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Vol. 60, Issue 4, 700-711, October 2001
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois (Y.Z., G.L.A., W.M., J.Z.Y., T.N.); and Merck Research Laboratories-San Diego, La Jolla, California (A.G., L.E.C.-N.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Alcohol is known to modulate the activity of a variety of
neuroreceptors and ion channels. Recently, neuronal nicotinic
acetylcholine receptors (nnAChRs) have become a specific focus of study
because not only are they potently modulated by alcohol but also they regulate the release of various transmitters, including
-aminobutyric acid (GABA) and dopamine, which play an important role
in the behavioral effects of ethanol. Whereas the potency of normal
alcohols (n-alcohols) to potentiate GABAA
receptors and to inhibit
N-methyl-D-aspartate receptors
increases with carbon chain length, we have found that n-alcohols, depending on the carbon chain length, exert
a dual action, potentiation and inhibition, on nnAChRs in primary
cultured rat cortical neurons. The mechanism of dual action of
n-alcohols on nnAChRs was further analyzed using human
embryonic kidney cells expressing the 
4
2 subunits. Shorter chain
alcohols from methanol to n-propanol potentiated
acetylcholine (ACh)-induced currents, whereas longer chain alcohols
from n-pentanol to n-dodecanol inhibited the currents. n-Butanol either potentiated or inhibited
the currents depending on the concentrations of ACh and butanol. The
parameters for both potentiation (log EC200) and
inhibition (log IC50) were linearly related to carbon
number, albeit with different slopes. The slope for potentiation was
0.299, indicating a change in free energy change (
G) of 405 cal/mol/methylene group, whereas the slope for inhibition was 0.584,
indicating a G of 792 cal/mol. These results suggest that
potentiating and inhibitory actions are exerted through two different
binding sites. Ethanol decreased the potency of
n-octanol to inhibit ACh currents, possibly resulting from an allosteric mechanism.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Alcohols
act on many neuronal receptors and ion channels in the central nervous
system. Some of them are inhibited by alcohols, including
N-methyl-D-aspartate (NMDA) receptors,
-amino-3-hydroxy-5-methy-4-isoxalone propionic acid
receptors, and voltage-gated calcium channels, whereas some others are
potentiated, including -aminobutyric acid (GABA) type A receptors,
glycine receptors, and type III 5-hydroxytryptamine receptors
(Mullikin-Kilpatrick and Treistman, 1993
; Crews et al., 1996; Lovinger,
1997, 1999; Mihic, 1999; Walter and Messing, 1999; Woodward, 1999). The
differential modulation of neuroreceptors embedded in the same neuronal
membrane points to the specific action of alcohol on these receptors.
Neuronal nicotinic acetylcholine receptors (nnAChRs) have recently
received much attention because the cholinergic system plays an
important role in modulating many other transmitter systems. nnAChRs
are located in postsynaptic, preterminal, and presynaptic regions of
GABAergic and other interneurons in the cortex and hippocampus. The
modulation of nnAChRs can lead to a cascade of synaptic events
involving multiple neurotransmitters. Ethanol has been found to
potently modulate nnAChRs (Covernton and Connolly, 1997; Aistrup et
al., 1999a; Cardoso et al., 1999; Narahashi et al., 1999). At
concentrations of 3 mM and above, ethanol potentiates -bungarotoxin
(-BuTX)-insensitive ACh-induced currents but weakly inhibits
-BuTX-sensitive currents in rat cortical neurons (Aistrup et al.,
1999a). Thus, -BuTX-insensitive nnAChRs might be an important target
of alcohol action.
The action of normal alcohols (n-alcohols) on
ligand-gated receptor channels depends on carbon chain length. Whereas
the potency of n-alcohols to modulate the activity of GABA,
glycine, and NMDA receptors increases with an increase in alkyl chain
length (Nakahiro et al., 1991, 1996; Mascia et al., 1996; Peoples and
Weight, 1999), the modulatory action of n-alcohols on
nonneuronal nicotinic AChRs, both skeletal muscle and Torpedo
californica nicotinic AChRs, changes from potentiation to
inhibition as the alcohol chain length increases (Bradley et al., 1984;
Wood et al., 1991). We also found that n-alcohols exerted a
dual action on nnAChRs depending on the carbon chain length.
