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NEUROPHARMACOLOGY
Departments of Pharmacology (M.E.H., L-X.L., T.K.M.) and Anesthesiology (T.K.M.), Texas Tech University Health Sciences Center, Lubbock, Texas; and Department of Molecular Physiology and Biophysics (J.S.S-S., D.M.L.), Vanderbilt University School of Medicine, Nashville, Tennessee
Received February 17, 2003; accepted May 2, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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;
Peters et al., 1994). To date,
two subunits of the 5-HT3 receptor, 5-HT3A
(Maricq et al., 1991;
Belelli et al., 1995;
Miyake et al., 1995;
Lankiewicz et al., 1998) and
5-HT3B (Davies et al.,
1999; Dubin et al.,
1999) have been cloned. Recent reverse transcriptase and
immunohistochemical studies demonstrated that the B subunit is largely
expressed in the peripheral nervous system, suggesting that the predominant
form in the brain is the A homomer
(Morales and Wang, 2002). The
most well documented actions of 5-HT3 receptors are to alter
gastrointestinal motility and to regulate the vomiting reflex
(Aapro, 1991). Moreover, the
5-HT3 receptor has been implicated in altering the voluntary intake
of ethanol in humans (Johnson et al.,
1993) and rodents (Knapp and
Pohorecky, 1992; Hodge et al.,
1993).
The 5-HT3 receptor is modulated by pharmacologically relevant
concentrations of n-chain alcohols and anesthetics. Alcohols and
volatile anesthetics, in the presence of low 5-HT concentrations, potentiate
native and heterologously expressed 5-HT3 receptors
(Lovinger and White, 1991;
Machu and Harris, 1994). The
mechanism for enhancement of receptor function by alcohols has been assessed
in NCB-20 cells, which express the 5-HT3 receptor endogenously.
Ethanol and 2,2,2-trichloroethanol (TCEt) enhanced peak currents evoked by a
maximally effective concentration of dopamine, which is a weak partial agonist
at the 5-HT3 receptor (Lovinger
et al., 2000). That alcohols would enhance receptor function under
conditions of full agonist occupancy suggests that they increase probability
of channel opening independent of any effect on agonist affinity. Whole cell
patch-clamp electrophysiological recordings conducted with rapid drug
superfusion suggest that alcohols increase the activation rate, decrease the
intrinsic desensitization rate, and decrease the deactivation rate of the
5-HT3 receptor channel. These effects act in concert to favor and
stabilize the open channel state (Zhou et
al., 1998).
The binding domain in the 5-HT3 receptor with which alcohols
and/or anesthetics interact remains unknown. On the basis of bidirectional
modulation of the nACh 
7 receptor and 5-HT3A receptors by
ethanol, a chimera of the two was used to define a candidate region for
ethanol sensitivity in these two receptors
(Yu et al., 1996). The chimera
was composed of the N terminus of the nACh 7 receptor, and the balance
5-HT3A receptor. Inhibition by ethanol was observed, consistent
with the hypothesis that the N terminus confers sensitivity. In contrast,
amino acids at position 15' of the second transmembrane domain (TM2) and
at position 16' of the third transmembrane domain (TM3) of other
ligand-gated ion channels, the 
-aminobutyric acid (GABA) type A and
rho1 receptors, as well as the glycine receptor, determine the
direction of alcohol and anesthetic modulation
(Belelli et al., 1997;
Mihic et al., 1997;
Amin, 1999). Alcohol cutoff,
which is the n-chain alcohol at which functional effects are lost
altogether or are reduced to that of the n-1 alcohol, was altered in
receptors containing mutations at these critical residues. Thus, mutant
glycine receptors had the cutoff of GABA rho1 receptors, and vice
versa (Wick et al., 1998).
These results, plus correlation of molecular volume of these critical residues
with direction or degree of modulation (Ye
et al., 1998; Koltchine et
al., 1999; Yamakura et al.,
1999) has led to the speculation that an alcohol/anesthetic
binding pocket may reside in a cavity between these amino acids in TM2 and
TM3. However, the possibility that these drugs bind elsewhere in the receptor,
with the mutations altering the transduction of the alcohol and anesthetic
signal, cannot be ruled out.
The present study was undertaken to examine alcohol action on
5-HT3A receptors containing mutations at Ile294 of TM2, a residue
that has recently been shown to face the channel pore
(Reeves et al., 2001;
Panicker et al., 2002). Ile294
is at position 16' of TM2, neighboring the amino acid that is critical
for n-chain alcohol and anesthetic modulation of the
GABAA, GABA rho1, and glycine receptors at position
15' (Belelli et al.,
1997; Mihic et al.,
1997; Amin, 1999).
We tested the hypothesis that mutation of Ileu294 in TM2 changes the response
to n-chain alcohol sensitivity in the absence of any actions on an
alcohol binding site. Ethanol and TCEt potentiation of receptor function is
largely lost and change in n-chain alcohol cutoff occurs with
mutation, suggesting that kinetics of channel gating have been altered rather
than the dimension and/or physicochemical characteristics of an
alcohol/anesthetic binding pocket.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Expression of 5-HT3A Receptors in Xenopus laevis Oocytes. Ovarian lobes were obtained from X. laevis frogs and placed in modified Barth's solution (MBS) containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, and 0.91 mM CaCl2 (pH 7.5). Stage V and VI oocytes were dissected with fine surgical forceps in a hypertonic isolation medium containing 108 mM NaCl, 2 mM KCl, 1 mM EDTA, and 10 mM HEPES (pH 7.5) and placed in MBS. Dissected oocytes were incubated for 10 min in buffer containing 0.5 mg/ml collagenase type IA and 83 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 10 mM HEPES (pH 7.5) to remove the follicular cell layer. After several rinses with MBS, oocytes were placed in incubation medium composed of ND96, containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.5), or MBS, plus 10 mg/l streptomycin, 50 mg/l gentamicin, 10,000 units/l penicillin, 0.5 mM theophylline, and 2 mM sodium pyruvate.
