Department of Molecular Pharmacology and Biological Chemistry,
Northwestern University Medical School, Chicago, Illinois
 |
Introduction |
The
importance of neuronal nicotinic acetylcholine receptors (nnAChRs) in
brain function and drug action is increasingly recognized. Because
nnAChRs are located on soma, preterminal, and presynaptic regions of
GABAergic and other interneurons in the cortex and hippocampus, their
modulation caused by various drugs could lead to a cascade of synaptic
events involving multiple neurotransmitters, resulting in complex
behavioral changes.
n-Alcohols have been shown to exert a dual action on nAChRs,
depending on the carbon chain length. In muscle nAChRs, ethanol and
other short-chain alcohols prolong the decay phase of the miniature
end-plated currents (EPCs) (Gage et al., 1975
), increase the peak EPC
amplitude, and prolong the channel lifetime (Bradley et al., 1980;
Linder et al., 1984). In contrast, longer chain alcohols
(n-butanol, n-hexanol, and n-octanol)
accelerate the EPC decay and reduce the peak EPC amplitude (Bradley et
al., 1984). At the single-channel level, butanol and pentanol increase
the burst frequency, resulting from an increase in the opening rate of
the ACh receptor channel (Dilger et al., 1994; Liu et al., 1994). This
effect would account for the observation that ethanol increased the
saturating response induced by high concentrations of ACh (Aistrup et
al., 1999; Zuo et al., 2001). In addition, n-alcohols reduce
single-channel conductance and shorten the mean channel open time in
muscle nAChRs, both of which are thought to be the basis for the
inhibitory action of alcohols (Murrell et al., 1991; Forman and Zhou,
1999).
Experiments performed with nnAChRs of native neurons have shown that
ethanol potentiates ACh-induced currents in 
4
2-type nnAChRs in
rat cortical neurons in primary culture (Aistrup et al., 1999). Using
the 42 nnAChRs stably expressed in human embryonic kidney (HEK)
cell line, we have previously found that shorter chain alcohols from
methanol to n-propanol potentiate the currents induced by
ACh, whereas longer chain alcohols from n-pentanol to
n-dodecanol (C5-C12) inhibit the currents (Zuo et al.,
2001). n-Butanol (C4) is at the transition position from
potentiation to inhibition and exerts a biphasic effect, either
potentiating or inhibiting the currents depending on the concentrations
of ACh and butanol. At low ACh concentrations, butanol exhibits a biphasic inhibition being accentuated by increasing butanol
concentration from 1 to 100 mM, but becoming less pronounced or even
being converted to potentiation at concentrations higher than 100 mM
(Zuo et al., 2001).
Because n-butanol is at the transition point from
potentiation to inhibition in the action of a series of aliphatic chain n-alcohols, and also because n-butanol itself
exhibits a dual action, it might hold a clue to the mechanism of the
dual action of alcohols. Several questions are asked. Is the relief of
inhibition observed at high butanol concentrations due to butanol
potentiation overcoming the inhibition? Alternatively, is it due to
butanol's direct activation of receptor as a partial agonist? Is
butanol less effective in blocking the butanol-activated receptor than the ACh-activated receptor?
It was found that n-butanol at high concentrations evoked
small currents that were blocked by both mecamylamine and
dihydro--erythroidine (DHE), suggesting that butanol acted as a
weak partial agonist on nnAChRs. n-Butanol also modulated
ACh-induced currents in a concentration-dependent manner. At a low
concentration (10 mM), butanol shifted the ACh dose-response curve
toward higher concentrations and suppressed the maximum response,
whereas at a high concentration (300 mM) butanol potentiated the
currents induced by low ACh concentrations and inhibited the currents
induced by high ACh concentrations.
These results could be simulated using a model in which butanol acted
both as a partial agonist and as an open channel blocker. In this
model, we assumed that receptors bound by two ACh molecules or two
butanol molecules were able to open the channel and that butanol was
able to block the two ACh-opened channel but not the butanol-activated channel.
| |
Materials and Methods |
Cell Preparations.
Human 42 AChR subunit combination
was stably expressed in the HEK293 cell line. Cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 2 mM
L-glutamine, 100 U of penicillin, 100 µg of streptomycin
(Invitrogen, Carlsbad, CA), 6% iron-supplemented calf serum
(Sigma-Aldrich, St. Louis, MO), and 100 µg/ml G418 (Mediatech,
Herndon, VA). Cells were kept at 34.7°C in air + CO2 (93 + 7%, by volume). For patch-clamp
experiments, cells were plated on glass coverslips coated with
poly-L-lysine and cultured for 1 to 5 days.
Electrophysiological Recording.
Whole-cell currents were
recorded with an Axopatch 200 patch-clamp amplifier (Axon Instruments,
Inc., Foster City, CA) at room temperature (20-25°C). Recorded
currents were directly digitized at 1 to 10 kHz via a Digidata 1200 ADC/DAC interfaced with a microcomputer under the control of the
ClampEx module of the pClamp6 software package (Axon Instruments,
Inc.). The holding potential was 
50 mV. The external solution
contained 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 5.5 mM HEPES acid, and
4.5 mM Na-HEPES, pH adjusted to 7.3 with HCl, and osmolarity 320 mOs. The internal solution contained 140 mM K-gluconate, 2 mM
MgCl2, 1 mM CaCl2, 11 mM
EGTA, 10 mM HEPES acid, 2 mM Mg2+ ATP, and 0.2 mM
Na+ GTP, pH adjusted to 7.3 with KOH, and
osmolarity 300 mOs. The patch-clamp pipettes were pulled from Clark
patch glass capillaries (PG120T-10, 1.2-mm o.d., 0.93-mm i.d., 10-cm
length; Warner Instrument, Hamden, CT) in two stages on a vertical
pipette puller (PP-830; Narishige, Tokyo, Japan) and lightly
fire-polished to a final resistance of 1.5 to 2 M
when filled with
internal solution. Recording was started about 5 to 10 min after the
rupture of the cell membrane to allow for the internal solution to
reach the equilibration with intracellular milieu.
