponds to the time between activation of different channels in the patch. For 0.2 µM
ACh, a long closed time of 60 ms indicates that there
are ~150 active channels in the patch.2 The small, brief
component of the control histograms occurring at
<100 µs most likely corresponds to the closing of a
channel followed by the rapid reopening of the same
channel. The bursting activity of the barbiturates is represented by a large number of gaps: the brief component of the closed time histogram. PB induces gaps
near 2 ms; Barb induces gaps near 70 µs. Neither barbiturate has a significant effect on the long closed component.3
The barbiturates decrease the open duration of single AChR channels but have little effect on the burst
duration (Fig. 3). Under control conditions, there are
two components in the open duration histogram; a
brief one at 200-500 µs comprising 20-50% of the
events and a long one at 4-5 ms. Because there are very few brief closures within a burst, the control burst duration histogram is very similar to the control open duration histogram. In the presence of either 100 µM PB or
500 µM Barb, the open duration histogram collapses to
a single component with a time constant of 1.2-1.4 ms.
In contrast, there is very little difference between the
control and barbiturate burst duration histograms both
in the number and time constants of the components.
The results from single channel experiments on 15 patches with PB and 6 patches with Barb are summarized in Figs. 4 and 5. We have plotted the concentration dependence of the open, open, and burst,
burst, durations (the longer component when there are two
components) (Figs. 4 A and 5 A), the number of openings per burst, Nopen/burst, (Figs. 4 B and 5 B), the gap
duration,
gap, (Figs. 4 C and 5 C), and current amplitude (Figs. 4 D and 5 D). Both barbiturates cause a
monotonic decrease in the open duration with 50 µM
PB or <200 µM Barb causing a 50% decrease in
open.
The burst duration is nearly constant except for [PB] > 100 µM. The number of openings per burst increases
to 1.5 and 5 at high concentrations of PB and Barb, respectively. The duration of PB-induced gaps varies between 1 and 4 ms. The gap duration at 25 µM PB may
be an underestimate because the closed time component probably consists of a mixture of PB-induced gaps
and the briefer channel closures seen in control. For
Barb, the gap duration is 40-60 µs. The single channel
current amplitude is independent of [PB] but there is a
decrease in the absolute value of the apparent current
amplitude with increasing concentrations of Barb.
For Barb, many gaps are not detected when a filter
frequency of 3 kHz and sampling time of 50 µs are
used. To verify that the correction procedure for accounting for missed events provides good estimates of
the mean open and gap durations, we studied six
patches using a cutoff frequency of 6 kHz, a sampling
time of 25 µs, and an applied voltage of 125 mV. Under these conditions, there was very little variation of
the current amplitude with Barb concentration; the
amplitude was 4% lower with 1 mM Barb than with control. The gap duration remained 50 µs. At the 6 kHz resolution, a greater number of gaps are resolved, but
after correcting for undetected events, the values of
Nopen/burst and
open are no different than at the 3 kHz
resolution. The Barb concentration dependence of the
burst duration was the same for both the 3 kHz and 6 kHz data.
If we assume that the Barb-induced bursts are composed of brief openings to the fully opened state and
brief closures to the fully closed state, we can use a beta
function analysis of the amplitude histogram to estimate the open and closed time within bursts (Yellen,
1984). To do this, we applied a 1-kHz Gaussian digital
filter to the single channel data and constructed an amplitude distribution from segments containing single
bursts. This distribution is then fit to a beta function
containing two parameters: the average open and closed
times within bursts. For four patches with 1 mM Barb,
the average open time was 150 ± 70 µs, and the average
closed time was 46 ± 17 µs. This estimate of the open time is shorter than the one obtained by analyzing single channel data with 1 mM Barb (540 ± 110 µs, Fig. 5
A), but the estimate of the closed time is similar to the
average gap duration from single channel analysis. This
suggests that even after correcting the single channel
data for unresolved events, we may overestimate the
open time and underestimate the number of openings
per burst.
The single channel data suggest that both PB and Barb act, at least qualitatively, as blockers of the AChR channel. In this interpretation, the two barbiturates differ in the duration of blocking events: the less potent drug, Barb, blocks for <0.1 ms, and the more potent drug, PB, blocks for ~2 ms. In the DISCUSSION, we make a quantitative test of models in which barbiturates block both open and closed AChR channels. Before doing so, we present data from macroscopic current experiments that provide additional information about the action of barbiturates on the channel.