Wild-type and mutant cDNAs were linearized with NotI, extracted with phenol-chloroform, precipitated with sodium acetate and ethanol, and resuspended in diethyl pyrocarbonate-treated water. The cDNAs were then transcribed with T3 mMESSAGE mMACHINE (Ambion, Austin, TX). An aliquot of cRNA was centrifuged at 15,000g and the ethanol was removed. The pellet was resuspended in RNase-free water, and 5 to 30 ng of cRNA were injected per oocyte. Oocytes were stored in incubation medium, and were recorded from days 2 through 7 after injection.
Two-Electrode Voltage-Clamp Electrophysiological Recordings. Oocytes
were perfused (2 ml/min) in a 100-µl volume chamber with MBS via a roller
pump (Cole-Parmer Instrument, Co., Vernon Hills, IL). Two glass electrodes
(1.2 mm outside diameter and 1–10 M
resistance) filled with 3 M
KCl were used to impale oocytes. A Warner Instruments model OC-725B or OC-725C
oocyte clamp (Hamden, CT) was used to voltage clamp oocytes to –70 mV.
Clamping currents were plotted on a strip chart recorder (Cole-Parmer
Instrument, Co.). Serotonin (Sigma-Aldrich, St. Louis, MO), ethanol (AAPER
Alcohol and Chemical Co., Shelbyville, KY), and other n-chain
alcohols (Sigma-Aldrich), and TCEt (>99% pure, Sigma-Aldrich) were
dissolved in MBS buffer. Serotonin was applied for 30 s. n-Chain
alcohols were perfused for 1 min before the application of 5-HT +
n-chain alcohol for 30 s; preapplication was used to ensure that the
relatively low potency n-chain alcohols were equilibrated with their
putative alcohol binding domains before exposure to 5-HT + n-chain
alcohol. Equipotent concentrations of 5-HT (
EC1) were used for
each receptor construct.
Data Analysis. The values in the 5-HT concentration response curves for wild-type and mutant 5-HT3A receptors were expressed as a percentage of the respective maximal 5-HT (10 µM) responses. Unless otherwise noted, in all other experiments, data were expressed as percent change from the control, baseline response. GraphPad Prism (GraphPad Software Inc., San Diego, CA) was used to calculate EC50 values, Hill coefficients, and two-way analysis of variance (ANOVA). GraphPad InStat was used to perform Student's t tests.
Expression of 5-HT3A Receptors in HEK 293 Cells.
Wild-type and mutant 5-HT3A cDNAs were subcloned into pcDNA3.1
(Invitrogen, Carlsbad, CA). HEK 293 cells grown in standard 35-mm-diameter
culture dishes were transfected by calcium phosphate precipitation as
previously described (Lovinger and Zhou, 1994). Cells were maintained in a 95%
O2/5% CO2 incubator, and recombinant receptor activity
was measured beginning 48 h after transfection.
Whole Cell Patch-Clamp Electrophysiological Recordings with Rapid Drug Application. In all experiments transfected cells were replated as single cells onto suspension dishes before initiating the recording. Cells were resuspended in a phosphate-buffered saline solution containing 3 mM EDTA. The cell suspension was centrifuged, and cells were resuspended with light trituration in the external medium used for electrophysiological recording (see below). Cells were then plated onto 35-mm-diameter suspension dishes and were allowed to settle on the dish for at least 5 min before recording was initiated.
Whole cell patch-clamp recordings were performed on transfected HEK 293
cells bathed in external solution containing (in mM): 150 NaCl, 2.5 KCl, 2.5
CaCl2, 10 HEPES, 10 D-glucose (pH adjusted to 7.4 with
NaOH and osmolality adjusted to 340 mOsmol/kg with sucrose) as described
previously (Zhou and Lovinger,
1996; Zhou et al.,
1998) with an Axopatch 200 amplifier (Axon Instruments, Union
City, CA). The solution constantly superfused cells at a rate of 2 to 3
ml/min. Patch pipettes had resistances of 1 to 2 M when filled with (in
mM): 140 CsCl, 10 cesium-EGTA, and 10 HEPES (pH adjusted to 7.4 with CsOH and
osmolality adjusted to 313–316 mOsmol kg–1
with sucrose). Cells were voltage-clamped with the membrane potential held at
–70 mV. After establishing a recording, the cell was lifted clear of the
bottom of the suspension dish and was placed into a solution stream coming
from the drug application micropipette. In selected experiments, alcohols were
applied alone for 30 s before application of agonist + alcohol.
Alcohol and other pharmacological agents were dissolved directly into
external solution, and applied to transfected HEK cells using two different
techniques. In the majority of experiments, agonist was applied with a
-tube whose lateral movement was controlled by a piezoelectric
manipulator, as previously described (Zhou
et al., 1998). The solution exchange time using this technique is
20 to 40 ms in the whole cell recording mode. In a few experiments drug
application was accomplished with a stepper-motor mounted on a
micromanipulator (Warner Instruments). In this system solution was applied
from glass tubes mounted on the manipulator, and solution exchange time was 22
± 5 ms. At the beginning of each experiment the cell was placed in
front of a solution stream of standard extracellular solution. Agonist
application was initiated under computer control, resulting in lateral
displacement of the application pipette by 100 to 200 µm such that the cell
was superfused by a solution containing agonist ± alcohol. Cessation of
the computer-generated pulse reversed the pipette displacement, returning the
cell to the standard external solution stream.
