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Vol. 296, Issue 3, 716-722, March 2001
Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado (E.L.G, T.V.D.); Veterans Affairs Medical Center, Denver, Colorado (T.V.D.); and Waggoner Center for Alcohol and Addiction Research and Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas (R.A.H.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Nicotinic acetylcholine receptors (nAChRs) are neurotransmitter-gated
ion channels and like most such channels, ethanol and longer chain
alcohols modulate their activity. In the present studies, the effects
of alcohols were characterized on defined combinations of human
neuronal nAChR subunits heterologously expressed in
Xenopus oocytes. Short-chain alcohols, such as ethanol,
propanol, and butanol potentiated ACh-induced currents in both
2
4 and 44
nAChRs. Longer chain alcohols, however, inhibited these receptor subtypes. Small increases in alcohol chain length were sufficient to
produce a "crossover" from potentiation to inhibition. For the
24 receptor subunit combination, butanol
clearly potentiated while pentanol inhibited ACh-induced current,
whereas for 44 nAChR, propanol
potentiated, butanol had no discernable effect, and pentanol inhibited
receptor function. Fluorinated analogs of ethanol, propanol, and
butanol were used to determine whether the effects of the alcohols were
dependent upon chain length or whether another related attribute, such
as molecular volume, was the defining characteristic. The experimental
results support the hypothesis that for both
24 and 44
receptor subtypes, molecular volume appears to be the most important
determinant of both the potency as well as the direction of modulation
of nAChR function by n-alcohols and related compounds.
Although it has been suggested that the inhibitory and facilitatory
effects of alcohols are mediated by actions at different sites on the receptor molecule, the present data suggest the possibility that there
may be a single site of alcohol action and that the nature of this
action is dependent upon the physical properties of the molecule.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Nicotinic
acetylcholine receptors (nAChRs) are members of a
neurotransmitter-gated ion channel superfamily that includes receptors for serotonin and the amino acids 
-aminobutyric acid (GABA) and glycine. These receptors are one of the most widely studied subgroups of this superfamily, and have contributed greatly to our understanding of the structure and function of these channels (for reviews, see
Albuquerque et al., 1997
; Arias, 1997; Gotti et al., 1997). Eleven
members of the neuronal nicotinic subunit gene family have been
identified and nine human homologs have been successfully cloned,
expressed, and pharmacologically characterized in the Xenopus oocyte expression system (Elliot et al., 1996;
Chavez-Noriega et al., 1997).
The effects of ethanol on this ligand-gated ion channel superfamily
have been extensively studied (Lovinger and Zhou, 1994; Mihic et al.,
1994; Mascia et al., 1996; Yu et al., 1996; Forman and Zhou, 1998;
Aistrup et al., 1999; Narahashi et al., 1999). Although the effects of
ethanol have been extensively characterized on native receptors, there
has been considerably less research done on recombinant receptors
comprised of defined combinations of human subunits, and particularly
those receptors expressed in the central nervous system. In general,
ethanol facilitates nAChR-mediated responses, although the magnitude of
the effect and the reported potency of ethanol vary considerably
between different systems. Early studies demonstrated biphasic effects of ethanol and longer chain alcohols on nAChRs (Murrell et al., 1991b;
Wood et al., 1991). Wood et al. (1991) reported that short-chain alcohols enhance ion flux through nAChRs in Torpedo
electroplaque vesicles, whereas longer chain alcohols appeared to cause
inhibition of ion flux, possibly via a channel blocking mechanism.
Intermediate-length alcohols had both facilitatory and inhibitory
effects that combined in an apparently additive manner.
In the present study, we sought to identify which molecular properties
of ethanol and a related series of alcohols determine the nature of its
interactions with nAChRs. To this end, the straight-chain n-alcohols, as well as fluorinated analogs of ethanol,
propanol, and butanol, were used to test the hypothesis that molecular
volume rather than acyl chain length is the key determinant of these kinds of modulatory effects. Some of this work has been presented previously in preliminary form (Gonzales et al., 1999).
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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cDNA and cRNA Preparation.
Clones of the human nAChR
subunits 2, 4, and
4 were kindly provided by Merck Research
Laboratories (Elliot et al., 1996). The cDNAs were transformed and
amplified in XL-1 Blue cells and purified in the QIAFilter Maxi kit. In
vitro transcripts were prepared using an mRNA capping kit (Stratagene,
La Jolla, CA).