We now report the results of analyses of the mechanism of the dual
action of n-alcohols on nnAChRs. In 42-type AChRs of cortical neurons and in human 4- and 2-containing AChRs expressed in human embryonic kidney (HEK) cells, we found that short-chain alcohols potentiated ACh-induced currents, whereas long-chain alcohols
inhibited the currents. The dual action of n-alcohols was
analyzed in detail using HEK cells expressing the human 42 nnAChRs. Both inhibition (log IC50) and
potentiation (log EC200) were linearly related to
carbon number, albeit with different slopes. These results suggest that
n-alcohols exert the two different effects by acting at two
different sites.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Preparations.
Rat cortical neurons were isolated and
cultured by a procedure slightly modified from that described elsewhere
(Marszalec and Narahashi, 1993). In brief, rat embryos were removed
from a 17-day pregnant Sprague-Dawley rat under methoxyflurane
anesthesia. Small wedges of frontal cortex were excised and
subsequently incubated in phosphate-buffered saline solution containing
0.25% (w/v) trypsin (type XI; Sigma, St. Louis, MO) for 20 min at
37°C. The digested tissue was then mechanically triturated by
repeated passages through a Pasteur pipette and the dissociated cells
were suspended in Neurobasal medium with B-27 supplement
(Invitrogen, Carlsbad, CA) and 2 mM glutamine. The cells were
added to 35-mm culture wells containing 3-ml aliquots at a
concentration of 100,000 cells/ml. Each well contained five 12-mm
coverslips [previously coated with poly-L-lysine]
overlaid with confluent glia that had been plated 2 to 4 weeks earlier.
Plated neurons were used for electrophysiological experiments after 4 to 9 weeks in culture. Both the neuron/glia cocultures and the HEK
cells were maintained in a humidified atmosphere of 93% air and 7%
CO2 at 37°C.
42 subunit combination was stably expressed in the HEK 293 cell line. Cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen), 6% iron supplemented calf serum
(Sigma), and 100 µg/ml G418 (Mediatech, Herndon, VA). Cells were kept
at 37°C in an 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,
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 to a microcomputer under control of the ClampEx module of
the PClamp6 software package (Axon Instruments). 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, at pH 7.3, and
osmolarity 320 mOsM. In addition, 0.1 µM tetrodotoxin was added to
block the sodium channel. 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, at pH 7.3 titrated with KOH, and osmolarity 300 mOsM. 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 long; Warner Instrument Corp., Hamden, CT) and lightly fire-polished to a final resistance of
1.5 to 2 M
when filled with internal solution.
Drug Application.
All drugs were applied to the cell by a
modified computer-operated U-tube system (Marszalec and Narahashi,
1993) having a solution exchange rise time of 10 to 15 ms as detected
by changes in junction potential or by U-tube application plus a 1- to
2-min bath preapplication. The solution exchange on the cell surface was 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). U-tube application exposure time was 250 ms, if not otherwise mentioned. The
test solution was applied at intervals of 2 min. In this study, the
term "coapplication" is referred to as the simultaneous application of alcohol and ACh through a U-tube, whereas the term
"preperfusion" is referred to as the application of alcohol through
the external bathing solution before coapplication of alcohol and ACh.
Data Analysis. Recorded currents were initially analyzed by the Clamp-Fit module of the PClamp6 to assess whole-cell current amplitudes and decay kinetics. Statistical analysis was performed with Excel, Office 2000. Data were expressed as the mean ± standard error of mean unless otherwise stated. The concentration-response data were subsequently compiled for graphical analysis in SigmaPlot 5.0. Student's t tests were performed to assess significance of differences between test and control measurements at the P value < 0.05.
Kinetic simulation of the receptor/channel activity was carried out with a C++ program for numerical solution. Short-chain alcohols were considered to modify the kinetic parameters, and long-chain alcohols were considered to reduce ACh binding to the receptor and to block the open ACh channel.| |
Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Comparison of n-Alcohols with Various Carbon Chain
Lengths.
Based on the reported relationship between the ability of
n-alcohols to modulate ion channel function and the length
of carbon chain (Nakahiro et al., 1991, 1996; Mascia et al., 1996;
Peoples and Weight, 1999), we selected concentrations of
n-alcohols that gave the equivalent efficacy to compare the
modulating action of various n-alcohols on
-BuTX-insensitive, 42-type currents in rat cortical neurons.
The concentration of each alcohol was decreased by a factor of 3 with
an addition of one carbon to the alcohol, and currents were induced by
10 µM ACh (3 × EC50).