The Effects of Barbital and Pentobarbital on Macroscopic AChR Currents
Both Barb and PB inhibit the macroscopic currents evoked by rapid perfusion of ACh. Fig. 6 presents examples of currents activated by 300 µM ACh in control and in the continuous presence of 50 and 400 µM PB (Fig. 6 A) or 2 and 20 mM Barb (Fig. 6 B). In the control traces, the current reaches a peak within <1 ms and then decays with a time constant of 50-60 ms due to desensitization. With 2 or 20 mM Barb, the peak currents are reduced to 60 or 5% of control and desensitization occurs with the same time course as in control. It appears that Barb interacts with the channels either before they are opened by ACh or very quickly after ACh is perfused. With 50 or 400 µM PB, an initial fast decay precedes desensitization. This suggests that, unlike Barb, PB is not very effective at interacting with closed channels. We explore this in more detail below. To determine the degree of inhibition of open channels by PB, we fit the data to a 2-exponential decay and extrapolate the slow component (desensitization) to t = 0 (Fig. 6 B, dotted lines). The extrapolated peak currents are reduced to 35% (50 µM) or 4% (400 µM PB) of control.
The results from experiments on nine patches with Barb (relative peak currents) and six patches with PB (relative extrapolated peak currents) are summarized in Fig. 7. These data were fit to the Hill equation (Fig. 7, solid lines):
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where [B] is the drug concentration, Ki is the drug concentration needed for 50% inhibition (IC50), and n is the Hill coefficient. For Barb: Ki = 1.9 ± 0.2 mM, n = 1.24 ± 0.07; for PB: Ki = 32 ± 2 µM, n = 1.09 ± 0.06. Thus, PB is 60 times more potent than Barb at inhibiting the AChR. Because the Hill slopes are close to unity, it is possible that only one barbiturate molecule is involved in the inhibition of a channel.
The fast decay that occurs in the macroscopic currents with PB provides information about the rate of
equilibration of PB with the channel. The decay is
faster with higher concentrations of PB. In Fig. 6 A, the
time constants are 3.2 and 1.9 ms for 50 and 400 µM
PB, respectively. Fig. 8 shows that the fast decay time
decreases monotonically with the concentration of PB.
The inhibitory effect of PB is not voltage dependent.
This is illustrated in Fig. 9 in which ACh-activated currents at three different voltages, 100,
50, and +50
mV, are compared for a single patch. The traces are
scaled so that the controls have the same peak current
level. Neither the time constant of the fast current decay, nor the extrapolated level of the residual current is
affected by voltage. There was no difference in the effect of Barb on macroscopic currents over this voltage
range either (not shown).
The macroscopic current data used for Figs. 6-9 were obtained with equilibrium drug concentrations; that is, the barbiturate was present in both the normal and test perfusion solutions. Information about the kinetics of current inhibition by the barbiturates can be obtained from experiments in which the drug is applied simultaneously with ACh. Fig. 10 shows that, for 100 µM PB, the current decay under equilibrium conditions and the current decay after rapid addition of the drug (onset) are identical. This supports the idea that PB has very little interaction with channels when they are closed; prior exposure to PB does not change the degree of inhibition either at the peak or during the decay of the current response.
Rapid addition of Barb also produces a current decay, but on a much faster time scale (Fig. 11). For this
experiment, the time resolution was increased by perfusing with 10 mM ACh (this saturates the ACh binding
sites within microseconds so that the 20-80% risetime of the current, 40 µs, is determined mainly by the channel opening rate [Liu and Dilger, 1991]), filtering at 15 kHz, sampling at 5 µs per point, and using +50 mV to
avoid channel block by ACh. Two pieces of qualitative
information about the effects of 5 mM Barb can be extracted from Fig. 11. The equilibrium trace shows that,
in contrast to PB, the inhibitory effect of Barb is
present at all times after perfusion with ACh. We conclude that Barb either interacts with closed channels to
the same degree as it interacts with open channels or, it
does not interact with closed channels but equilibrates
with open channels extremely quickly, on the order of
tens of microseconds or faster. The second observation, that the onset current trace exhibits a 60-µs decay,
probably reflects both the binding kinetics of Barb and
the time course of the Barb concentration jump. Similarly, the kinetics of recovery from block by Barb show a
relaxation from the equilibration level of inhibition to
control with a time course of 50 µs (not shown). The
time resolution of these experiments is not sufficient to determine if this represents the kinetics of Barb dissociation from its inhibitory site or simply the diffusion of
Barb away from the patch.