Electrophysiological Recording of Xenopus Oocytes. Oocytes were obtained from mature Xenopus laevis frogs, obtained from Xenopus I (Ann Arbor, MI) or Nasco (Fort Atkinson, WI). Frogs were kept in aquarium tanks at 18-20°C on a 12-h light/dark cycle and fed frog brittle (Nasco or Xenopus I) three times a week. Frogs were anesthetized by immersion in a 0.12% 3-aminobenzoic acid ethyl ester solution for approximately 30 min before surgical removal of a small fold of ovary. The ovarian tissue was placed in modified Barth's solution [88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 2.4 mM NaHCO3, 0.91 mM CaCl2, 0.33 mM Ca(NO3)2, 10 mM HEPES, pH 7.5] until just before isolation. Each frog was subjected to this procedure at most once a month.
To facilitate manual isolation of oocytes, the ovarian tissue was placed in a hypertonic isolation medium (108 mM NaCl, 2 mM KCl, 2 mM EDTA, 10 mM HEPES, pH 7.5) to cause the oocytes to shrink within the encapsulating membrane. Using surgical forceps, mature oocytes (stages V/VI) with uniform animal/vegetal poles were isolated by peeling them out of the epithelium and underlying theca layer. To remove the follicular cell layer, isolated oocytes were treated for 10 min with 0.5 mg/ml collagenase 1A in a buffer containing 83 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES adjusted to pH 7.5. Alternatively, oocytes were treated with 2 mg/ml collagenase B in OR2 buffer (825 mM NaCl, 25 mM KCl, 10 mM MgCl2·6H2O, 50 mM HEPES, pH 7.6) for 1.5 h until they had dissociated from the ovarian membrane and follicular cell layer. Dissociated oocytes were then rinsed with fresh OR2 buffer and transferred to incubation medium [modified Barth's solution: 88 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.82 mM MgSO4, 2.4 mM NaHCO3, 0.91 mM CaCl2, 0.33 mM Ca(NO3)2, pH 7.5; supplemented with 10 mg/l streptomycin, 10,000 U/l penicillin G, 50 mg/l gentamicin, 2 mM sodium pyruvate, 0.5 mM theophylline]. Oocytes were injected with 50 nl of diethyl pyrocarbonate-treated water containing 2.5 to 10 ng ofxy subunit
combinations of cRNA in a 1:1 ratio. Oocytes were incubated at 18°C
in incubation medium and typically expressed nAChRs 2 to 5 days after injection.
Electrophysiological recordings were performed as follows. Oocytes were
placed in a 100-µl rectangular recording chamber, perfused (1.4 ml/min) with ND96 buffer (96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, 10 mM
HEPES, pH 7.4) containing 1 µM atropine sulfate through 18-gauge
polyethylene tubing (Clay Adams Co., Parsippany, NJ), and impaled at
the animal pole using two glass electrodes filled with 3 M KCl. Oocytes
were clamped at a membrane potential of 
70 mV using a Warner
Instruments (Hamden, CT) model OC-725A oocyte clamp. Currents were
continuously recorded using a strip-chart recorder (Cole-Parmer
Instrument Co., Chicago, IL).
ACh was applied for 20 s at 5-min intervals. Hexanol, octanol,
decanol, and dodecanol were first dissolved in dimethyl sulfoxide (DMSO), diluted in ND96 to a final concentration not exceeding 0.05%
DMSO, and sonicated to disrupt micelles and equilibrate the solution.
This concentration of DMSO did not affect ACh responses. Experimental
concentrations of the n-alcohols were based on work from
Alifimoff et al. (1989) and concentrations of fluorinated analogs were
based on in vivo minimum alveolar concentration values (Eger et al.,
1999). All alcohols were preapplied for 2 min to achieve complete
equilibration in the recording chamber and then coapplied with agonist
for 20 s. The washout interval was extended to 15 min for
long-chain alcohols: hexanol, octanol, decanol, and dodecanol.
Corrections for evaporative loss of octanol, decanol, and dodecanol
during oocyte perfusion were made with the data obtained by
Dildy-Mayfield et al. (1996) using an identical superfusion system.
Alcohol concentrations given in the figures represent final bath
concentrations. Molecular volumes of all alcohols used in this study
were determined by computer optimization of molecular structures using
Spartan (version 5; Wave form Inc., San Diego, CA) and AM1 variables.