;
Wood et al., 1991), the effects of n-alcohols on nnAChRs in
cortical neurons were not monophasic in nature. For short-chain alcohols, a potentiating action was observed, whereas for longer chain
alcohols, an inhibitory action was observed. The currents were
potentiated by 1000 mM methanol, 300 mM ethanol, 100 mM propanol, and
30 mM butanol, although the degree of potentiation was decreased by
increasing the alcohol carbon chain length (Fig.
1). In contrast, the currents were
suppressed by 10 mM pentanol, 3 mM hexanol, 1 mM heptanol, and 0.3 mM
octanol (Fig. 1). Thus, the current potentiation was converted to
inhibition when the carbon chain was lengthened from butanol to
pentanol.
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42 subunit combination. This combination was selected because it constitutes the
major component of nnAChRs in the vertebrate brain (Gotti et al.,
1997). Three types of analyses were performed. First, the dose-response
relationship for ACh activation of the receptor was obtained and the
EC50 value was determined. Second, the
dose-response relationship for ACh current potentiation by each of the
short-chain alcohols was measured, and the concentration to increase
the current to 200% of control (EC200) was
estimated. Third, the dose-response relationship for ACh current
suppression by each of the long-chain alcohols was obtained, and the
IC50 and Hill coefficient
(nH) were determined.
ACh Dose-Response Relationship for 42 HEK Cells.
The
42 AChRs expressed in HEK cells behaved similarly to the
-BuTX-insensitive AChR of cortical neurons. Both of them were very
sensitive to ACh, and the 42 receptor responded to even 1 µM
ACh with an appreciable inward current (Fig.
2A). The currents reached a maximum at an
ACh concentration of 3 mM and decreased at higher ACh concentrations.
The bell-shaped dose-response relationship was also observed with
nonneuronal nicotinic AChRs (Tonner et al., 1992; Wu et al., 1994). At
ACh concentrations higher than 1 mM, the current decayed rapidly and a
tail current was generated upon termination of ACh application (Fig.
2A).
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n-Alcohols Exert Either Potentiating or Inhibiting
Action on ACh-Induced Currents.
To examine the direct modulating
action of alcohols on the 42 nnAChRs expressed in HEK cells,
n-alcohols were coapplied with ACh or preapplied for 2 min
before coapplication because a prolonged exposure to alcohol using bath
application techniques increases the possibility of indirect effects
via intracellular regulatory systems (Diamond and Gordon, 1997). ACh
(30 µM) was coapplied with different concentrations of various
alcohols for 250 ms at intervals of 2 min. The 2-min interval in
general gave the receptor enough time to recover from a previous
exposure to ACh and alcohols, unless otherwise stated. The types of
alcohols used and the concentration ranges covered in the experiment
are given in Table 1.
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1000 mM), ethanol (300 mM; Fig. 3A),
and propanol (100 mM). These potentiating effects were completely
reversible after a 2-min washout as shown for ethanol in Fig. 3A.
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42 receptors. An
example for octanol is shown in Fig. 3B. After inhibition by long-chain
alcohols at very high concentrations, it sometimes took a long time for
the current to return to the original amplitude. In each case washing
for 15 to 20 min was necessary. For example, octanol at 3 mM sometimes
needed 15 min for recovery, and in several other cases it could recover
only about 75% of the control.
Figure 4 illustrates the dose-response
relationship for the potentiating and inhibitory actions of various
n-alcohols. The potency of potentiation for the short-chain
alcohols (from methanol to propanol) increased as the carbon number
increased, with the EC200 values of 1900 ± 500 mM for methanol, 1000 ± 200 mM for ethanol, and 500 ± 100 mM for propanol (Table 1).
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5%
inhibition) and reached the largest inhibition at 100 mM (50%
inhibition). Above this concentration, butanol caused less inhibition
at high concentrations (Fig. 4), and in some cases increased the
current beyond the control level (Fig. 5). It seemed that the
potentiating action overcame the inhibitory action, a result suggesting
that the two actions are not independent of each other.
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Chain Length Dependence of Potentiating and Inhibitory
Effects.