Drug Application.
All drugs were applied to the cell by a
modified computer-operated U-tube perfusion system (Marszalec and
Narahashi, 1993). The speed of solution exchange was measured by the
junction potential change using the patch pipette placed at a distance
from the opening of the U-tube or near the U-tube opening. The rise
time (10-90%) of junction potential change was found to be 50 ms when
the patch pipette was placed 200 µm from the opening of the U-tube
and was reduced to less than 10 ms when the patch pipette was placed
near the opening of the U-tube. When the recording cell was placed at
200 µm from the opening of the U-tube, the solution exchange on the
cell surface was, however, found to complete within around 200 ms by
measuring the rate of changes in ACh-induced current in response to
changes in sodium ion concentration (Liu and Dilger, 1991; Mori et al.,
2001). In a few experiments, the recording cell was brought near the
opening of the U-tube. The rise time of the ACh response was less than
10 ms. In the present experiments, short-pulse drug exposure time was
250 ms unless otherwise stated, and long-pulse experiments were
performed with 1 to 10 s pulses. In all these cases, the intervals
between pulses were 2 min to avoid current reduction due to
agonist-induced desensitization. Control currents (30 µM ACh alone)
were usually checked before and after each application of a test drug
to ensure the stability of control responses. n-Butanol was
purchased from Aldrich Chemical Co. (Milwaukee, WI). DHE and
mecamylamine were purchased from Sigma/RBI (Natick, MA).
Data Analysis.
Recorded currents were initially analyzed by
the Clamp-Fit module of the pClamp6 to assess whole-cell current
amplitudes and decay kinetics. Data were then exported from pClamp6 to
a Microsoft Excel program (Microsoft Office 2000) for statistical
analysis. The concentration-response data were subsequently fitted to a single or double Hill logistic equations and compiled for graphical analysis in SigmaPlot 5.0 (SPSS Science, Inc., Chicago, IL). Data were
expressed as mean ± S.E.M. unless otherwise mentioned.
The ACh dose-response curve was fitted by a single Hill equation or by
the sum of two Hill equations:
where y is the normalized peak currents, and
x is the agonist concentration. EC50H
and EC50L are the half-effective concentrations for the high- and low-affinity receptors, respectively.
nH1 and nH2 are the Hill coefficients of the
high- and low-affinity receptors, respectively, and
p1 is the percentage of receptors in
the high-affinity state.
Simulation.
The kinetic simulation was carried out with
C++ programs for numerical solution of
differential equations based on a kinetic scheme (Scheme I in Fig. 9).
Butanol is assumed to have two kinds of action on nnAChRs, as a partial
agonist and as a channel blocker.
| |
Results |
Both ACh and n-Butanol Activate nnAChR
Currents.
The currents of the 42 AChRs expressed in HEK
cells rose rapidly, reaching a peak in less than 100 ms at low ACh
concentrations, and its rising phase became faster with increasing ACh
concentrations. At 3 mM, the time to peak is less than 10 ms (Figs.
1A and 2A). During application of 10-s
pulse of 3 mM ACh, the ACh-induced currents decayed with two
exponential phases (Fig. 1A), reflecting receptor desensitization. The
time constants (
) of fast and slow desensitization were 350 ± 30 ms (fast) and 2270 ± 70 ms
(slow), respectively (n = 7).
About 22% of the current was associated with the fast component,
whereas the rest was associated with the slow one.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Dose-response relationship for ACh-induced currents
in the 42 receptors expressed in HEK cells. Currents were
recorded at a holding potential of 50 mV. A, currents recorded from
one cell in response to 10 s perfusion of 30 and 3000 µM ACh. B,
simulated currents induced by 30 and 3000 µM ACh. C, dose-response
relationship of peak currents. The dotted line represents the best fit
using a single Hill equation (EC50 of 48 ± 9 µM and
nH of 0.7 ± 0.1). A better fit is
obtained with the sum of two Hill equations (solid line; see equation,
Materials and Methods). In this case, 20%
(p1) of the receptors have a high affinity
for ACh, with an EC50H of 1.0 ± 2.5 µM and an
nH1 of 0.8 ± 0.7, whereas the
remaining 80% receptors have a low affinity, with an EC50L
of 63 ± 14 µM and an nH2 of 1.3 ± 0.2 (n = 7-28) (mean ± S.E.M.,
n = 7-28). D, simulated dose-response relationship
of peak currents of the low-ACh-affinity receptors. Kinetic parameters
are given in Scheme I (Fig. 9) and Fig. 9 legend and text. The
simulated current amplitudes at various ACh concentrations were
normalized to the peak current obtained at 3000 µM ACh. The fit to
the simulated data gives an EC50 of 61 µM and an
nH of 1.4.
|
|
When the dose-response relationship was fitted to a single Hill
equation (Fig. 1C, dotted line), an EC50 value of
68 µM and an nH of 0.7 ± 0.1 were obtained (n = 7-28). This
EC50 value is much higher than that measured on
the -bungarotoxin-insensitive nnAChRs in rat cortical neurons
(EC50 of 2.7 µM; Aistrup et al., 1999). The
high EC50 value and a small
nH of the ACh dose-response relationship for the expressed 42 nnAChRs has also been reported previously (Chavez-Noriega et al., 1997; Zuo et al., 2001; Buisson and
Bertrand, 2001).