Solution applications ranged in duration from 1 to 30 s. Data were filtered at 5 kHz with a 3-pole Bessel filter and digitized at 10 kHz. In the majority of experiments, data were acquired using an Axopatch 200 amplifier and digitized using an ITC-16 analog/digital interface linked to a MacIntosh IIfx computer by means of IGOR software (Wavemetrics Inc., Lake Oswego, OR). In a few experiments data acquisition was accomplished using a TL-1-125 interface (Scientific Solutions, Mentor, OH) linked to a Pentium microcomputer running pClamp v5.5 software (Axon Instruments). Data were analyzed offline using either IGOR v3.0 or pClamp v8.0 software. Current amplitudes were determined using a cursor-based system to measure current at a particular time point, and these amplitudes were then subtracted from the baseline, preagonist current. Peak current was the maximal agonist-induced current, and was defined as the difference between the maximal current and the baseline preagonist holding (trough) current. Steady-state current was the stable current observed during prolonged (30-s) agonist application. Steady-state currents were measured at 30 s. It must be noted that at 2 µM 5-HT (see Figs. 2B and 5A) the Ile294Thr mutant receptor reached an apparent steady state at 30 s. Very slight decrements in current were observed between 25 and 30 s of agonist exposure. Nonlinear curve-fitting and estimates of the time course of exponential decay were obtained using IGOR software. The initial current slope was calculated for data points between 10 and 20% of the maximal current on the downward-sloping initial phase of current. Measurement of 10% of the peak response eliminates the contributions of nonlinear components of current at the bottom of a presumed sigmoidal-shaped response. Measurement at 20% of the peak current, rather than at higher percentages of peak current, ensures that slope can be measured accurately, with the least possible contamination from any other current component. All current amplitudes were normalized to whole cell capacitance and are reported as pA pF–1. All averaged measurements are expressed as mean ± S.E.M. values.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In HEK 293 cells expressing wild-type and Ile294Thr 5-HT3A receptors, 5-HT concentration response curves were generated (Fig. 1B). Two-way ANOVA revealed that the curves were not significantly different (F(1,26) = 3.36, p = 0.08). EC50 values (in µM) and Hill coefficients were 3.82 ± 0.11 and 2.2 ± 0.13 (wild-type) and 2.73 ± 0.18 and 1.7 ± 0.2 (Ile294Thr). It should be noted that the difference in 5-HT potency in oocytes compared with HEK 293 cells is due to the relatively slow agonist application in oocytes where current is measured after many of the receptors have undergone desensitization, in which case apparent agonist affinity and potency are increased. In addition, the observation in the oocyte expression system that the Hill coefficient of the Ile294Thr mutant was greater than that of the wild-type receptor may reflect an underestimation of the peak current, and hence a steeper curve.
Currents generated in the presence of 5-HT in cells expressing wild-type or
mutant receptors are presented in Fig.
2. In Fig. 2A,
representative currents obtained from oocytes expressing wild-type, Ile294Thr,
and Ile294Leu receptors are shown. Similar desensitization of the responses to
10 µM 5-HT were observed between wild-type and Ile294Leu receptors. In
contrast, the most striking aspect of the currents generated in the Ile294Thr
mutant expressed in oocytes is the slow decay toward baseline of current
during the prolonged application of 10 µM 5-HT
(Fig. 2A). Rapid current decay
was observed in the wild-type receptor; however, recovery of currents in the
Ile294Thr mutant was only 33% at the end of the 30-s application of 5-HT
(10 µM). Generally, a minimum of 4 min was required for the holding current
to reach the baseline, pre-5-HT application levels. In
Fig. 2B, current traces
obtained in wild-type and Ile294Thr 5-HT3A receptors expressed in
HEK 293 cells are shown. The most pronounced effect is the reduction in the
amount of desensitization upon prolonged exposure to 10 µM 5-HT (30 s) in
the Ile294Thr mutant. Furthermore, 5-HT-mediated current activated by a low
concentration of agonist (2 µM) exhibited a sustained current in the
Ile294Thr mutant, in contrast to the rapid peak and desensitization of
currents produced by the same agonist concentrations in the wild-type receptor
(Fig. 2B). The insets in
Fig. 2B are presented with an
expanded time scale and highlight the differences in current onset and
desensitization of the wild-type and mutant receptors.
With the rapid drug application protocol we measured several of the
parameters of whole cell current in receptors expressed in HEK 293 cells that
provide information about macroscopic kinetics and modulation of ion channel
function by alcohols, as shown previously
(Zhou et al., 1998). These
include the initial slope of current during the activation phase, the 20 to
80% rise-time, the steady state/peak (SS/Peak) current ratio, and the rate of
current decay during prolonged agonist application. The Ile294Thr mutation did
not produce a significant increase in initial current slope (101.2 ± 35
pA pF–1 s–1,
n = 5) relative to the wild-type receptor (89.3 ± 62.7 pA
pF–1 s–1, n =
3), (Student's t test, 
p = 0.86). The 20 to 80%
rise-time of the Ile294Thr mutant (1.7 ± 0.46 s, n = 5) was
not significantly greater than that of the wild-type receptor (0.37 ±
0.22 s, n = 3), (Student's t test, p =
0.09), given the large standard error in the measurements. However, a longer
rise-time would indicate that during this phase of activation the mutant is
slower than the wild-type receptor. In the example shown in
Fig. 2B in the inset at the
extreme right of the figure, wild-type and Ile294Thr receptor current traces
in response to 2 µM 5-HT are overlaid. The slower rise time in the mutant
receptor is apparent. A greater than twofold increase in SS/Peak current ratio
was observed in the Ile294Thr mutant relative to the wild-type receptor upon
application of 5-HT (10 µM) (Fig.