Values are presented as van der Waals volumes since this measurement
corresponds best with volumes of putative binding sites calculated from
molecular modeling techniques (Ueno et al., 1999).
Materials. Collagenase type 1A, streptomycin/penicillin, gentamicin, acetylcholine chloride, atropine sulfate, n-alcohol series from propanol to dodecanol, and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Collagenase type B was purchased from Boehringer-Mannheim (Indianapolis, IN). Ethanol was obtained from Aaper Alcohol and Chemical (Shelbyville, KY). Fluorinated analogs of ethanol, propanol, and butanol were kindly provided by Drs. James R. Trudell (Department of Anesthesia and Program for Molecular and Genetic Medicine, Stanford University, Stanford, CA) and Edmond I. Eger II (Department of Anesthesia, University of California, San Francisco). XL-1 Blue cells and the mRNA capping kit were purchased from Stratagene (La Jolla, CA). The QIAFilter Maxi kit was from Qiagen (Chatworth, CA).
Statistical Analysis. Results are presented as normalized percentages using control responses bracketing each alcohol application to define the baseline. All results are presented as mean ± S.E.M. Data for each drug tested was obtained from oocytes from at least two different frogs and n refers to the number of different oocytes used. Linear regressions were performed using GraphPad Prism software (San Diego, CA).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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As has been reported in previous studies (Cardoso et al., 1999),
superfusion with moderate concentrations of ethanol (25-100 mM)
enhanced inward ACh current responses elicited by an
EC30 concentration of ACh in oocytes expressing
both 24 and
44 combinations of
human nAChR subunits (Fig. 1A). The
24 and
44 nAChR combinations
were selected for these studies because both receptor combinations
demonstrated comparable levels of expression, as well as current
responses that were indistinguishable with respect to the kinetics of
the response, which facilitated comparisons between these two receptor
combinations. Responses to ethanol were readily reversible upon washout
and were repeatable as well. With a longer chain alcohol (hexanol),
inward currents induced by ACh were decreased for both combinations of
AChR subunits (Fig. 1B). Again, these responses were readily reversible
and repeatable.
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To determine where the crossover from potentiation to inhibition
occurred, the effects of the straight chain n-alcohols
through dodecanol were determined on both the
24 and
44 receptor subunit combinations. EC50 concentrations in tadpoles
(Alifimoff et al., 1989) are presented in Table
1; these concentrations were used to
determine preliminary ranges of alcohol concentrations used in
this study and concentration-response curves were extended in both
directions beyond these values. The potentiating effect of the
short-chain alcohols (methanol, ethanol, propanol, and butanol) on both
receptor subtype compositions are shown in Fig. 2. Significant enhancement of currents
mediated by the 24
nAChR subtype was observed for all of these, with the rank order
potency C4 > C3> C2 > C1 (Fig. 2A). In contrast, ethanol
and propanol significantly potentiated the
44 receptor subtype
with similar potencies, methanol was significantly weaker in this
respect, and butanol had no effect (Fig. 2B). Higher concentrations of alcohols than those illustrated were not examined systematically in
these studies because they appeared to compromise the viability of the
oocyte. Following exposure to higher concentrations, there was often a
deterioration in the quality of the recordings, or agonist responses
did not recover to control values. This was especially found to be the
case with methanol.
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Alcohols with chain lengths longer than butanol inhibited currents
mediated by both subtypes of the nAChRs in a concentration-dependent manner (Fig. 3). The long-chain alcohols
pentanol through decanol showed increasing potency with increased chain
length for both 24
and 44 receptor
subtypes (Fig. 3, A and B, respectively). Dodecanol was somewhat more
potent than decanol on the
44 nAChR, but did not
show any further increase in potency over that of decanol on the
24 nAChR, suggesting
that the "cutoff" had been reached for this receptor subtype [we
have used the definition of cutoff adopted by Wick et al. (1998), i.e.,
the point at which increases in alkanol chain length have no further
effect on potency]. Because of the limited solubilities of longer
chain alcohols, it was not possible to determine the alcohol
cutoff for the
44 combination using
alcohols longer than dodecanol. Occasionally, low concentrations of
pentanol, octanol, and decanol potentiated the
44 receptor (Fig.
3B), but this effect was somewhat variable and quantitatively very
small.
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To explore the relationship between the physical properties of alcohols
and their effects on nAChRs in further detail, fluorinated analogs of
several members of the n-alcohol series were tested as well.