Interactions between Barbital and Pentobarbital
To determine whether PB and Barb inhibit the AChR
channel by binding to a single site on the AChR protein, we performed experiments with both barbiturates.
Fig. 12 is an example with 5 mM Barb and 100 µM PB.
In the left panel, 100 µM PB decreases the extrapolated peak current to 22% of control. In the right
panel, 5 mM Barb decreases the current to 34% of control (note different current scale). When both barbiturates are present, the current decreases to 40% of the 5 mM Barb current (14% of control). Thus, PB is less effective when applied in combination with Barb than
when applied by itself. If the two drugs acted independently, the current would have been 22 × 34% or 7.5%
of the control. If the two drugs compete for the same
binding site, the predicted current is 15% of control
(see DISCUSSION). The time constant of the fast decay component is 1.6 ms for PB alone and 1.8 ms for both
barbiturates in combination.
Inhibition curves for PB alone and PB + 5 mM Barb
are shown in Fig. 13. The latter data are normalized to
the relative current observed in the presence of 5 mM
Barb alone, 0.30 ± 0.02 (n = 25). Fits of the data to the
Hill equation (Eq. 2), give Ki = 28 ± 2 µM for PB alone
and Ki = 53 ± 3 µM for PB + 5 mM Barb. For both
data sets, the Hill coefficient was close to unity: 1.09 ± 0.07 and 0.96 ± 0.07, respectively. Thus, there is a considerable decrease in the effectiveness of PB when Barb
is present. However, this decrease is not as great as
would be expected if PB and Barb were competing for
a single binding site (Fig. 13, dotted line ; see DISCUSSION). In the presence of 5 mM Barb, the time constant of the fast decay seen with PB is decreased at 25 and 50 µM [PB] but is unchanged at [PB] 100 µM (Fig. 14).
Effects Seen with One Barbiturate
The bursting effect of the barbiturates on single AChR
channels suggests a model in which drug molecules
bind to the AChR and block the flow of ions through
the channel. This bursting cannot be explained by a
model in which drug molecules bind to and block only
the open state of the AChR (purely open channel block) because the expected increase in burst duration
(Neher, 1983) is not seen (Figs. 4 A and 5 A). We will
test the adequacy of the model shown as SCHEME I
(Murrell et al., 1991
; Dilger et al., 1992
) in which barbiturate molecules (B) can bind to both the open (O)
and closed (C) conformations of the channel.
Scheme I.
In SCHEME I, the various closed states of the AChR
(unliganded, singly liganded, and doubly liganded) are
represented by a single state. The effective opening
rate, , depends on agonist binding, agonist concentration, and the channel opening rate. The channel closing rate is
. A barbiturate molecule may bind to either
the closed state (to form CB) or open state (to form OB); the association (f and f
) and dissociation (b and
b
) rates may depend on the channel conformation.
The gating transition rates between drug-bound open
and closed states (
and
) may differ from the normal gating transition rates. In SCHEME I, a single barbiturate molecule is sufficient to inhibit one channel; this
is supported by the concentration-inhibition curves for
PB and Barb (Fig. 7) that have Hill coefficients close to
unity.
SCHEME I makes quantitative predictions about the
drug concentration dependence of open,
burst,
gap, and
Nopen/burst (Dilger et al., 1992
).
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The relative peak current induced by rapid perfusion of saturating concentrations of ACh can also be calculated from SCHEME I.
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SCHEME I predicts that the time constant of the current decay induced by a jump in drug concentration is:
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Note that, in SCHEME I, the time constant of the macroscopic current decay is dependent on the same parameters as the kinetics of channel flickering. Hence, the decay is the multi-channel correlate of single channel
flickering. Neither nor
appear in the macroscopic current expressions (Eqs. 7 and 8) because, with saturating concentrations of ACh, dissociation of one molecule of ACh is quickly followed by binding of another.
Under these conditions, the concept of burst loses its
meaning.
The Ki values (from Fig. 7) for PB (32 µM) and Barb
(1.9 mM) determine the equilibrium between open
and open-blocked channels (b/f) in SCHEME I. These
values are about twofold greater than those reported
for PB and Barb inhibition of flux in Torpedo AChR (deArmendi et al., 1993). For PB, the association rates
are given by fitting the concentration dependence of
the single channel open time (Eq. 3; Fig. 4 A, solid line);
f = 6.5 × 106/M/s. The dissociation constant can be
calculated from f × b/f; b = 210/s. Very similar values
for the association and dissociation constants for PB
are obtained by fitting the concentration dependence of the fast decay time constant (Eq. 8, Fig. 8); f = 4.0 ± 0.6 × 106/M/s and b = 210 ± 20/s.