The IC50 and
EC200 values estimated from the dose
relationships for potentiating and inhibitory effects of alcohols
illustrated in Fig. 4 were plotted as a function of the chain length of
n-alcohols (Fig. 6). The
IC50-carbon chain relationship from pentanol to decanol is shown in Fig. 6A. The plot of log IC50
versus alcohol carbon number had a slope of 0.58 ± 0.04. The
IC50 value decreased by a factor of 3.3 per
methylene increase, which is equivalent to a change in Gibb's free
energy change (G) of 790 ± 50 cal/mol/CH2 group as calculated from
G0 = RT · lnK, where R is the gas
constant, T is the absolute temperature, and K is the dissociation
constant (R = 1.987 cal · K
1 · mol1; T = (273.16 + 23) K; RT = 588.47 cal · K1 · mol1). This
value of G is similar to that obtained from muscle nAChRs (881 cal/mol/CH2) (Wood et al., 1991). Thus, the
potency of the long-chain alcohols to inhibit ACh current increased
with an increase in carbon chain, reached a maximum at decanol, and
then declined at dodecanol. The last effect is called cut-off. From
methanol to propanol, the log EC200 values
decreased linearly with the carbon chain length with a slope of
0.30 ± 0.02 (Fig. 6B). The decrease in
EC200 by a factor of 1.99 per methylene moiety
gives a G of 400 ± 30 cal/mol/CH2
group.
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0.70 ± 0.06 and a G of
950 ± 70 cal/mol/CH2 group (Fig. 8B). These
values are similar to those obtained by coapplication only, which
yielded a slope from pentanol to octanol of 0.58 ± 0.04 and a
G of 790 ± 50 cal/mol/CH2 group.
However, for these longer chain alcohols, some differences in
IC50 values and nH values were noted between coapplication and pre- and coapplication, depending on the concentration of ACh. At a low ACh concentration of 30 µM, the IC50 values were slightly lower and the
nH values were slightly higher with pre-
and coapplication than with coapplication (Table
2). However, there is about 10-fold
difference in alcohol blocking potency between coapplication only and
coapplication together with preperfusion at 3 mM ACh. This is because
no equilibrium was reached in the coapplication experiments at the time
when the current induced by 3 mM ACh reached the peak, 30 to 100 ms, which was much shorter than the time to peak of 200 ms of the 30 µM
ACh-induced currents. Because the time required to reach the
equilibrium in our experiments is around 200 ms, the peak amplitude
measured in the coapplication experiment does not reflect the full
action of alcohol.
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Potentiating Action of Short-Chain Alcohol.
To further
elucidate the mechanism of enhancement of ACh-induced currents by
short-chain alcohols, their effects on the agonist dose-response curve
must be evaluated. Thus, we studied the effects of ethanol (100 and 300 mM) and propanol (100 mM) on the ACh dose-response curve. Alcohol was
coapplied with different concentrations of ACh for 250 ms using the
U-tube system at 2-min intervals. The ACh dose-response curves with and
without ethanol are plotted by normalizing all currents to the current
induced by 3 mM ACh in the same cell (Fig.
9). Both the affinity and efficacy of ACh increased with the increase in ethanol concentration. The
EC50 values for ACh were 43.5 ± 8.1 µM without
ethanol, 24.0 ± 4.1 µM with 100 mM ethanol
(P < 0.10), and 19.0 ± 6.9 µM with 300 mM
ethanol (P < 0.05). The maximum response was
increased by 13.0 ± 4.0% (P < 0.05) by 300 mM ethanol. A 2-fold reduction in EC50 value and a small
but significant increase (5.0 ± 1.2%, P < 0.05) in the maximum response were observed in the presence of 100 mM propanol (data not shown). Alcohol enhancement of current amplitude at
high concentrations of ACh is qualitatively similar to that observed
with the 42-type ACh receptor of cortical neurons (Aistrup et
al., 1999a). The results that the short-chain alcohols cause a
reduction in EC50 values of ACh to activate nnAChRs
accompanied with an increase in the maximum response differ from their
potentiating action on GABAA receptors. In the latter case,
alcohols reduce GABA EC50 values without changing the
maximal response (Marszalec et al., 1994).
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Interactions of Ethanol and Octanol. The potentiating and inhibitory actions exerted by butanol suggest that there is an interaction between the potentiating site and the inhibitory site. Experiments were performed to test such interactions using ethanol as a potentiator and octanol as an inhibitor at nnAChRs. A protocol of 2-min alcohol bath-application followed by 250-ms alcohol and ACh coapplication was used. Ethanol at 300 mM potentiated the current induced by 30 µM ACh to 140 ± 2% of control, 30 µM octanol inhibited the current to 34 ± 3% of control, and coapplication of ethanol and octanol inhibited the currents to only 68 ± 10% of control. However, if octanol inhibited the nnAChRs independently of ethanol potentiating action, one would have expected that coapplication of ethanol and octanol would reduce the ACh current to 48% of the control. Similar experiments were also performed at 3 mM ACh, which produced a saturating response. Ethanol (300 mM) potentiated the current to 113 ± 4% of control, 30 µM octanol inhibited it to 28 ± 2% of control, and the coapplication of ethanol and octanol inhibited it to 61 ± 4% of control, which was much higher than the estimated 32% of control. These results suggest that octanol is less effective in inhibiting the ethanol-potentiated nnAChR currents.