Molecular volumes, EC50 values, and the minimum
alveolar concentrations of these analogs necessary to induce anesthesia are presented in Table 1. By replacing the hydrogens with fluorines, it
is possible to increase the molecular volume of the alcohol molecule
without adding additional hydrophobic alkyl groups (cf. C4: 111.00 Å3 versus FC4: 157.90 Å3). For both types of nAChRs, the
pharmacological actions of the fluorinated analogs were more accurately
predicted by their calculated molecular volumes than by acyl chain
length (Fig. 4). For example, the effect
elicited by fluorinated butanol was comparable to that of hexanol and
unlike that for the nonhalogenated butanol (Fig. 4, A and B). Thus,
molecular volume appears to be the best predictor of 1) the direction
of the modulatory effect, 2) the potency of the alcohol, and 3) the
apparent efficacy of the modulation of nAChR activity.
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Estimating the potencies of the various alcohols was difficult because
there were no clearly definable maximal responses and hence, no
EC50 values could be determined. Instead,
"threshold" concentrations for each compound were estimated by a
novel method of plotting the log of the alkanol concentration versus
the log of the response amplitude in the presence of the alkanol
expressed as a percentage of control, which generally resulted in
linear concentration-response curves (Fig.
5). The "threshold" for each agent
was then defined as the intercept of the regression line with 2.0 (i.e., 100% of control response, no effect). Additionally, data points
that fell within the range of the error bars of the next higher
concentration tested were not included in the regression analysis. This
was done to prevent "no effect" points from skewing the threshold
concentration determinations. Since butanol did not have any
appreciable effect on the
44 nAChR, no
threshold concentration could be determined. There was a highly
significant correlation between the logs of these threshold values and
the calculated molecular volumes for the longer chain alcohols pentanol through dodecanol, (r2 = 0.93, p < 0.0001 for
24 nAChR, and
r2 = 0.94, p < 0.0001 for 44 nAChR; Fig. 5,
C and D, solid line). When both short- and long-chain alcohols were
included in the regression analysis, the results remained highly
significant and did not change appreciably from the more limited
analysis (r2 = 0.94, p < 0.0001 for 24
nAChR, and r2 = 0.95, p < 0.0001 for
44 nAChR; Fig. 5, C
and D, dashed line).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The results of the present experiments demonstrate that as with
other neuronal nAChRs, ethanol significantly enhances responses to
brief application of ACh (Nagata et al., 1996; Aistrup et al., 1999;
Cardoso et al., 1999). As has been reported previously for non-neuronal
nAChRs (Murrell et al., 1991b; Wood et al., 1991), longer chain
alcohols had an inhibitory effect on these receptors, suggesting a
fundamental similarity in this respect between these different types of
nAChRs. However, the present findings also demonstrate that the
molecular volumes of a series of n-alcohols are better
predictors of the pharmacological properties of these agents with
respect to modulation of nAChR function than are alkyl chain lengths.
Thus, for both the 24
and 44 nAChR
combinations there was a high degree of correlation between the
threshold concentration of alcohol required to affect the nAChR and the
van der Waals molecular volume of the alcohol. Additionally, these
effects were fully reversible to control values and were
concentration-dependent, suggesting a specific interaction with the
nAChR at an alcohol binding pocket.
Consistent with previous reports involving neuromuscular or
Torpedo nAChRs, the n-alcohol series demonstrates
a biphasic and concentration-dependent effect on the nAChRs (Murrell et
al., 1991b; Wood et al., 1991). Whereas the other members of the
nicotinic superfamily of neurotransmitter-gated ion channels are either consistently potentiated by all of the straight-chain alcohols that
have been tested (5-HT3-R: Fan and Weight, 1994;
Gly-R: Mascia et al., 1996; GABAA-R:
Dildy-Mayfield et al., 1996) or consistently inhibited
(GABA
-R: Mihic and Harris, 1996), the
nicotinic acetylcholine receptors deviate from this pattern.
Short-chain alcohols potentiated receptor function, and as the chain
length was increased from one acyl group (methanol) to three to four acyl groups, there was a consistent increase in potency. Longer chain
alcohols beginning with pentanol inhibited current responses in both
24 and
44 receptor
combinations in our system. This result is consistent with many
previous reports on other nicotinic receptors (Bradley et al.,
1984; Murrell and Haydon, 1991a; Murrell et al., 1991b; Wood et al.,
1995; Forman, 1997).