For Barb, analysis of the amplitude distribution suggests the open duration is more severely affected by the
limited time resolution of the recording system than
are the gap durations. Assuming for the moment that
b >> , the observed gap duration gives b = 2 × 104/s
(Eq. 5, Fig. 5 C). Combining this with the equilibrium
dissociation constant gives f = 1 × 107/M/s. This is
faster than the value obtained by fitting the Barb concentration dependence of the open duration (f = 4 × 106/M/s) but within the range obtained from analysis
of the amplitude distribution (6-12 × 106/M/s).
Estimates for the remaining undetermined parameter in SCHEME I, , can be obtained by fitting the concentration dependence of either the burst duration or
the number of openings per burst. However, the burst
duration may be the better measurement to fit because
unresolved events will affect the number of openings
per burst more than the burst duration. For PB, the
burst duration is very sensitive to the value of
; only
values in the range 150-220/s provide a good description of the data at
100 µM PB. With
= 200/s, the
predicted number of openings per burst do not differ
very much from the observed values (Fig. 4 B, solid line).
For Barb,
is not as well defined; values in the range 100-800/s all predict a fairly flat concentration dependence of the burst duration. With all of these values of
, the predicted number of openings per burst is
much higher than the observed values at 1,000 µM
Barb: Nopen/burst = 32 with
= 100/s and 16 with
= 800/s. This range of values for
satisfies the assumption that b >>
, validating the estimate of b from the gap duration. The predictions of SCHEME I are shown
with solid lines in Figs. 4 and 5 using the best fitting values (or intermediate values when there is a range of acceptable values) of f, b, and
(Table I).
In their single channel study of PB on ACh receptors in denervated mouse muscle, Gage and McKinnon found quantitatively similar results on the open, gap, and burst durations. From the concentration dependence of the open duration, they calculated f = 3.4 × 106/M/s (16°C). They found a fivefold increase in the gap duration over the range of 10-500 µM PB from 1 to 5 ms. They considered this latter result as definite evidence against a sequential open channel blocking mechanism (SCHEME I without the CB state) but did not explore any additional models.
A state dependence for barbiturate binding to Torpedo AChRs was observed by deArmendi et al. (1993).
They found that the open state is preferred over the
closed state by a factor of 4.7 (PB) and 3.2 (Barb).
These values were determined by comparing the concentration of barbiturate needed to inhibit flux with
the concentration needed to displace [14C]amobarbital
bound to the resting receptor (Dodson et al., 1990
). The ~100 µs time resolution of our patch clamp experiments limits our ability to quantify the degree of barbiturate binding to the closed state. Fig. 10 indicates that
there is no more than a 10% block of the closed channel with 100 µM PB. This implies a binding affinity to
the closed state on the order of 1 mM and an open/ closed state preference of about 30-fold for PB in
AChRs from BC3H-1 cells. We cannot determine the
state preference of Barb for our experiments (Fig. 11).
Interactions between Barbital and Pentobarbital
The experiments illustrated in Figs. 12-14 address the question of whether PB and Barb compete for a single binding site on the AChR channel. If binding of the two drugs were absolutely competitive, the inhibition curve for PB in the presence of Barb, would be described by Eq. 9.
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With 5 mM Barb, Eq. 9 predicts a 3.3-fold shift ([Barb]/KBarb) of the PB inhibition curve to a half maximum effect at 94 µM PB (Fig. 13, dashed line). The observed shift of the half maximum concentration is only 1.9-fold. Thus, the binding of PB does not exclude the binding of Barb. The binding of the two drugs is not independent either. The presence of Barb decreases the binding affinity of PB. This is also apparent from measurements of onset kinetics (Fig. 14). In the presence of Barb, PB exhibits faster kinetics. Fits of the data to Eq. 8 indicate that, in the presence of 5 mM Barb, the association rate of PB is decreased (from 4.8 ± 0.6 × 106/M/s to 3.5 ± 0.8 × 106/M/s), and the dissociation rate of PB is increased (from 200 ± 25/s to b = 340 ± 70/s). One interpretation is that the binding sites for PB and Barb are the same but when both drugs bind, they have to move to nearby, less stable positions. Alternatively, there could be two distinct binding sites for the drugs and these sites interact allosterically. Our data cannot distinguish between these two possibilities.