One possibility of the low nH for the
ACh dose-response curve is due to the existence of two receptor pools
with different affinities for ACh, as suggested by Buisson and Bertrand
(2001). To examine this possibility, additional experiments covering a wider range of ACh concentrations were performed and the dose-response relationship was fitted with the sum of two Hill equations. In this
case, 20% (p1) of the receptors have
a high affinity for ACh, with an EC50H value of
1.0 ± 2.5 µM and an nH1 of
0.8 ± 0.7, whereas the remaining 80% receptors have a low
affinity, with an EC50L value of 63 ± 14 µM and an nH2 value of 1.3 ± 0.2 (n = 7-28) (Fig. 1C, solid line). The large
standard deviations for the EC50L and
nH2 for the high-affinity receptor are
probably due to the fact that it constitutes a minor component of the
total responses. These results resemble those of Buisson and Bertrand (2001) in that high-affinity receptors have an
EC50H value of 1.60 µM and an
nH1 of 0.92, whereas the low-affinity
receptors have an EC50L value of 68 µM and an
nH2 of 1.60.
To test an alternative hypothesis that the small Hill coefficient is
due to rapid receptor desensitization, an improvement in our perfusion
system was made by bringing a smaller cell to the site close to the
opening of the U-tube so that the solution exchange was greatly speeded
up to less than 10 ms. Figure 2 depicts examples of current traces and the dose-response relationship. Even at
low ACh concentrations, the ACh-induced currents reached the peak in
less than 20 ms. The decay time constant estimated from the current
induced by 3 mM ACh was 310 ± 40 ms (n = 5). No
decay time constant faster than 100 ms was detected. When the data were
fitted to the single Hill equation, the EC50 and
nH were estimated to be 38 ± 9 µM and 0.8 ± 0.2, respectively (Fig. 2B, dotted line). Thus, it
is rather unlikely that the small nH is due to the complication from the receptor desensitization because the peak of ACh current was measured at less than 20 ms and because the
receptor underwent desensitization with a time constant of more than
300 ms. However, when one assumed 80% of the data coming from the
low-affinity receptor with 20% from the high-affinity receptor, an
EC50 and an nH
of 60 ± 13 µM and 1.3 ± 0.3, respectively, were obtained
for the low-affinity receptor (Fig. 2B, solid line). The 20%
high-affinity receptor was based on the analysis of Fig. 1C. To
simplify the simulation, we simulated the effect of butanol only on the
low-affinity receptor, because the low-affinity receptor represents the
majority of the receptors in our cell lines, and also because most of
the experiments were performed at high concentrations of ACh (>10
µM).
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
Dose-response relationship for ACh-induced currents
in the 42 receptors expressed in HEK cells using an improved
perfusion system. Cells were lifted to near the opening of the U-tube
so that the solution exchange was greatly speeded up. A, currents
recorded from one cell in response to 500 ms of perfusion of 3 to 3000 µM ACh. The current induced by 3000 µM ACh reached the peak in less
than 10 ms with a rise time of 3 ms (see inset). Currents were recorded
at a holding potential of 50 mV. The decay phase of the current
induced by 3000 µM ACh was fitted by a single exponential function to
obtain the fast time constant of desensitization of 340 ± 40 ms
(n = 5). B, dose-response relationship of peak
currents. Current amplitudes were normalized to the current obtained at
3000 µM ACh (mean ± S.E.M., n = 6). The
dotted line represents the best fit using a single Hill equation
(EC50 of 38 ± 9 µM;
nH = 0.8 ± 0.2). The solid line
represents the best fit of the low-affinity receptors only, assuming
80% of the data coming from the low-affinity receptor with 20% from
the high-affinity receptor (EC50 of 60 ± 13 µM and
nH = 1.3 ± 0.3). The 20%
high-affinity receptor was based on the analysis of Fig. 1C.
|
|
In the absence of ACh, application of long pulses (2-5 s) of
n-butanol generated inward currents in a dose-dependent
manner at a holding potential of 50 mV (Fig.
3A). The currents induced by
n-butanol were small compared with ACh-induced currents. For example, the currents induced by 300 mM butanol ranged from 40 to 80%
of the currents induced by 30 µM ACh. Unlike ACh-induced currents,
the butanol-induced currents decayed with a single exponential time
course (Fig. 3A). For currents induced by 300 mM butanol, the time
constant of decay was around 2 s. A logistic equation with three
parameters was used to fit the butanol data, giving an
EC50 value of 230 ± 90 mM, an
nH of 1.8 ± 0.4, and the
saturation currents estimated to be 1.6 ± 0.5 times that of the
currents induced by 300 mM butanol (n = 6) (Fig. 3B).
The butanol-induced current had a much slower onset (200-300 ms)
compared with the ACh-induced current, suggesting either a lower
binding rate of butanol to nnAChRs or a slower open rate of
butanol-bound receptors. Furthermore, no current was evoked by butanol
in the cells that failed to respond to ACh. These results suggest that
n-butanol is capable of activating nnAChRs albeit with less
potency and less efficacy than ACh.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Dose-response relationship for
n-butanol-induced currents in the 42 receptors
expressed in HEK cells. A, currents recorded from one cell in response
to 1 to 300 mM butanol, which was applied for 5 s at intervals of
2 min using the U-tube system. B, dose-response relationship of the
peak currents. Current amplitudes were normalized to the current
obtained in 300 mM butanol. A maximum response could not be evoked
because of the limited solubility of butanol in water, but fitting the
data to a logistic equation gave the estimated maximum response of 1.5 times of the response induced by 300 mM butanol. Three parameters were
used to fit the data: EC50 of 230 ± 70 mM;
nH of 1.8 ± 0.4; and the saturation
current estimated to be 1.6 ± 0.5 times that of the currents
induced by 300 mM butanol (mean ± S.E.M., n = 4). C, simulated dose-response relationship of peak currents. Kinetic
parameters are given in Scheme I (Fig. 9), and Fig. 9 legend and text.