If the apparent effect of alcohols represents a mixture of inhibition and potentiation, potentiation is diminished at high concentrations of ACh that give saturating responses so that the underlying inhibition is disclosed. Ethanol-octanol interactions were studied by using two concentrations of ACh (Fig. 10). The potency of octanol inhibition did not change with the ACh concentration, with an IC50 value of 14.3 ± 1.3 µM at 30 µM ACh, and an IC50 value of 18.3 ± 2.7 µM at 3 mM ACh. This suggests that octanol neither acts as a pure open channel blocker nor as a simple competitive antagonist on ACh receptors. With increasing agonist concentration, the IC50 value of octanol would increase with a pure receptor antagonist model and decrease with an open channel block model. However, at 30 µM ACh coapplication of 300 mM ethanol significantly decreased the potency of octanol inhibition by increasing the IC50 value from 14.3 ± 1.3 to 31.9 ± 4.3 µM (P < 0.002) (Fig. 10A), whereas no significant shift was observed at 3 mM ACh (0.10 < P < 0.20) (Fig. 10B). The latter result agrees with that of AChRs of T. californica membrane vesicles (Wood et al., 1991); 1.0 M ethanol had no effect on the inhibitory action of
octanol over 15 ms of 86Rb+
flux measurement at 1 mM ACh. The ACh concentration-dependent ethanol
effect on octanol inhibition of ACh currents further suggests that
ethanol and octanol do not act independently. This point will be
elaborated by model simulation as described under
Discussion.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bell-Shaped Dose-Response Relationship of ACh-Induced
Currents.
The ACh-induced current increased in amplitude with
increasing ACh concentration, reached a peak, and then declined at very high concentrations of ACh. Such a bell-shaped dose-response
relationship was seen with nicotine, as reported previously on T. californica nicotinic receptors (Tonner et al., 1992; Wu et al.,
1994) and depicted in Scheme 1.
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Multiple Effects of n-Alcohols on ACh
Receptors.
Two major actions of n-alcohols observed
in nnAChRs are similar to their actions on muscle nACh receptors in the
following respects. 1) The potentiating effect: this effect was seen
with short-chain alcohols and the potency increased with an increase in
carbon chain length. The slope for the log EC200 and carbon number indicates a G of 400 cal/mol/CH2. 2) The
inhibitory effect: this effect was observed with long-chain alcohols
and the potency increased with carbon chain length. The slope for the
log IC50-carbon number relationship gave a G of 790 cal/mol/CH2. This value is close to the energy required to
move one CH2 group from hydrophilic environment to
hydrophobic environment (800 cal/mol/CH2). The larger free
energy change involved in inhibitory action suggests that the site of
inhibitory action has a stronger hydrophobic component.
G for the inhibition of the 42 nnAChRs by
longer chain alcohols (790 cal/mol/CH2) is
comparable with that for the inhibition of muscle nAChRs (880 cal/mol/CH2) (Wood et al., 1991), suggesting the
possibility of a similar binding site. Ethanol was suggested to bind to
muscle nAChRs at a site in the M2 region of nAChR channel based on
mutagenesis studies (Forman et al., 1995; Forman, 1997; Zhou and
Forman, 1998). The G value for the inhibition of the 42
receptors is also comparable with the G for the
n-alcohol potentiation of the GABAA
receptors (880 cal/mol/CH2) (Peoples and Weight,
1999). This may be related to the fact that nnAChRs and
GABAA receptors belong to the same ligand-gated
receptor superfamily. The G for the potentiation of the 42
nnAChRs (400 cal/mol/CH2) is close to that for
the inhibition of the NMDA receptors (300 cal/mol/CH2) (Peoples and Weight, 1999), yet the
significance of this similarity remains to be seen.
The 42 nnAChRs and muscle nAChRs differ in their responses to
propanol. nnAChR currents induced by ACh were potentiated by 100 mM
propanol, whereas muscle nAChRs were inhibited by propanol with a
Ki of 270 mM (Wood et al., 1991). This
suggests that alcohol actions on nAChRs are subunit-dependent. This was
indeed the case from our preliminary studies: the ACh-induced currents
in the 42, 44, and 34 subunit combinations expressed
in HEK cells were potentiated by ethanol, whereas the 32 subunit
combination was inhibited (Aistrup et al., 1999b). A more detailed
investigation of subunit dependence of alcohol action is in progress.