Allosteric Model
An alternative interpretation of the bursting behavior
induced by barbiturates is to consider bursting to arise
from the control burst activity at rates modified by barbiturates. SCHEME II is a model that is often used to describe the normal kinetics of AChR single channels
(Auerbach, 1993).
Scheme II.
In this scheme, R represents the receptor and A represents ACh. Channel activation results from the binding to two molecules of ACh followed by a conformational change from the doubly-liganded closed state
(A2R) to the open state (A2R*). At low concentrations of ACh, a burst consists of one or more transitions between A2R and A2R* terminated by the dissociation of
the agonist at a rate k2. The open time is given by 1/
,
the gap duration by 1/(
+ k
2) and the number of
gaps per burst by
/k
2. Under control conditions,
= 0.5/ms, b
k
2
30/ms (Auerbach, 1993
), so that the
average burst consists of two 2-ms openings and one 20-µs gap (with the time resolution of our experiments, very few of these gaps are detected). Assume that the
binding of barbiturates (with microsecond kinetics)
modifies these rates to produce the observed burst kinetics. We have calculated the rates at each concentration of barbiturate. For both PB and Barb,
increases
as a function of concentration and is on the order of
1/ms for 100 µM PB and 250 µM Barb. 100 µM PB decreases both
and k
2 by a factor of 100. The effects of
250 mM Barb are more moderate;
decreases by a factor of 2 and k
2 decreases by a factor of 5. The difficulty
with SCHEME II, however, is that it cannot account for
the fast decay seen in macroscopic currents with rapid
perfusion of 300 µM ACh in the presence of PB (Fig.
10). Moreover, SCHEME II predicts that the onset of
macroscopic currents would have an onset time (at
high concentrations of ACh) of 1/(
+
), which is predicted to be 0.8 ms at 100 µM PB. Experimentally, we
do not see any decrease in onset time (Fig. 10). We
conclude that an allosteric model such as SCHEME II is
not viable explanation for the effects of barbiturates.
Extension of the Blocking Model
In SCHEME I, the single channel gap duration (Eq. 5) is
inversely proportional to the sum of b and and is independent of the barbiturate concentration. For PB,
the predicted gap duration is 2.2 ms (Fig. 4 C, solid
line). The observed gap durations vary from 1.1 to 3.4 ms. The measured burst durations at high concentrations of PB also differ from the predictions of the
model. One could argue that these deviations from the
predicted values are unimportant because they occur at
concentrations greater than three-times the Ki (after
all, even the archetypal AChR open channel blocker, QX-222, shows deviations from predictions at high concentrations [Neher, 1983
]). However, we wanted to determine whether we could use this information to gain
further insights into the mechanism of action of the barbiturates. Several pieces of evidence suggest that the observed deviations from SCHEME I may be due to the binding of a second molecule of PB: (a) an increase in gap
duration with [PB] is expected if the second molecule
binds with low affinity and postpones the re-opening
(unblocking) of the channel, (b) the interactions seen
when both PB and Barb are present suggest that two
barbiturate molecules may bind simultaneously, and
(c) when the macroscopic current inhibition data (Fig.
7) is fit to a two-site inhibition function (Eq. 10), the
two binding affinities are K1 = 34 ± 3 µM and K2 = 800 ± 500 µM (the dashed line in Fig. 7 is the prediction for
two binding sites with affinities of 38 and 460 µM).
![]() |
(10) |
We then considered whether SCHEME III could be
used to quantitatively predict the observed single channel gap and burst distributions. SCHEME III contains
one additional state with two barbiturate molecules bound. We used Mathematica (version 2.2; Wolfram
Research, Inc., Champaign, IL) to numerically evaluate
the relevant matrix operations (Colquhoun and
Hawkes, 1995b) for this model.
Scheme III.