2C) (Student's t test, p = 0.0006). This
increase reflects a decrease in the extent of desensitization; i.e.,
steady-state levels were achieved after receptors had desensitized 60%
upon prolonged exposure to 5-HT (10 µM). In contrast, in the wild-type
receptor, currents achieved steady state after 95% desensitization.
Furthermore, the rate of desensitization (1/
) was reduced in the presence
of the Ile294Thr mutation, as measured by the time constant for current decay
() following the peak response in the continued presence of agonist
(Fig. 2D) (Student's t
test, p = 0.0006). The mutation decreases the rate and extent
of desensitization of the 5-HT3A receptor. One probable outcome of
the changes in desensitization is the open state of the mutant receptor
channel would likely be stabilized relative to the wild-type receptor.
We next examined the modulatory actions of ethanol on wild-type and mutant
5-HT3A receptors expressed in Xenopus oocytes and HEK 293
cells (Fig. 3). The function of
the wild-type and Ile294Leu 5-HT3A receptors expressed in oocytes
was similarly enhanced by ethanol over the range of concentrations tested
(Fig. 3A). Stimulation
increased from an average of 15% at ethanol (50 mM) to 55% at ethanol
(200 mM). In contrast, the Ile294Thr 5-HT3A receptor was inhibited
by ethanol, but the concentration-response curve for inhibition was relatively
flat, ranging from 15 to 27%. Two-way ANOVA demonstrated that mutation
had a significant effect (F(3,54) = 228, p <
0.0001). The Ile294Thr 5-HT3A receptor was significantly different
from the wild-type receptor at all ethanol concentrations tested (Tukey's post
hoc test, p < 0.05). In HEK 293 cells
(Fig. 3B), 5-HT-mediated
currents were measured in the absence and presence of ethanol in the wild-type
and Ile294Thr receptors. Stimulation of 17 and 26% was observed in the
wild-type receptor with 50 and 100 mM ethanol, respectively. No effects on
receptor function were obtained in the Ile294Thr 5-HT3A receptor
with ethanol (10–100 mM). The percentage changes in response to ethanol
were not significantly different between wild-type and Ile294Thr receptors,
two-way ANOVA, (F(1,22) = 3.47, p = 0.08).
Ethanol applied alone did not elicit any detectable current in the wild-type
or Ile294Thr 5-HT3A receptors expressed in oocytes (n = 5)
or HEK 293 cells (n = 3–4) (data not shown).
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In previous studies we have demonstrated that wild-type 5-HT3A
receptor function is more robustly enhanced by TCEt than by ethanol (Lovinger
and Zhou, 1996; Zhou et al.,
1998), and we have also performed extensive kinetic analysis of
TCEt's actions at this receptor (Zhou et
al., 1998). Therefore, it was of interest to examine the actions
of TCEt on Ile294Thr receptors and to compare the receptor-channel macroscopic
kinetic parameters of channel function in the presence of TCEt in wild-type
and Ile294Thr receptors. We first examined the effect of TCEt on wild-type and
Ile294Thr 5-HT3A receptors expressed in Xenopus oocytes;
the Ile294Leu mutant was not studied because of the lack of change in
modulation by ethanol due to this mutation
(Fig. 4). A
concentration-dependent enhancement of 5-HT-evoked currents was seen in
wild-type receptors, ranging from 55 to 2646% with 0.25 to 10 mM TCEt. A
biphasic response was observed in oocytes expressing the Ile294Thr mutant.
Receptor function was inhibited by 58 and 34% at 0.25 and 5 mM TCEt,
respectively. No effects were observed at 1 and 2 mM TCEt, whereas stimulation
occurred at 5 and 10 mM TCEt. Two-way ANOVA revealed that the curves were
significantly different (F(1,78) = 106, p <
0.0001). TCEt applied alone did not elicit any detectable current in the
wild-type or Ile294Thr 5-HT3A receptors expressed in oocytes
(n = 4–6) (data not shown).
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In studies with rapid drug application, we focused on measuring kinetic
parameters of wild-type and Ile294Thr 5-HT3A receptors expressed in
HEK 293 cells in the absence and presence of 3 mM TCEt. No detectable currents
were observed in wild-type or Ile294Thr 5-HT3A receptors when TCEt
was applied alone (n = 3–4) (data not shown). Representative
tracings of 5-HT evoked currents generated in wild-type and mutant receptors
are shown (Fig. 5A). As
demonstrated previously (Zhou et al.,
1998), TCEt enhanced wild-type receptor function (panel a).
However, Ile294Thr receptor currents were slightly inhibited by TCEt, with an
apparent decrease in the slope of activation (panel b). The insets (panels a
and b) in Fig. 5A are presented
using an expanded time scale and highlight the onset of current in the
wild-type and mutant receptors. In Fig.