One-Site versus Two-Site Model.
The dual action of
n-alcohols has been extensively studied on muscle nicotinic
receptors. Two models were proposed to explain the dual action of
alcohols: a single-site model and a two-site model. In the single-site
model proposed by Bradley et al. (1984), both potentiating action and
inhibitor action of all alcohols arise from their interaction with one
hydrophobic site within the channel lumen. Long-chain alcohols are
large enough to block the channel, masking the potentiating action,
whereas short-chain alcohols are too small to block, exhibiting as the
potentiating action. The alcohols can stabilize the channel at the open
state, resulting in a potentiating action (Bradley et al., 1984). This theory was challenged by the two-site model for the following reasons
(Wood et al., 1991): 1) ethanol does not compete with octanol for the
inhibitory site on the receptor; 2) alcohol chain length dependence for
flux enhancement differs greatly from that of flux inhibition; and 3)
all-or-none inhibition of ACh-induced flux was observed when alcohol
was switched from ethanol to propanol. Work on T. californica nAChRs has also shown that the alkanol site that
modulates the apparent agonist affinity for channel opening is distinct
from the site that results in inhibition of cation flux through the
channel (Alifimoff et al., 1993).
; Murrell and Haydon,
1991; Liu et al., 1994), it was concluded that alcohols have both
inhibitory and excitatory actions on nAChR channels. The inhibitory
action was well explained by a model in which drug molecules bind to
the channel protein and block the flow of ions through the channel
(Murrell et al., 1991; Dilger and Brett, 1991; Dilger et al., 1993),
resulting in a decrease in the apparent single-channel conductance or a
decrease in the number of conducting channels. The potentiating action
of ethanol, butanol, and pentanol may at least partially be due to an
increase in burst frequency as observed in muscle nAChRs by Liu et al.
(1994). Consistent with this view is the observation that the gating
modulation and the reduction in the single-channel conductance can be
differentially modified. Mutations of the inhibitory site within the
channel lumen enhanced the sensitivity to ethanol inhibition without
altering the ethanol-induced gating modulation (Forman et al., 1995;
Forman, 1997; Zhou and Forman, 1998). Another mutation near the agonist binding domain increased ethanol-induced potentiation, but did not
affect the reduction of single-channel conductance by alcohol (Forman
and Zhou, 1999). It remains to be seen whether these notions are also
applicable to nnAChRs.
The difference in G as shown in present study strongly suggests
that the inhibitory action and potentiating action are exerted at
different sites. Our studies of interaction between ethanol and octanol
suggest that these two types of actions may interfere with each other.
Although the potentiating and inhibitory actions occur at different
sites, they may allosterically affect each other, rendering the
potentiated receptors less likely to be inhibited. This notion is
further supported by the following simulation.
Simulation of Ethanol and Octanol Action.
Different kinetic
models have been previously applied to analyze the drug action on
muscle nAChRs, and the rate constants for channel kinetics of muscle
nicotinic ACh channels were estimated from single-channel studies
(Dilger and Brett, 1991; Franke et al., 1993; Liu et al., 1994; Dilger
et al., 1995). In the present study, the kinetic model of AChR was used
to simulate the octanol inhibition and
ethanol potentiation is shown in Schemes 2 and 3. The parameters used to simulate
ACh-induced currents in the absence and presence of alcohols are given
in the legend of Fig. 11.
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; Franke et al., 1993). However, some
modifications were made of rate constants to find the best fit with the
experimental results. The simulation yielded an ACh dose-response curve
with an EC50 value of 57.2 ± 2.0 µM and
an nH value of 1.40 ± 0.05. The
EC50 value is near the experimental result
(38.8 ± 9.6 µM), but the nH is
larger than the experimental result (0.65 ± 0.10). The simulation
results are consistent with the kinetic model in which two ACh
molecules are required to open the channel. The reason for the
discrepancy in Hill coefficient is not clear, yet it should be noted
that the interpretation of Hill coefficient in reference to
stoichiometry is a matter of controversy, and that the observed values
for Hill coefficient in nAChRs and GABAA
receptors reported in the literature are quite variable.