Although many nicotinic receptors show similar sensitivities to
different alkanols, there are notable differences as well. In terms of
the receptors examined in the present study, butanol enhanced the
function of the 24
receptor, but had no effect on the
44 receptor; in
addition, the cutoff (see below) for the
24 receptor appeared
to be C10, whereas the cutoff for the 44 combination was
C12. These differences are relatively minor compared with those
involving other -subunits. For example,
3-containing receptors appear to be
insensitive to ethanol, whereas 7-receptors are inhibited by ethanol (Aistrup et al., 1999; Cardoso et al., 1999).
Thus, relatively minor differences in amino acid sequences can produce
profound effects on the actions of ethanol, which is consistent with
the hypothesis that there is a hydrophobic site on this receptor that
has specific requirements for alkanol activity. The present results
with short-chain alcohols also differ somewhat from what has been
reported for non-neuronal nAChRs, such as the native nAChRs in
Torpedo membrane vesicles (Wood et al., 1991). In
Torpedo, ethanol did not enhance ion flux and
intermediate-length alcohols such as propanol, butanol, and pentanol
caused both flux enhancement followed by inhibition as the
concentrations were increased.
There are several possible explanations that could account for the fact
that alcohols can both facilitate and inhibit ACh-evoked currents. One
possibility is that there is a single site, such as a hydrophobic
pocket, at which alcohols interact, and that short-chain alcohols act
at this site to increase the probability that ACh can induce the
conformational shift associated with channel opening, whereas
long-chain alcohols interact with the same site to reduce the
likelihood of this occurring. This model would predict a gradual
progression from potentiation to inhibition with increasing chain
length. Consistent with this possibility is the observation that for
the most part, the type of effect of any given alcohol was consistent
across all concentrations (either potentiation or inhibition, but not
both). Furthermore, the highly linear correlation between alcohol
modulation and molecular volume, which was maintained across the
transition between facilitation and inhibition, would be consistent
with the hypothesis that both types of effects are mediated by
interactions with a common (possibly allosteric) site. An alternative
possibility is that there may be two independent sites: the short-chain
alcohols generally would have higher affinities for the facilitatory
site than for the inhibitory site, and the longer chain alcohols would
have higher affinities for the inhibitory site. Bradley et al. (1984)
presented evidence from studies with neuromuscular nicotinic receptors
that is consistent with this hypothesis. They observed that low
concentrations of short-chain alcohols such as ethanol and propanol
enhanced ACh-induced ion flow, whereas higher concentrations diminished
peak current responses elicited by ACh, and we have made similar
observations with butanol (E. L. Godden and T. V. Dunwiddie,
unpublished). These types of effects are somewhat difficult to explain
with a single-site model. However, it should be noted that a
hydrophobic pocket that could accommodate octanol is large enough for
two ethanol molecules; double occupancy might occur at high
concentrations of ethanol, and might have the same effect as octanol,
i.e., inhibition. This single-site mechanism could account for the
facilitatory effects of low concentrations of short-chain alcohols, and
inhibitory effects of high concentrations. However, other groups (Wood
et al., 1991) have also provided evidence that the nicotinic receptors from Torpedo have two separate and distinct sites of action
for alcohol molecules, one associated with inhibitory effects, and the
other with facilitatory effects. This would provide a possible explanation for the markedly different slopes of the concentration effect curves for different alcohols, particularly those that produced
inhibition (Fig. 5, A and B). If the potencies at the two sites are
similar for a given alcohol, this might lead to a very flat
concentration-response curve, whereas if they are very different, the
slope would be steeper. Whether this is the case, and whether different
types of nicotinic receptors interact with alcohols in similar ways are
issues that are yet to be resolved. Furthermore, size (molecular
volume) is only one of several determinants of an interaction with a
putative binding site; other factors include the shape and flexibility
of the interacting ligand and/or the site itself (steric constraints).