5A, panels c and d, wild-type and Ile294Thr receptor current
traces in response to 5-HT or 5-HT + TCEt are overlaid; these are the same
traces as presented in panels a and b, but are overlaid to illustrate the
differences between the constructs. Activation is slower in the mutant (panel
c, inset), but is even slower in the mutant in the presence of TCEt (panel d,
inset). Both peak and isochronal (4.5 s) measurements were made in each
receptor construct (Fig. 5B),
and data were expressed as
((ITCEt/Icontrol) x 100), where
ITCEt is the current obtained in the presence of TCEt and
Icontrol is the current obtained in its absence. Both peak
and isochronal measurements were performed to take into account the fact that
peak currents occur at different time points and that desensitization is also
altered in the two constructs. Clearly, in the wild-type receptor both peak
(Student's paired t test, *p = 0.04) and isochronal currents
(Student's paired t test, *p = 0.04) were increased, whereas
in the mutant receptor an insignificant decrease was observed in peak
(Student's paired t test, p = 0.37) and isochronal currents
(Student's paired t test, p = 0.17). The wild-type and
Ile294Thr receptor peak (Student's t test, +p = 0.03) and
isochronal currents (Student's t test, +p = 0.01) were
different from each other.
Measurements of initial slope and rise-time (20–80% of the response),
which represent activation of the receptor, in HEK 293 cells were performed in
the presence of TCEt and expressed as a percentage of that obtained in its
absence (Fig. 5C). Activation
rate of the wild-type receptor was enhanced 70% in the presence of TCEt.
The slope of 61.4 ± 18 pA pF–1
s–1 in the absence of TCEt was significantly
different from the slope of 87.8 ± 20.6 pA
pF–1 s–1 in the
presence of TCEt (Student's paired t test, *p = 0.03). No
effect was observed in the mutant receptor (Student's t test,
p = 0.42). The slopes were significantly different between the
wild-type and Ile294Thr receptors (Student's t test, +p =
0.004). Rise-time was unaffected by the presence of TCEt in either construct
(wild-type: Student's paired t test, p = 0.26; mutant:
Student's paired t test, p = 0.81). The rise-times of the
wild-type and mutant receptors were not significantly different from one
another (Student's t test, p = 0.66). Finally, SS/Peak
current ratios were compared in the absence and presence of TCEt in the
wild-type and mutant receptors (Fig.
5D). As seen in Fig.
5A, wild-type receptors desensitize rapidly in the presence of 2
µM 5-HT and thus the SS/Peak current ratios are less than 0.1. Addition of
TCEt increases both peak and steady-state currents in the wild-type receptor,
and the increase in the SS/Peak current ratio in the presence of TCEt is
significant (Student's paired t test, p = 0.03). Minimal
desensitization takes place in the Ile294Thr 5-HT3A receptor
(Fig. 5A), which is reflected
by an SS/Peak current ratio that is greater than 0.8. The addition of TCEt
does not further increase the SS/Peak current ratio in the mutant
(Fig. 5D), (Student's paired
t test, p = 0.13). The SS/Peak current ratios are different
between wild-type and Ile294Thr 5-HT3A receptors in the presence of
2 µM 5-HT (Student's t test, +p < 0.0001) or 2 µM
5-HT + 3 mM TCEt (Student's t test, + p < 0.0001).
Alcohol cutoffs were determined in wild-type and mutant 5-HT3A
receptors expressed in Xenopus oocytes. The alcohol cutoff for a
protein is the n-chain alcohol at which functional effects are lost
altogether or are reduced to that of the n-1 alcohol. Jenkins et al.
(1996) previously showed that
hexanol and lower chain alcohols stimulate 5-HT3A receptor evoked
currents but that octanol and higher chain alcohols produce inhibition. A
series of n-chain alcohols were examined. In
Fig. 6A, pentanol (0.25–4
mM) enhanced the function of both wild-type and Ile294Leu 5-HT3A
receptors, although the mutant receptor was less sensitive as determined by
two-way ANOVA (F(1,39) = 53, p < 0.0001). The
actions of hexanol on wild-type and mutant receptors were examined. In B and
C, hexanol (0.25–10 mM) enhanced the function of wild-type receptors,
but inhibited or had no effect on Ile294Leu and Ile294Thr receptors. Where
responses to hexanol (0.1–1 mM) were measured, two-way ANOVA yielded
significant effect of mutation (F(6,52) = 198, p
< 0.0001), and two-way ANOVA also revealed a significant effect of mutation
for hexanol at the 5 and 10 mM concentrations (F(2,25) =
54.96, p < 0.0001) (Tukey's post hoc test, *p < 0.05,
compared with wild-type). These results suggest that the alcohol cutoff for
the Ile294Leu 5-HT3A receptor is hexanol. Actions of heptanol
(0.1–1 mM) and octanol (0.03125–1 mM) were evaluated in wild-type
and Ile294Thr receptors (Fig.
6D). Minimal potentiation of 5-HT-mediated currents (<27%) was
observed in wild-type receptors over the range of heptanol concentrations
tested, suggesting that the alcohol cutoff for the wild-type receptor is
heptanol. Robust inhibition of the Ile294Thr mutant was produced with
heptanol, and the wild-type and Ile294Thr receptors' concentration response
curves were significantly different, two-way ANOVA
(F(1,24) = 112.5, p < 0.0001). The Ile294Thr
mutant was modulated by octanol to an extent similar to the wild-type
receptor, two-way ANOVA (F(1,25) = 2.45, p =
0.1302). Inhibition that is observed with higher concentrations of octanol in
both of the receptors has been previously reported for the wild-type receptor
and is believed to represent a distinct alcohol site(s) from the potentiating
site(s) (Jenkins et al.,
1996). Given that the Ile294Thr 5-HT3A receptor is
inhibited by all n-chain alcohols, it is difficult to ascertain
alcohol cutoff for the inhibition observed in the mutated receptor.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Thus, we propose that the loss of enhancing actions of
TCEt in the Ile294Thr receptor is not due to a change in an alcohol-binding
domain, but instead occurs as a consequence of kinetic changes that favor the
open channel state and likely occlude the effects of alcohol.