The simulated current amplitudes were normalized to the current
obtained at 300 mM butanol. The same fit used for experimental results
to the simulated data gave an EC50 value of 180 mM and an
nH of 1.7, and saturation currents was
estimated to be 1.4 times that of the currents induced by 300 mM
butanol.
|
|
To further test the hypothesis that butanol-induced current was due to
the activation of nnAChRs, we tested whether specific AChR inhibitors
could block the currents. DHE has been widely used as a nonselective
and competitive nAChR antagonist. It has a nanomolar affinity for
42 nnAChRs (Dwoskin and Crooks, 2001). The previous study of
human 42 nnAChRs showed that the inhibitory action of
mecamylamine is voltage-dependent and noncompetitive, suggesting that
it acts as an open channel blocker (Papke et al., 2001). Both 1 µM
DHE (ACh receptor blocker) and 50 µM mecamylamine (ACh channel
blocker) blocked the currents induced by either 300 mM butanol or 30 µM ACh when preperfused in the bath for 2 min and coapplied with the
agonist for 2 or 5 s (Fig. 4). Both
DHE and mecamylamine blocked the peak ACh- and butanol-induced
currents almost to the same extent (Table 1).
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of DHE and mecamylamine on ACh-induced
currents (A and B) and butanol-induced currents (C and D). The blockers
were preperfused for 2 min and then coapplied with the agonist. DHE
(1 µM) suppressed the 30 µM ACh-induced currents to 28 ± 1%
of control at the peak and to 12 ± 3% at the end of drug
application pulse (n = 3) (A) and the 300 mM
butanol-induced currents to 26 ± 3% of control at the peak and
33 ± 1% at the end of drug application pulse
(n = 3) (C). Mecamylamine (50 µM) suppressed the
30 µM ACh-induced currents to 21 ± 3% of control at the peak
and 1.0 ± 0.4% at the end of drug application pulse
(n = 5) (B) and the 300 mM butanol-induced currents
to 17 ± 4% of control at the peak and 29 ± 2% at the end
of drug application pulse (n = 3) (D). All these
inhibitions were reversible after washing with inhibitor-free solution
for 2 to 10 min. The U-tube application time was 2 or 5 s with
2-min intervals between each application.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 1
Block of ACh and/or butanol-induced currents by DHE and mecamylamine
Data are given as percentages of current amplitude relative to control
with the number of experiments in
parentheses.
|
|
The blocking kinetics in the presence of DHE and mecamylamine were
significantly different between the ACh-opened (Fig. 4, A and B) and
the butanol-opened channels (Fig. 4, C and D). In the presence of 1 µM DHE, the butanol-induced current rose more slowly than the
butanol control current (Fig. 4C), whereas the ACh-induced current was
simply scaled down without changing kinetics (Fig. 4A). When the block
was measured at the end of agonist pulse, ACh-induced currents were
more sensitive than butanol-induced currents to both blockers (Table
1). DHE at 1 µM reduced butanol-induced currents at the end of
agonist pulse to 33 ± 1% (n = 3) of control, while reducing ACh-induced currents to 12 ± 3%
(n = 3) of control. Similarly, 50 µM mecamylamine
reduced butanol-induced currents to 29 ± 2% (n = 3) of control compared with 1.0 ± 0.4% (n = 5) for ACh-induced currents (Table 1). The slow rise in the
butanol-induced current in the presence of DHE is consistent with
the notion that butanol at high concentrations might compete against
DHE for binding to the receptors. Thus, the DHE-bound receptors
become unblocked as more butanol molecules competitively bind to the receptors to open the channel. Without doing more detailed Schild analysis of competition between butanol and DHE, one could not rule
out an alternative explanation, namely, a different conformational change of the receptor occurs in the presence of butanol and DHE. Mecamylamine blocked the ACh-induced current in a time-dependent manner
(Fig. 4B), leading to a near complete block at the end of 2 s
pulse (Table 1). The time-dependent enhancement of block of
mecamylamine was not seen with butanol-induced current (Fig. 4D).
Thus, the butanol-induced current seems to be generated by the
activation of AChRs, and butanol acts as a partial agonist for nnAChRs.
However, the butanol-opened channels seem to be less sensitive to
DHE and mecamylamine than the ACh-opened channels. The reduced
sensitivity to DHE is probably due to the displacement of DHE
from the binding site by high concentrations of butanol, whereas the
reduced sensitivity to mecamylamine block may represent a less
sensitivity of the butanol-activated channel to open channel blockers.
Modulation of ACh Dose-Response Curve by Butanol.