In this study, we attempted to determine the alcohol cutoff for the
24 and
44 nAChR combinations
using the long-chain alcohols C5, C6, C8, C10, and C12. The term
alcohol/anesthetic cutoff, as defined by Franks and Lieb (1987), has
been used to denote the alcohol chain length at which alcohols have no
effect. This is consistent with the alcohol binding pocket being
completely filled such that hydrophobic groups are forced into the
aqueous, and therefore unfavorable environment. Because this definition of cutoff may depend on the alcohol solubility, Wick et al. (1998) modified this to define cutoff as the point at which the potency of the
n-alcohol no longer increases (left-shift on a dose-response curve) with increasing carbon chain length. These longer chain alcohols, C5 through C12, demonstrated a continued left shift in their
concentration-response curves (increase in potency) up to at least C10
for both subunit combinations. The potency of C12 on the
24 receptor
combination did not appear to be any greater than C10 (Fig. 3A),
whereas C12 was more potent than C10 on the
44 combination (Fig.
3B). Longer alcohols were not tested because of their extremely low
water solubility. Thus, the cutoff was C10 for the
24 receptor
combination and C12 for the
24 receptor
combination, which is clearly larger than that reported for a number of
other receptors within the nicotinic (5-HT3-R: C5, Fan and Weight, 1994; GABA-R: C7, Mihic
and Harris, 1996; Gly-R: C10, Mascia et al., 1996;
GABAA-R: C10, Dildy-Mayfield et al., 1996) and
other ligand-gated ion channel families (P2X-R: C3, Weight et al.,
1999; NMDA-R: C6, Peoples and Weight, 1995; KA-R: C8, and AMPA-R: C9,
Dildy-Mayfield et al., 1996).
The present studies clearly support the hypothesis that molecular
volume is an excellent predictor of the potencies of a variety of
alcohols for either potentiation or inhibition of nAChR function. Previous work using a series of sterically constrained
cycloalkanemethanols as well as n-alcohols on synaptic
membranes enriched in nAChRs from T. nobiliana demonstrated
that the potencies of these agents corresponded with their respective
molecular volumes (Wood et al., 1993). Once an alcohol molecule, either
straight chained or the constrained ring, exceeded a molecular volume
of ~340 Å3, there was a complete loss of
effect on the native receptor. This molecular volume cutoff is in
contrast to what we report in that 1) we do not observe a loss of
effect but rather a failure to increase the potency of the alcohol
(Fig. 4, A and B); and 2) the molecular volumes corresponding to the
point at which there is no further left shift in the
concentration-response curves for these data are ~234 and 276
Å3, for the
24 and
44 receptors,
respectively. It should be noted, however, that Wood and colleagues
report molecular volumes derived from molar volumes (molecular weight
per density) divided by Avogadro's number, whereas the molecular
volumes in this report are calculated based on molecular modeling
techniques using the van der Waals radii. Furthermore, the fluorinated
alcohols used in this study provided a different series of agents that
can be compared with the n-alcohols in terms of their
pharmacological properties. The results support the conclusion that the
molecular volume occupied by an alcohol is a key determinant of its
effects on these nAChRs.
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Acknowledgments |
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We gratefully acknowledge Merck Research Laboratories, San Diego (previously SIBIA Neurosciences Inc.), for providing the human nAChR subunit clones and Virginia Bleck for technical assistance.
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Footnotes |
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Accepted for publication December 1, 2000.
Received for publication September 18, 2000.
This study was supported by National Institutes of Health Grants AA03527 (to T.V.D.), AA06399 (to R.A.H.), and AA03527 (to R.A.H.). This work was presented in preliminary form at the Society for Neuroscience meeting in Miami Beach, FL, October, 1999.
Send reprint requests to: Thomas V. Dunwiddie, Ph.D., Department of Pharmacology, Box C236, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. E-mail: Tom.Dunwiddie{at}UCHSC.edu
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Abbreviations |
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nAChR, neuronal nicotinic acetylcholine
receptor;
GABA, -aminobutyric acid;
DMSO, dimethyl sulfoxide;
ACh, acetylcholine;
NMDA, N-methyl-D-aspartate;
HT, hydroxytryptamine;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
R, receptor;
KA, kainic acid.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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22, h24, h32, h34, h42, h44 and h7 expressed in Xenopus oocytes.
J Pharmacol Exp Ther
280:
346-3562-7 and 2-4 nicotinic acetylcholine receptor subunits and functional expression of the 2, 3, 4, 7, 2 and 4 subunits.
J Mol Neurosci
7:
217-228[Medline].
1 receptor GABAergic currents by alcohols and volatile anesthetics.
J Pharmacol Exp Ther
277:
411-4167 receptors involves the amino-terminal domain of the receptor.
Mol Pharmacol
50:
1010-1016[Abstract].
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