Each of the members of the superfamily of ligand-gated ion channels has
been mutated in the TM2 domain, but the difficulty in studying such receptors
lies with the fact that the pore-lining domains are critical in participating
in the conformational changes that occur when the channels open. This gating
process may be fundamentally disturbed in a variety of ways (reviewed in
Colquhoun, 1998). Changes in
desensitization are demonstrated in the present study, where the Ile294Thr
5-HT3A receptor shows a marked reduction in extent and rate of
desensitization. Other mutation-induced changes that have been reported are
changes in affinity, shift from antagonism to agonism
(Bertrand et al., 1992), loss
of allosteric modulation (Findlay et al.,
2001), and production of tonically open channels
(Findlay et al., 2001).
Collectively, these data suggest that mutations in TM2 alter the
state-dependence of these channels by shifting the probability of closed
open desensitized transitions. Such changes would have a high
likelihood of interfering with allosteric changes in channel gating such as
those produced by alcohols.
Mutation at Ile294 produced a number of changes in gating and allosteric
modulation in the 5-HT3A receptor. The EC50 value for
the Ile294Thr 5-HT3A receptor was significantly reduced relative to
the wild-type receptor when oocytes were used as an expression system.
Although not significantly lower, the EC50 value for the Ile294Thr
5-HT3A receptor was reduced in HEK 293 cells. In contrast, the
Ile294Leu mutant, when expressed in oocytes, had a greater EC50
value than the wild-type receptor. The Ile294Thr and not the Ile294Leu mutant
lost positive allosteric modulation by ethanol. These results are consistent
with the findings of others that demonstrate that a TM2 mutation-induced
increase in agonist potency correlates with a decrease in alcohol or
anesthetic modulatory actions (Yamakura et
al., 1999). A mutant 5-HT3A receptor with greater
agonist potency than the wild-type receptor is more likely to open at any
given 5-HT concentration. From a mechanistic standpoint, the resting
conformation of such a mutant is more thermodynamically favored to make a
transition from a closed to an open state. Positive allosteric modulation is
typically lost when probability of opening is increased, as exemplified by
loss of ethanol modulatory actions at high concentration of 5-HT in the
wild-type receptor (Machu and Harris,
1994).
The most striking change produced by the Ile294Thr mutation was the
reduction in the rate and extent of desensitization compared with wild-type
receptors. Changes in desensitization in the mutant receptor suggest that once
activated by 5-HT, it is more likely than the wild-type receptor to remain in
the open channel state. The most parsimonius explanation for the reduction in
enhancing effect of ethanol and TCEt on the Ile294Thr 5-HT3A
receptor that we have observed is that the open channel state is already
favored in the absence of alcohols and that the mutation actually mimics the
actions of alcohols, albeit to a greater extent, on desensitization. Thus, the
Ile294Thr mutation may actually mask the stimulatory effects of alcohols. Our
data support this conclusion in that TCEt significantly increases steady-state
to peak current ratios in the wild-type receptor, but not the
Ile294Thr5-HT3A receptor. Further supporting evidence comes from
work in the GABAA receptor. Mutations in either TM2 or in the N
terminus have been described that produce tonically open channels, an extreme
case of increased probability of channel opening wherein a certain fraction of
receptors are constantly open. These receptors have very reduced or no
sensitivity to allosteric modulation by alcohols and anesthetics
(Thompson et al., 1999;
Findlay et al., 2001;
Zhang et al., 2002).
Two contrasting effects on activation were observed in the Ile294Thr 5-HT3A receptor. Both the initial slope and the 20 to 80% rise-time were increased, although not significantly, in the Ile294Thr mutant relative to the wild-type receptor. An increase in activation rate would favor the open channel state in the mutant relative to the wild-type receptor. However, an increase in rise-time means that the time required for opening is longer in the mutant, suggesting that the wild-type receptor is more favored to open when this parameter is considered. It is important to note that the 20 to 80% rise-time is a less precise measure of activation, since it may be contaminated with other components of the whole cell current. We suggest that the desensitization of some of the mutant channels may be occurring while others are still opening. Although it is the summation of all factors that contribute to the stabilization of the open state, we suggest that the effects of the mutation on rate and extent of desensitization are likely to be the most important. That is, once the Ile294Thr receptor opens, it stays open, which prolongs the later stages of current onset, as is evidenced by the overlaid tracings in Fig. 5A, panel c.
TCEt had contrasting effects on 5-HT-mediated currents in the wild-type and Ile294Thr 5-HT3A receptors. In the wild-type receptor, TCEt increased the initial slope, but had no effect on 20 to 80% rise-time. In the mutant receptor, the initial slope of current is reduced, although nonsignificantly, by TCEt. Rise-time in the mutant receptor was unaffected by TCEt. Taken together, these data suggest that inhibition of activation of channel opening is the third mechanism through which the potentiating effects of alcohol are lost in the mutant receptor. In fact, inhibition of activation may in part account for blockade of mutant receptor function (oocyte expression system) with TCEt at lower concentrations and of mutant receptor function (HEK 293 cell expression system) at 3 mM TCEt. Thus, slower current activation, combined with reduced desensitization, likely contribute to the loss of alcohol effects. The biphasic response observed over the TCEt concentrations tested (0.125–10 mM), with potentiation occurring at 5 and 10 mM, may reflect TCEt binding at lower affinity sites whose allosteric modulation is less affected or unaffected by the mutation.