In our
previous study (Zuo et al., 2001), butanol was thought to have a direct
inhibitory action and its IC50 value was
estimated to be 6.8 mM by extrapolation of the relationship between the IC50 and carbon number of longer chain
n-alcohols. Because the estimated IC50
value of butanol is far lower than its EC50 value of 230 mM, one could evaluate its direct inhibitory action on ACh
dose-response relationship. Figure 5A
shows the effects of a low concentration (10 mM) of butanol on the
currents induced by various concentrations of ACh. Butanol inhibited
currents induced by 10 to 3000 µM ACh about 30%. The
EC50 value of ACh dose-response curve was
slightly and insignificantly (P > 0.5) shifted in the direction of higher ACh concentrations by 10 mM butanol coapplication, without change in the Hill coefficient (EC50 of
66 ± 25 µM; nH of 0.6 ± 0.1 without butanol versus EC50 of 96 ± 45 µM; nH, 0.6 ± 0.1 with 10 mM
butanol; Fig. 6A). At lower
concentrations, butanol clearly suppressed the ACh-induced current, but
the suppression was less than that predicted by
IC50 value of 6.8 mM. These results suggest that
butanol is not a simple channel blocker.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of n-butanol at concentrations
of 10 mM (A) and 300 mM (B) on ACh-induced currents. A and B were
obtained from two different HEK cells expressing 42 nnAChRs. ACh
(3-3000 µM) was coapplied with butanol by U-tube system for 1 s
at an interval of 2 min. Butanol at 10 mM exerts an inhibitory action
at all ACh concentrations tested (A). Butanol at 300 mM potentiates the
currents induced by low ACh concentrations, but inhibits the currents
induced by high ACh concentrations (B). The shaded currents in A and B
are controls without butanol.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
The ACh dose-response curve is differentially
modified by 10 and 300 mM butanol in the 42 HEK cells. A,
experiment data. ACh (3-3000 µM) was coapplied with butanol for 250 ms at intervals of 2 min. The peak current amplitudes were normalized
to the current obtained at 3 mM ACh alone. Data are presented as
mean ± S.E.M. (n = 5-6). The
EC50 value for ACh dose-response curve without butanol is
66 ± 25 µM with an nH of 0.6 ± 0.1 and the EC50 value for that with 10 mM butanol is
96 ± 45 µM with an nH of 0.6 ± 0.1. Butanol at 300 mM produces a dual action: potentiating action at
low ACh concentrations and a biphasic inhibition at moderate-to-high
ACh concentrations. B, simulation. The symbols represent the simulated
values, which were fitted by a single Hill equation. The
EC50 value for ACh dose-response curve without butanol is
61 µM with an nH of 1.4, and the
EC50 value with 10 mM butanol is 66 µM with an
nH of 1.3. Both experimental data and the
simulated value in the presence of 300 mM butanol were connected by a
line. See the legend of Fig. 9 and the text for kinetic parameters and
further explanation.
|
|
The effect of n-butanol on the ACh dose-response
relationship was also evaluated in the presence of high butanol
concentration at which it could act as a partial agonist. Butanol at
300 mM modulated ACh-induced currents in a complex manner (Fig. 5B). Currents induced by low concentrations of ACh (3 and 10 µM) were potentiated by butanol, whereas those induced by higher concentrations of ACh (30-3000 µM) were inhibited, with the maximum inhibition occurring at 100 µM ACh (Fig. 6A). Again, this V-shape (Fig. 6A) ACh-dose-response relationship in the presence of 300 mM butanol is not
consistent with a simple partial agonist model, which predicts a
monophasic ACh dose-response curve with butanol raising the foot at low
ACh concentrations without altering the maximum response.
Interaction between Butanol and ACh.
Because butanol acts as a
partial agonist only at higher concentrations and as a channel blocker
at lower concentrations, one would expect that the interaction between
butanol and ACh on the 42 receptor depends on the concentrations
of both butanol and ACh. Thus, the dose-response relationship for
butanol action was examined in the presence of 30 µM and 1 mM ACh.
The inhibition of ACh-induced currents by low-to-medium butanol
concentrations up to 100 mM was slightly dependent on the ACh
concentration (Figs. 7 and
8A). However, the effect of butanol at
300 mM was greatly dependent on ACh concentration: either no change or
further inhibition was observed. Butanol exhibited a biphasic
inhibitory dose-response curve at low ACh concentrations (30 and 100 µM ACh, around ACh EC50) (Figs. 7A and 8A). At
1 mM ACh, which activates most receptors, butanol exhibited a
monophasic dose-response relationship for inhibiting the ACh-induced
current (Figs. 7B and 8A), because butanol not only competed with ACh for the binding site but also acted as a blocker. Thus, the inhibition by low-to-medium butanol concentrations up to 100 mM was slightly dependent on the ACh concentration (Figs. 7 and 8A). At 300 mM butanol
one would not expect to see much current because butanol could
effectively abolish the current by competing with ACh for binding to
the receptor and by blocking the open channel. Because a substantial
current actually remains, butanol must be less effective in blocking
the butanol-opened channel than the ACh-opened channel, a result
consistent with the weak blocking action of mecamylamine (Fig. 4D). At
higher concentrations of ACh, a monophasic inhibition is expected to be
observed because both the partial agonist action and the channel
blocking action would contribute to the butanol inhibitory action.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Butanol inhibition of currents evoked by 30 µM (A)
and 1 mM (B) ACh in the 42 HEK cells. Butanol caused a biphasic
inhibition on the current induced by 30 µM ACh (A) and a monophasic
inhibition on the currents induced by 1 mM ACh (B). ACh and butanol
were coapplied by U-tube system for 250 ms at intervals of 2 min.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Butanol inhibitory dose-response curves at 30 µM
and 1 mM ACh in the 42 HEK cells. A, ACh and butanol were applied
by U-tube system for 250 ms at intervals of 2 min. Current amplitudes
were normalized to the current induced by ACh alone. Data are presented
as mean ± S.E.M. (ACh 30 µM, n = 6; and 1 mM, n = 5). B, similar dose-response relationships
obtained by simulation. See text for kinetic parameters and further
explanation.
|
|
Experimentally, the coapplication of 30 µM ACh and 300 mM butanol
produces highly variable results compared with the control current
produced by 30 µM ACh. The examples shown in Figs. 7 and 10 represent
extremes of the inhibitory and potentiating responses. This is due to
the fact that butanol is a weak agonist with an EC50 value around 230 mM and with a large S.E.M.
of 90 mM.