Our experimental results showed that ethanol potentiated the ACh
currents evoked by both low and high concentrations of ACh, but with
different efficacies. At 30 µM ACh, the enhancement of current was
49 ± 4% (n = 28), whereas at 3 mM ACh, the
enhancement was 13 ± 4% (n = 8). These results
could not be accounted for by a classical description of dose-response
relationship as depicted by y = (100% × [c]n)/([c]n + [EC50]n), where c is
ACh concentration, EC50 is the ACh concentration to activate 50% of the maximum response, 100%. Alcohol reduces ACh
EC50, which would increase ACh response
activated by low ACh concentrations but not high ACh concentrations.
The modern version of dose-response relationship for an agonist to
activate receptor to open the channel can be depicted as
y = x × [c]2/[1 + 2 × ([c]/K) + ([c]2/K2) + (x × [c]2/K2)],
where K is the binding constant and x determines
the open equilibrium (according to Scheme 2 without desensitization and agonist block). The results suggest that alcohols favor equilibrium to
the open state. One possibility of ethanol potentiation is to increase
the channel opening rate, leading to an increase in the open
probability. When the opening rate () was increased from 2000 to
3500/s, ethanol at 300 mM caused a potentiation of 52% at 30 µM ACh
and a potentiation of 15% at 3 mM ACh. Both values are similar to
those of the experimental results. However, another possibility that
cannot be overlooked is a decrease in the closing rate (). The
decrease of from 1000 to 600/s could also cause a potentiation of
47% at 30 µM ACh and a potentiation of 14% at 3 mM ACh. Thus,
similar results of simulation are obtained by either increasing the
open rate constant () or decreasing the closing rate constant ().
However, these two possibilities cannot be distinguished at the
whole-cell level and must be resolved at a single-channel level.
The difference in the free energy change involved in ethanol
potentiation and that in octanol inhibition suggests that octanol and
ethanol act at different sites. Whereas ethanol may affect the gating
step to exert its potentiating action, octanol could act at two sites
to exert its inhibitory action. One site is the channel pore where
octanol could block when the channel is open, and the other is the ACh
binding site where octanol could reduce ACh binding to its own
receptor. The two models would make different predictions as to ACh
dependence of octanol blocking action. An open channel block model
predicts that the IC50 value of octanol would
decrease with an increase in ACh concentration, whereas the opposite
prediction is expected with the ACh binding model. The simulation based
on an open channel block model with a blocking rate
(b+1) of 2.5 × 107
M1 · s1, and an
unblocking rate (ub1) of 300/s showed that the IC50 value of octanol inhibition would be 67.8 µM at 30 µM ACh (nH = 1.00) and 19.8 µM at 3 mM ACh (nH = 1.00) (Fig. 11B).
These blocking and unblocking rates are similar to the values obtained from single-channel study with muscle nAChRs, b = 3.2 × 107 M1
s1, and ub = 480/s (Dilger and Brett,
1991). Only the IC50 value at 3 mM ACh is close
to the experimental result (18.3 ± 2.7 µM). On the other hand,
if octanol reduced kon for ACh in a
dose-dependent manner with an IC50 value of 25 µM, this would give an IC50 value of octanol of
17.7 µM at 30 µM ACh (nH = 1.22) and
1097 µM at 3 mM ACh (nH = 1.23). The
IC50 value of octanol obtained at 30 µM ACh is
very close to the experimental result (14.3 ± 1.3 µM) (Fig.
11A), whereas the IC50 value of octanol obtained
at 3 mM ACh differs drastically from the experimental result. The
increase in the simulated IC50 value of octanol
at 3 mM ACh is due to the prediction that the reduction in ACh binding
rate constant, kon, is overcome by the high
concentration of ACh.
Neither simulation for reduction of kon nor
that for open channel block fits the ACh-dependent
IC50 for octanol block. When octanol block of the
open ACh channel and slowing of the ACh binding are incorporated in the
model, the simulation produces a satisfactory result: an
IC50 value of 14.5 µM at 30 µM ACh
(nH = 1.18) (Fig. 12A) and an IC50
value of 19.7 µM at 3 mM ACh (nH = 1.00)
(Fig. 12B). Both sets of simulated values are similar to the
experimental results.
|
1 · s1;
ub1 = ub2 = 300/s), the
overall affinity for octanol block would be enhanced greatly, giving
the IC50 value of octanol less than 1 µM even
in the coapplication-only situation (Fig. 11C), which disagrees with
the experimental results. If close channel block occurs with the same
affinity as open channel block but with lower blocking and unblocking
rates (b+2 = 2.5 × 105 M1 · s1 and ub2 = 3/s),
during preapplication of 30 µM octanol, the percentage of the blocked
receptor (B) before ACh application would reach 71% of total ACh
receptors with the blocking time constant of 95 ms. In the subsequent
coapplication, an additional open channel block occurs. As a result,
the simulation still produces a greater blocking effect than the
experimental result.