The kinetic data that we have shown provide us with a compelling argument
that the lack of stimulatory action of alcohols on the Ile294Thr
5-HT3A receptor is not due to an alteration of a binding pocket for
alcohols and anesthetics. Another supporting piece of evidence is the finding
that Ile294 faces the pore of the channel
(Reeves et al., 2001;
Panicker et al., 2002). Given
the fact that alcohols enhance 5-HT3A receptor function, it would
be difficult to reconcile the notion of an alcohol binding domain that
included amino acids in the pore. A pore-lining pocket would be more
consistent with an inhibitory, channel-blocking action of alcohols.
Furthermore, in the face of other evidence presented, our observation that the
Ile294Leu mutant has a reduced cutoff relative to the wild-type receptor does
not lend credence to the idea of a binding pocket at Ile294, and is likely a
coincidental finding.
In summary, we have shown that the Ile294Thr 5-HT3A receptor has a markedly altered kinetic profile in comparison with the wild-type receptor. Changes in the extent and rate of desensitization and increased activation rate, as well as slowing of activation in the presence of alcohols, can explain the loss of stimulatory effects of ethanol and TCEt. These results, coupled with the finding that Ile294 faces the pore of the channel, cast doubt that Ile294 is part of an alcohol-binding domain. Our results suggest that loss of alcohol and anesthetic modulation of function and changes in alcohol cutoff should not be used as the sole criteria for identifying a putative alcohol-binding domain. Kinetics of channel function should be evaluated in a series of mutants at an apparently critical residue before an alcohol-binding site should be invoked.
|
Footnotes |
|---|
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; TCEt, 2,2,2-trichloroethanol; nACh, nicotinic acetylcholine; TM2, transmembrane 2; MBS, modified Barth's solution; ANOVA, analysis of variance; HEK, human embryonic kidney.
1 Current address: Dept. of Pharmacology, Duke University Medical Center,
Research Drive, Durham, NC 27710. 
2 Current address: Laboratory of Integrative Neuroscience, National Institute
of Alcohol Abuse and Alcoholism, Room 158H Park 5 Bldg., 12420 Parklawn Drive,
Rockville, MD 20852.
Address correspondence to: Dr. Tina K. Machu, Dept. of Pharmacology, Texas Tech University Health Sciences Center, 3601 Fourth St., Lubbock, TX 79430. E-mail: tina.machu{at}ttuhsc.edu
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References |
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|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aapro MS (1991) 5-HT3 antagonists: an overview of their present status and future potential in cancer therapy-induced emesis. Drugs 42: 551–568.[Medline]
Amin J (1999) A single hydrophobic residue confers
barbiturate sensitivity to -aminobutyric acid type C receptor.
Mol Pharmacol 55:
411–423.
Belelli D, Balcarek JM, Hope AG, Peters JA, Lambert JJ, and Blackburn TP (1995) Cloning and functional expression of a human 5-hydroxytryptamine type 3As receptor subunit. Mol Pharmacol 48: 1054–1062.[Abstract]
Belelli D, Lambert JJ, Peters JA, Wafford K, and Whiting PJ
(1997) The interaction of the general anesthetic etomidate with
the -aminobutyric acid A receptor is influenced by a single amino acid.
Proc Natl Acad Sci USA
94:
11031–11036.
Bertrand D, Devillers-Thiery A, Revah F, Galzi J-L, Hussy N, Mulle
C, Bertrand S, Ballivet M, and Changeux J-P (1992) Unconventional
pharmacology of a neuronal nicotinic receptor mutated in the channel domain.
Proc Natl Acad Sci USA
89:
1261–1265.
Colquhoun D (1998) Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125: 924–947.[Medline]
Davies PA, Pistis M, Hanna MC, Peters JA, Lambert JJ, Hales TG, and Kirkness EF (1999) The 5-HT3B subunit is a major determinant of serotonin-receptor function. Nature (Lond) 397: 359–363.[CrossRef][Medline]
Derkach V, Surprenant A, and North RA (1989) 5-HT3 receptors are membrane ion channels. Nature (Lond) 339: 706–709.[CrossRef][Medline]
Dubin AE, Huvar R, D'Andreas MR, Pyati J, Zhu JY, Koy KC, Wilson
SJ, Galindo JE, Glass CA, Lin L, et al. (1999) The
pharmacological and functional characteristics of the serotonin
5-HT3A receptor are specifically modified by a 5-HT3B
receptor subunit. J Biol Chem
274:
30799–30810.