Simulation of Butanol Action.
We previously performed a
kinetic simulation for the dual action of n-alcohols on
nnAChRs (Zuo et al., 2001). The original scheme based on that study was
expanded to Scheme I (Fig. 9) to include
the assumption that butanol acts as a partial agonist on nnAChRs. In
addition, we assumed that the ACh receptor-channel can be opened by two
ACh molecules or two butanol molecules, but not by one ACh molecule and
one butanol molecule. In addition, butanol is able to block the two
ACh-opened channel but not the butanol-activated channel. Despite some
indication of ACh "self-block" at higher concentrations (3 mM;
Figs. 1A and 10, F and G), it was not included in the kinetic
simulation. This is because most of butanol and ACh interaction studies
were performed at ACh concentrations less than 3 mM. To simplify the
simulation, we focused on the effect of butanol on the low-affinity
receptor, which constitutes at least 80% of the total current we
measured.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
A kinetic scheme based on the assumption that
n-butanol acts as a partial agonist and as an open
channel blocker. R, nnAChR; A, ACh; B, n-butanol; RA and
RB, receptors bound by one agonist molecule; RA2, RAB and
RB2, receptors bound by two agonist molecules;
RA2* and RB2*, agonist-induced activated
receptors; RA2D1, RB2D,
and RA2D2, desensitized receptors;
RA2B, butanol-blocked receptors;
kon1 and kon2,
ACh and butanol binding rates (molar per second);
koff1 and koff2,
ACh and butanol unbinding rates (per second); 1 and
2, channel closing rates (per second); 1
and 2, channel opening rates (per scond);
 1, 2, and 3, the rates of
entry into desensitization (per second);  1,
2, and 3, the exit rates from
desensitization (per second); b1, butanol blocking rate for
ACh-opened channels (molar per second); and ub1, unblocking
rates of butanol (per second). These rate constants were chosen as
described in the text.
|
|
Because data on single-channel recording and kinetic analysis of
42 nnAChRs are limited, most of the parameters used in our
simulation (Fig. 9) were chosen based on previous publications on
muscle nAChRs (Colquhoun and Sakmann, 1985; Franke et al., 1991, 1993).
We started with the parameters from Colquhoun and Sakmann (1985):
kon1 = 108
M
1 s1,
koff1 = 8,000 s1, = 700 s1,
and = 30,000 s1. Their rate constants
were modified based on our experimental results and for best fitting to
our data. The modification of parameters was performed in several
steps. First, the rate constants for ACh binding and unbinding rates
used in our simulation were as follows: a
kon1 of 108
M1 s1 and a
koff1 of 6,000 s1. These values gave a
Kd of 60 µM for ACh binding, very
close to the observed EC50L. Second, for butanol
binding, the slower onset of butanol-induced currents compared with
ACh-induced currents (200-300 ms versus less than 20 ms) can be
explained by either the slow binding of butanol
(kon), or the slow opening () of the butanol-bound channel. In our model, butanol binds to the receptor
much more slowly than ACh, whereas the opening rates are assumed to be
the same in both cases. The rate constants
kon2 (400 M1
s1) and koff2
(100 s1) were chosen to generate a butanol
activation dose-response relationship similar to experimental
observation (Kd = 250 mM for each
binding step). Both rate constants for butanol were smaller than those of ACh, because butanol induced currents with a much slower onset. Third, Two-step desensitization process,
RA2D1 and
RA2D2, was used because the
ACh-induced current decayed with a biexponential time course (Fig. 1A).
The desensitization rates 1 and
3 were chosen as 3 and 0.4 s1, respectively, with the resensitization
rates () 1/10 of desensitization rates. Fourth, desensitization
occurred much more slowly for butanol-activated currents than for
ACh-activated currents. The decay for the current induced by 300 mM
butanol is well fitted with one exponential time course, with around 2 s. Thus, 2 is estimated to be
around 0.5 s1, with 2
1/10 of 2. Fifth, single-channel study of the
muscle receptor showed a very high open probability
[Popen = /(+)] of about
0.98 (Colquhoun and Sakmann, 1985). Previous data suggest that the
nnAChRs have a much lower open probability, although no maximum
Popen value is available (Weaver and
Chiappinelli, 1996). Because no such data are available, we chose 0.67 for Popen of ACh-opened channels, as
in our previous simulations (Zuo et al., 2001). The same opening and
closing rate constants are assigned to butanol-opened channels to
simplify the simulation.
The currents were simulated from Scheme I (Fig. 9) at various
concentrations of ACh. The simulated current traces are illustrated in
Fig. 1B and the peak amplitude normalized to that evoked by 3 mM ACh is
plotted against ACh concentration (Fig. 1C). The
EC50 and nH
values were estimated to be 61 µM and 1.4, respectively. The
simulated butanol dose-response relationship exhibited an EC50 value of 180 mM and an
nH of 1.7 (Fig. 3C). The
EC50 values for both ACh and butanol are close to
those of the experimental results.
Butanol effects on ACh dose-response curve were simulated with the
incorporation of the blocking action of butanol on the ACh-activated
currents. The following blocking parameters were used: the rate
constant for butanol block of the two ACh-opened channel
(b1), 1.5 × 104
M1 · s1; and the
butanol unbinding rate constant (ub1), 100 s1. These values give us the
Kd around 6.7 mM, similar to the
extrapolated IC50 value for butanol block (Zuo et
al., 2001). Based on weak blocking action of the open channel blocker
mecamylamine on the butanol-activated currents, we assumed that the
butanol-opened channels are not sensitive to butanol block at all. The
simulated results are plotted in Fig. 6B, which captures many features
of the observed ACh dose-response curves with both 10 and 300 mM butanol.