Numerical evaluation of SCHEME III requires values
for nine independent constants. The channel activation rate, , which depends on ACh binding, channel
isomerization and the number of active channels, was
set to 0.02/s to correspond to a typical control interburst interval of 400 ms. The previously determined values of
and f were used (Table I). The values of
and
b were adjusted to account for the gap duration at low
concentrations of PB; the best agreement was obtained
with
= 400/s and b = 300/s. We assumed that the
poor interaction of PB with closed channels results from a low association rate and a normal dissociation
rate: f
= f/10, b
= b. Detailed balancing was used to
evaluate
. We also assumed that the binding of a second molecule of PB has a normal association rate and a
fast dissociation rate: f2 = f, b2 = 12 × b (giving b2/f2 = 460 µM which is near the lower limit of the range obtained from a two-site fit). The results of the numerical
evaluation are shown with dashed lines in Fig. 4 A, B,
and C.4 SCHEME III quantitatively predicts the PB concentration dependence of the gap and burst durations
and the number of openings per burst (the prediction
for open duration is not shown because it is identical to
that of SCHEME I). Similar results are obtained when
different assumptions are used (second binding site
having a slow association rate and a normal dissociation
rate: f2 = f/12, b2 = b; binding to closed channel having a normal association rate and a fast dissociation
rate: f
= f, b
= 10*b). As might be expected, as the affinity of the second binding site is decreased, higher
concentrations of PB are needed to obtain comparable
changes in the predicted gap and burst durations (e.g.,
the predictions for b2/f2 = 920 µM at 1,000 µM PB are
similar to the predictions for b2/f2 = 460 µM at 500 µM
PB). The predictions of SCHEME III for macroscopic
currents and kinetics do not differ significantly from
the predictions of SCHEME I.
Summary
The action of PB and Barb differs primarily in the dissociation rate; Barb dissociates 80 times faster than PB.
This is a greater difference than would be expected if
lipid solubility were the only factor that determines barbiturate potency; the octanol:buffer partition coefficients
are 106 (PB) and 4.5 (Barb), giving a ratio of 23 (Firestone et al., 1986a). The same conclusion was reached,
based on flux experiments with 14 barbiturates, by deArmendi et al. (1993). Our experiments suggest that
the inhibitory binding site is not identical for PB and
Barb. This is a plausible explanation for the poor correlation between potency and lipid solubility. Interestingly, the potency ratio for Barb and PB anesthesia in
tadpoles is also large: 14.6 mM/0.16 mM = 90 (Lee-Son
et al., 1975
).
The kinetic experiments described here do not directly address the question of the location of the barbiturate binding site(s). The close temporal association
between the duration of inhibitory events seen at the
single channel level (the gap duration in Figs. 4 and 5)
and the kinetics of macroscopic current inhibition after rapid perfusion of barbiturate (the onset time in
Figs. 10 and 11) suggest that barbiturate binding and
channel inhibition are inseparable. This favors a steric
blocking mechanism over an allosteric effect. This has
also been observed with other anesthetics acting on the
AChR channel (Dilger et al. 1994). Allosteric mechanisms cannot be completely dismissed, though. One
possibility is that the barbiturates bind and dissociate
on the microsecond time scale and induce a conformational change to a new closed state of the channel. In
this scenario, the transition rates between the open and
new closed states determine bursting and relaxation kinetics. These rates would have to be exquisitely sensitive to the difference in chemical structure between PB
and Barb to account for the 100-fold difference in the
kinetic actions of these drugs.
The question of the location of the barbiturate binding site(s) might be answered more convincingly by
site-directed mutagenesis experiments as has been done
for open channel blockers such as QX-222 (Charnet et
al., 1990). Yost and Dodson (1993)
have argued that the
site of action for amobarbital does not involve amino- acids at the 10
level (near the center of the membrane)
of the M2 transmembrane region of the channel. This
does not, however, rule out other sites within the pore
of the channel, nor does it rule out the 10
level as being part of the binding site for other barbiturates. Inhibition of AMPA-selective glutamate receptor channels
by PB is influenced by amino acids within the M2 region of the channel pore (Yamakura et al., 1995
). However, so far there is no kinetic evidence that PB acts as
blocker of this channel.
Original version received 19 July 1996 and accepted version received 20 December 1996.
Address correspondence to Dr. James P. Dilger, Department of Anesthesiology, University at Stony Brook, Stony Brook, NY 11794-8480. Fax: 516-444-2907; E-mail: jdilger{at}ccmail.sunysb.edu
This research was supported in part by a grant from the National Institute of General Medical Sciences (GM 42095); Klinik für Anästhesiologie, Universität Bonn; Department of Anesthesiology, University at Stony Brook; and an institutional grant from the School of Medicine, University at Stony Brook.We thank Ms. Claire Mettewie for culturing cells.