Findlay GS, Susumu U, Harrison NL, and Harris RA
(2001) Allosteric modulation in spontaneously active mutant
-aminobutyric acidA receptors. Neurosci
Lett 305:
77–80.[CrossRef][Medline]
Hodge CW, Samson HH, Lewis RS, and Erickson HL (1993) Specific decreases in ethanol but not water-reinforced responding produced by the 5-HT antagonist ICS 205–930. Alcohol 10: 191–196.[CrossRef][Medline]
Jenkins A, Franks NP, and Lieb WR (1996) Actions of general anesthetics on 5-HT3 receptors in N1E-115 neuroblastoma cells. Br J Pharmacol 117: 1507–1515.[Medline]
Johnson BA, Campling GM, Griffiths P, and Cohen PJ (1993) Attenuation of some alcohol-induced mood changes and the desire to drink by 5-HT3 receptor blockade: a preliminary study in healthy male volunteers. Psychopharmacology 112: 142–144.[CrossRef][Medline]
Knapp DJ and Pohorecky LA (1992) Zacopride, a 5-HT3 receptor antagonist, reduces voluntary ethanol consumption in rats. Pharmacol Biochem Behav 41: 847–850.[CrossRef][Medline]
Koltchine VV, Finn SE, Jenkins A, Nikolaeva N, Lin A, and Harrison
NL (1999) Agonist gating and isoflurane potentiation in the human
gamma-aminobutyric acid type A receptor determined by the volume of a second
transmembrane domain residue. Mol Pharmacol
56:
1087–1093.
Lankiewicz S, Lobitz N, Wetzel CHR, Rupprecht R, Gisselmann G, and
Hatt H (1998) Molecular cloning, functional expression and
pharmacological characterization of 5-hydroxytryptamine3 receptor
cDNA and its splice variants from guinea pig. Mol
Pharmacol 53:
202–212.
Lovinger DM, Sung KW, and Zhou Q (2000) Ethanol and trichloroethanol alter gating of 5-HT3 receptor-channels in NCB-20 neuroblastoma cells. Neuropharmacology 39: 561–570.[CrossRef][Medline]
Lovinger DM and White G (1991) Ethanol potentiation of 5-hydroxytryptamine3 receptor-mediated ion current in neuroblastoma cells and isolated adult mammalian neurons. Mol Pharmacol 40: 263–270.[Abstract]
Machu TK and Harris RA (1994) Alcohols and anesthetics
enhance the function of 5-hydroxytryptamine3 receptors expressed in
Xenopus laevis oocytes. J Pharmacol Exp Ther
271:
898–905.
Maricq AV, Peterson AS, Brake AJ, Myers RM, and Julius D (1991) Primary structure and functional expression of the 5-HT3 receptor, a serotonin-gated ion channel. Science (Wash DC) 154: 432–434.
Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, et al. (1997) Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature (Lond) 389: 385–389.[CrossRef][Medline]
Miyake A, Mochizuki S, Takemoto Y, and Akuzawa S (1995) Molecular cloning of human 5-hydroxytryptamine3 receptor: heterogeneity in distribution and function among species. Mol Pharmacol 48: 407–416.[Abstract]
Morales M and Wang SD (2002) Differential composition
of 5-hydroxytryptamine3 receptors synthesized in the rat CNS and
peripheral nervous system. J Neurosci
22:
6732–6741.
Panicker S, Cruz H, Arrabit C, and Slesinger PA (2002)
Evidence for a centrally located gate in the pore of a serotonin-gated ion
channel. J Neurosci 22:
1629–1639.
Peters JA, Lambert JJ, and Malone HM (1994) Electrophysiological studies of 5-HT3 receptors, in 5-Hydroxytryptamine3 Receptor Antagonists (King FD, Jones BJ, and Sanger GJ eds), pp 115, CRC Press, Boca Raton.
Reeves DC, Goren EN, Akabas MH, and Lummis SC (2001)
Structural and electrostatic properties of the 5-HT3 receptor pore revealed by
substituted cysteine accessibility mutagenesis. J Biol
Chem 276:
42035–42042.
Thompson S-A, Smith MZ, Wingrove PB, Whiting PJ, and Wafford KA (1999) Mutation at the putative GABAA ion-channel gate reveals changes in allosteric modulation. Br J Pharmacol 127: 1349–1358.[CrossRef][Medline]
Wick MJ, Mihic SJ, Ueno S, Mascia MP, Trudell JR, Brozowski SJ, Ye
Q, Harrison NL, and Harris RA (1998) Mutations of -amino
acid and glycine receptors change alcohol cut-off: evidence for an alcohol
receptor? Proc Natl Acad Sci USA
95:
6504–6509.
Yamakura T, Mihic SJ, and Harris RA (1999) Amino acid
volume and hydropathy of a transmembrane site determine glycine and anesthetic
sensitivity of glycine receptors. J Biol Chem
274:
23006–23012.
Ye Q, Kolthcine VV, Mihic SJ, Mascia MP, Wick MJ, Finn SE, Harrison
NL, and Harris RA (1998) Enhancement of glycine receptor function
by ethanol is inversely correlated with molecular volume at position
267. J Biol Chem
273:
3314–3319.
Yu D, Zhang L, Eiselé J-L, Bertrand D, Changeux J-P, and
Weight FF (1996) Ethanol inhibition of nicotinic acetylcholine
type 7 receptors involves the amino-terminal domain of the receptor.
Mol Pharmacol 50:
1010–1016.[Abstract]
Zhang L, Hosoi M, Fukuzawa M, Sun H, Rawlings RR, and Weight FF
(2002) Distinct molecular basis for differential sensitivity of
the serotonin type 3A receptor to ethanol in the absence and presence of
agonist. J Biol Chem
277:
46256–46264.
Zhou Q and Lovinger DM (1996) Pharmacologic
characteristics of potentiation of 5-HT3 receptors by alcohols and diethyl
ether in NCZB-20 neuroblastoma cells. J Pharmacol Exp
Ther 278:
732–740.
Zhou Q, Verdoorn TA, and Lovinger DM (1998) Alcohols
potentiate the function of 5-HT3 receptor-channels on NCB-20
neuroblastoma cells by favouring and stabilizing the open channel state.
J Physiol 507.2:
335–352.
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