A similar simulation was also performed for the butanol inhibitory
dose-response relationship (Fig. 8B). A biphasic inhibitory dose-response curve was obtained using 30 µM ACh with a maximal inhibition at 100 mM butanol, whereas a monophasic inhibitory relationship was observed at 1 mM ACh. Thus, simulation yields the
butanol dose-response relationships similar to those of experimental results.
Butanol as Channel Blocker.
n-Butanol also acted as
a channel blocker for nnAChRs, in a way similar to other long-chain
alcohols (Murrell and Haydon, 1991). Although butanol was a weak
agonist with an EC50 value around 200 mM, it was
much more potent as a channel blocker of the ACh-opened channels. The
blocking rate constant of 1.5 × 104
M1 · s1 for the two
ACh-opened channel, combined with the unbinding rate constant of 100 s1 gave rise to a
Kd of 6.7 mM. This difference between
the agonist EC50 value and the blocking
Kd was reflected in currents induced by coapplication of ACh and butanol (Fig.
10). Butanol at 10 mM showed hardly any
agonist action (Fig. 10A), but caused a significant inhibition of
currents induced by 30 and 3000 µM ACh (Fig. 10, D and G). Butanol at
300 mM had a significant agonist action (Fig. 10B), potentiated the
current evoked by 30 µM ACh (Fig. 10E), and inhibited the current
evoked by 3000 µM ACh (Fig. 10H).
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 10.
Long-pulse (5-s) application of ACh,
n-butanol, or both to the nnAChRs stably transfected in
the HEK293 cells. Drugs were applied through U-tube and the recordings
were performed at a holding potential of 50 mV at intervals of 2 min.
A, 10 mM butanol alone. B, 300 mM butanol alone. C, 30 µM ACh alone.
D, 30 µM ACh plus 10 mM butanol. E, 30 µM ACh plus 300 mM butanol.
F, 3000 µM ACh alone. G, 3000 µM ACh plus 10 mM butanol. H, 3000 µM ACh plus 300 mM butanol.
|
|
| |
Discussion |
Multiple Action of n-Butanol.
The present study
showed that n-butanol exerted multiple actions on the
42 nnAChRs stably expressed in HEK293 cells. In the absence of
ACh, n-butanol generated a small current, and in the
presence of ACh, it either potentiated or inhibited ACh-induced currents, depending on the concentrations of ACh and butanol. Most of
the features of these multiple actions could be simulated by a model
based on the hypothesis that n-butanol acts both as a
partial agonist to induce currents and as an open-channel blocker (Fig.
9).
Contributions of Different Actions of Butanol to the Biphasic
Dose-Response Relationship.
To understand the contributions of two
major actions of butanol as a partial agonist and as a channel blocker
to the overall action of butanol on nnAChRs, we analyzed the biphasic
nature of the ACh dose-response curve at 300 mM butanol in detail. At low concentrations of ACh, 3 µM for example, we observed over 3-fold
potentiation by coapplication of 300 mM butanol (Fig. 6A). This is
mainly due to the agonist action of butanol at such a high
concentration to open the ACh channel. Based on our simulation (Fig.
11), 300 mM butanol itself can open
about 40% of the total AChR channels, similar to what 60 µM ACh
does. Thus, with 3 µM ACh and 300 mM butanol, over 99.8% of the open
channels are occupied by two butanol molecules. However, because the
affinity of ACh for the receptor is more than 4000 times higher than
that of butanol (ACh Kd value of 60 µM versus butanol EC50 value of 250 mM),
butanol's contribution as a partial agonist decreases dramatically as
the ACh concentration increases, because more and more receptors are bound by ACh instead of butanol. The most significant change in the
composition of the overall open channels occurs between the ACh
concentrations of 30 and 100 µM: the fraction of the two ACh-open channel increases from 10% of total opened channels at 30 µM ACh to
more than 99% at 100 µM ACh, and at the same time, the two butanol-opened channel drops from 90% of total open channels to less
than 1%. This trend continues until the concentrations are increased
to 3000 µM ACh, when almost all the open channels are occupied by two
ACh molecules, whereas butanol hardly opens any channel by itself
(<0.01%).
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 11.
Compositions of different receptor states at the
peak of the simulated currents induced by ACh and butanol. The
parameters used for simulation are given in Fig. 9. A, effects of 300 mM butanol on ACh dose-response relationship (1-3000 µM). B, butanol
inhibition (1-300 mM) at 30 µM ACh. Filled circles,
RA2*, channel opened by two ACh molecules; open circles,
RB2*, channel opened by two butanol molecules; triangles,
RA2B, the two ACh-opened channel blocked by
butanol; and squares, R* (total conducting channels), the total open
channels considering both the partial agonist action and the channel
blocking action of butanol.
|
|
Figure 11 depicts the results of simulation of the fraction of various
receptor states at the peak current in the presence of various
concentrations of ACh (Fig. 11A) and butanol (Fig. 11B). Complex
dose-response curves are obtained showing an apparent potentiating
action of butanol at low ACh concentrations and a biphasic inhibitory
action at moderate-to-high ACh concentrations (Fig. 11A, open squares)
as seen experimentally (Fig. 6A). Figure 11A illustrates that the
initial level (~35%) occurring at 1 µM ACh is mainly due to the
activation of the receptors by butanol. Because butanol blocks the
ACh-opened channels only, the blocking action takes place only when
there are many ACh-opened channels. This is shown by the fact that the
percentage of butanol block of the total active receptors is 1% at 1 µM ACh compared with 82% at 30 µM ACh. Because the blocking action
of butanol takes place at a rate much lower than that of ACh to open
the channel, as the concentration of ACh increases, the current peaks
sooner and butanol blocks less. Thus, only 48% block occurs at 3000 µM ACh compared with 82% block at 30 µM ACh. Thus, the combination of both the partial agonist action and the kinetics of channel blocking
action generates a V-shaped ACh dose-response curve at 300 mM butanol
(Fig. 11A, squares).