Simulation of Ethanol-Octanol Interaction. Ethanol at 300 mM reduced octanol inhibitory potency by shifting the IC50 value to a higher concentration. To simulate ethanol-octanol interaction, we first assumed that there was no interaction between the potentiating site and the inhibitory site. Namely, when octanol and ethanol were coapplied with ACh, each alcohol performed its action independently. In this model, the IC50 value was 14.3 µM (nH = 1.16) at 30 µM ACh and 17.1 µM at 3 mM ACh (nH = 1.00). There was hardly any shift of IC50 value of octanol by 300 mM ethanol. If there is a shift at all, the shift is in the direction opposite the experimental result.
Thus, we went on to test an alternative hypothesis that there is an interaction between ethanol and octanol. Ethanol (300 mM) reduced octanol inhibitory action by decreasing octanol inhibitory action on ACh kon and the blocking rate constant for octanol. The IC50 value of octanol was increased to 34.8 µM at 30 µM ACh (nH = 1.09) (Fig. 12A) and to 28.5 µM at 3 mM ACh (nH = 1.00) (Fig. 12B), very similar to our experimental results of 31.9 ± 4.3 µM at 30 µM ACh (nH = 0.93 ± 0.13) (Fig. 10A) and 26.9 ± 3.4 µM 3 mM ACh (nH = 1.17 ± 0.18) (Fig. 10B). This simulation suggests that there is an allosteric interaction between ethanol and octanol at the42 receptor.
Mechanism of Cut-Off Phenomenon.
The inhibitory action of
long-chain alcohols on nnAChRs reached a maximum at decanol and
declined on further lengthening of the carbon chain. This phenomenon is
called "cut-off". The similar cut-off effect was also seen in
nnAChRs of isolated Lymnaea stagnailis neurons (McKenzie et
al., 1995) and in other systems such as NMDA receptors (Peoples and
Weight, 1999) and GABAA receptors (Nakahiro et
al., 1996). The cut-off effect has been interpreted in terms of both
lipid and protein theories of alcohol action. In terms of the lipid
theory, the cut-off effect was previously interpreted as being due to
the limited ability of long-chain alcohols to partition into lipid
bilayers because of the low solubility (Pringle et al., 1981), but
subsequent direct measurements revealed no such cut-off in membrane
partition (Franks and Lieb, 1986). In terms of the protein theory, the
affinity of short- to medium-chain alcohols for the receptor continues
to increase as the fitting to the hydrophobic pocket improves. For
longer chain alcohols, the affinity does not continue to increase
because the additional increase in chain length does not contribute to
binding to the hydrophobic pocket.
| |
Acknowledgments |
|---|
We thank Nayla Hasan for technical assistance, Brian O'Neil
Claeps for technical assistance with the 42 cell line culture, Julia Irizarry for secretarial assistance, and Min-John Lee for writing
the C++ program for numerical solution of kinetic simulation. HEK cell
lines stably expressing the 42 AChR subunits were provided by
SIBIA Neurosciences, Inc. (now Merck Research Laboratories-San Diego),
La Jolla, CA.
| |
Footnotes |
|---|
Received March 23, 2001; Accepted June 13, 2001
This work was supported by National Institutes of Health Grant AA07836.
Dr. Toshio Narahashi, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. E-mail: tna597{at}northwestern.edu
| |
Abbreviations |
|---|
NMDA, N-methyl-aspartate;
GABA, -aminobutyric acid;
nnAChR, neuronal nicotinic acetylcholine
receptor;
-BuTX, -bungarotoxin;
n-alcohol, normal
alcohol;
AChR, acetylcholine receptor;
HEK, human embryonic kidney;
ACh, acetylcholine.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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269:
157-163-aminobutyric acid receptor-channel complex by alcohols.
J Pharmacol Exp Ther
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235-240 current by a series of n-alcohols disappears at a cutoff point of a longer-chain n-alcohol in rat dorsal root ganglion neurons.
Neurosci Lett
205:
127-130[Medline].This article has been cited by other articles:
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) and by pre- and coapplication (
) in 