The simulation of the V-shape dose-response curve for butanol
inhibition at 30 µM ACh (Fig. 8B) could be similarly accounted for as
illustrated in Fig. 11B (squares). At low concentrations of butanol
(1-30 mM), more than 99% of the total open channels bind two ACh
molecules and are very sensitive to butanol blocking action. However,
as the butanol concentration increases, butanol starts competing with
30 µM ACh to open the channel. For example, 100 and 300 mM butanol
contribute 11 and 90%, respectively, to the total channel conductance
in the presence of 30 µM ACh and butanol. Thus, a biphasic inhibitory
dose-response relationship was obtained at 30 µM (Fig. 8).
When the 42 receptors were activated by 1 mM ACh, butanol exerted
a monophasic inhibition, yielding an apparent
IC50 value around 100 mM. A similar dose-response
relationship was simulated by the kinetic model in which butanol acted
as a partial agonist and as an open channel blocker. In the simulation,
however, the IC50 value for open channel block
was assumed to be 6.7 mM, a value similar as the extrapolated
IC50 value from the previous study of the
chain-length dependence of long-chain alcohols to inhibit the
whole-cell current of 42 receptors (Zuo et al., 2001).
The obvious discrepancy between the measured IC50
value of 100 mM and the extrapolated one of 6.7 mM is due to two
factors, both of which would reduce IC50 value
measured at the whole-cell level. One factor is the butanol's dual
action because the channel blocking action could not be cleanly
separated from its partial agonist action at the whole-cell level.
Another factor contributing to the lesser potency of block is the
kinetic effect of channel gating on the equilibrium block. Given the
blocking and unblocking rate constants used in the simulation, butanol
should reach an equilibrium block at the peak of ACh current. However,
in the constant presence of ACh during coapplication of ACh and
butanol, the rate constants governing channel opening and closing would reduce the butanol block. This gating effect on the open channel block
was recognized in open channel block of other channels as well
(Coronado and Miller, 1979; French and Shoukimas, 1981).
Comparison of multiple actions with previous studies.
Multiple
actions have been reported previously for the n-octanol
action on the GABAA receptor (Kurata et al.,
1999); the actions of d-tubocurarine (Steinbach and Chen,
1995), metocurine, and atracurium (Fletcher and Steinbach, 1996) on the
fetal nAChRs; and the actions of d-tubocurarine on nnAChRs
containing 4 subunit (Cachelin and Rust, 1994). However, in most
cases, all the observations were satisfactorily explained by a model in
which the drug acted as a weak agonist to open the channel either by
itself or by cobinding with another agonist. The latter example was
used in the model to explain the dual action of atropine (Zwart and
Vijverberg, 1997) and d-tubocurarine (Cachelin and Rust,
1994) on nnAChRs because the receptors occupied by two atropine or
tubocurarine molecules do not conduct current. In our case, butanol
acts as a partial agonist at high concentrations and as channel blocker at low concentrations on the 42 receptor.
Inasmuch as channel opening and block are such distinct actions, the
multiple effects of the agents suggest multiple sites of action on
nnAChRs. Our previous simulation based on a model in which long-chain
alcohols both block the open ACh receptor-channel and interfere with
ACh binding explains satisfactorily the alcohol's inhibitory action
(Zuo et al., 2001). It was also suggested that the inhibitory action of
long-chain alcohols is due to both alcohol block of open channels and
alcohol interference with ACh binding (decrease in
kon). One possible mechanism of
kon reduction is that long-chain
alcohols can compete with ACh for the ACh binding site. Because butanol
has a similar size to ACh, it is capable of opening the channel after
binding to ACh binding site. This competition between long-chain
alcohols and ACh for binding to the agonist sites would manifest as a
slowing in kon, the rate constant for
ACh to bind the agonist site in the previous simulation (Zuo et al.,
2001).
We thank Nayla Hasan for technical assistance and Julia Irizarry
for secretarial assistance. HEK cell lines expressing the 42
nnAChR subunits were provided by SIBIA Neurosciences, Inc. (La Jolla,
CA) (now Merck Laboratories, San Diego, CA).
nAChR, nicotinic acetylcholine receptor;
EPC, end-plated current;
ACh, acetylcholine;
nnAChR, neuronal nAChR;
HEK, human embryonic kidney;
DHE, dihydro--erythroidine.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
4
2 Acetylcholine Receptors
Top
Abstract
Introduction
30 µM), while inhibiting the currents induced by high
concentrations of ACh (100-3000 µM). In addition, butanol at a low
concentration (10 mM) suppressed the currents evoked by 10 to 3000 µM
ACh, a result consistent with a channel-blocking action. Most features of n-butanol effects were satisfactorily simulated by a
model in which butanol acts as a partial agonist and as a channel blocker.
![y=[p<SUB>1</SUB>/(1+(<UP>EC<SUB>50H</SUB></UP>/x)<SUP>n<SUB><UP>H1</UP></SUB></SUP>)]+[(1−p<SUB>1</SUB>)/(1+(<UP>EC<SUB>50L</SUB></UP>/x)<SUP>n<SUB><UP>H2</UP></SUB></SUP>]](/content/vol304/issue3/fulltext/1143/img001.gif)
