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NEUROPHARMACOLOGY

Arginine 246 of the Pretransmembrane Domain 1 Region Alters 2,2,2-Trichloroethanol Action in the 5-Hydroxytryptamine_3A Receptor

Department of Biomedical Sciences, College of Health Sciences, Marquette University, Milwaukee, Wisconsin

Received for publication August 30, 2007
Accepted December 18, 2007.

	Abstract

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Ligand-gated ion channels participate in synaptic transmission, and they are involved in neurotransmitter release. The functions of the channels are regulated by a variety of modulators. The interaction of 2,2,2-trichloroethanol, the active hypnotic metabolite of chloral hydrate, with the 5-hydroxytryptamine (5-HT) (serotonin) type 3 receptor results in a positive allosteric modulation. We have demonstrated previously that arginine 246 (R246) located in the pretransmembrane domain 1 is critical for coupling agonist binding to gating. In this study, we examined the role of R246 in the action of trichloroethanol with a combination of mutagenesis and whole-cell patch-clamp techniques. The R246A mutation converted the partial agonist dopamine into a full agonist at the 5-HT_3A receptor, and it facilitated activation of the mutant receptor by dopamine, suggesting an enhanced gating process due to the mutation. The positive modulation of the 5-HT_3A receptor by trichloroethanol was dramatically reduced by the R246A mutation. Trichloroethanol had little agonist activity in the wild-type receptor (<1% of maximal 5-HT response). However, the R246A mutation significantly increased the direct activation of the receptor by trichloroethanol in the absence of agonist (10% of maximal 5-HT response). The current activated by trichloroethanol could be blocked by the competitive 5-HT₃ receptor antagonist tropanyl 3,5-dichlorobenzoate (MDL 72222), and it had a similar reversal potential to those of current activated by 5-HT. In addition, predesensitization of the mutant receptor by trichloroethanol prevented 5-HT from activating the receptor. These data suggest that R246 is a crucial site for mediating the actions of both agonists and modulators.

5-HT₃ receptors are expressed in both the central and peripheral nervous systems, and they are thought to participate in a variety of physiological functions such as cognitive processing, sensory transmission, regulation of autonomic function, integration of the vomiting reflex, pain processing, and control of anxiety (Barnes and Sharp, 1999). The 5-HT_3A subunit was first cloned in 1991 (Maricq et al., 1991). Since then, an additional four subunits, 3B, 3C, 3D, and 3E, have been added to the 5-HT₃ receptor family (Davies et al., 1999; Niesler et al., 2003). Only the 3A and 3B subunits have been investigated in detail, whereas the functional role of the 3C, 3D, and 3E is not clear at present. The homomeric assembly of the 3A subunits, but not the 3B subunits, can form functional channels. Coexpression of the 3A and 3B subunits leads to formation of a heteromeric receptor that displays different biophysical properties from the homomeric 5-HT_3A receptor (Davies et al., 1999; Dubin et al., 1999; Hapfelmeier et al., 2003). The functions of the 5-HT₃ receptor can be tuned by allosteric modulators such as ions, alcohol, and anesthetics (Lovinger and Zhou, 1993; Hu and Lovinger, 2005). Chloral hydrate is a widely used hypnotic and anesthetic agent; and trichloroethanol is the active metabolite of chloral hydrate and an analog of ethanol. Allosteric modulation of Cys-loop ligand-gated ion channels by trichloroethanol has been observed in the 5-HT₃ (Lovinger and Zhou, 1993), GABA_A (Krasowski et al., 1998a), and glycine (Krasowski et al., 1998a) receptors. However, the critical elements in the Cys-loop ligand-gated ion channels for trichloroethanol action are not fully understood. There is a cluster of three positively charged arginine residues at the C-terminal end of the extracellular N terminus of the 5-HT_3A receptor (Fig. 1A). The last arginine residue, R246, has been found to be one of the transducing elements in coupling agonist binding to channel gating (Hu et al., 2003). The goal of this study was to investigate the role of R246 in the action of 2,2,2-trichloroethanol. Our results reveal that the R246 residue of the mouse 5-HT_3A receptor is a pivotal site for both coupling agonist binding to channel gating and mediating the action of trichloroethanol.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Effects of the R246A mutation on dopamine (DA)-activated responses. A, sequence alignment of the preTM1 and TM1 of the 5-HT3A receptor. B, traces show currents activated by 5-HT and DA in HEK 293 cells expressing wild-type (top) and R246A (bottom) receptors. The arrow indicates the application of 5-HT for 250 ms, and the bar indicates the application time for DA. C, concentration-response curves for dopamine in the WT and R246A receptors. Data were normalized to peak current activated by 30 µM 5-HT for each cell. Each data point represents mean ± S.E.M. from six to nine cells. D, traces show receptor activation elicited by 3 mM DA in the WT and R246A receptors. DA-activated responses were normalized to peak current for comparison. The bar indicates the time of DA application. E, averaged data show the activation time constants for DA in the WT and R246A receptors. Each bar represents mean ± S.E.M. from 11 to 12 cells.

	Materials and Methods

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Cell Culture and Transient Receptor Expression. HEK 293 cells (American Type Culture Collection, Manassas, VA) were grown in minimum essential medium (Invitrogen) supplemented with 10% horse serum, and they were maintained in a humidified incubator at 37°C in 5% CO₂. HEK 293 cells were transiently transfected with the wild-type or mutant 5-HT_3A receptor cDNA using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Green fluorescent protein (pGreen Lantern; Invitrogen) was coexpressed with the 5-HT_3A receptor subunits to permit selection of transfected cells under fluorescence optics. Each 35-mm dish was transfected with 3 µg of cDNA encoding the wild-type or mutant receptors along with 1 µg of green fluorescent protein cDNA.

Whole-Cell Patch-Clamp Recording. Whole-cell recordings were performed in HEK 293 cells 1 to 3 day after transfection. HEK 293 cells were replated on the day of the experiment to ensure that recordings were only made from single, isolated cells. Cells were continuously superfused with a solution containing 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1.2 mM MgCl₂, 5 mM glucose, and 10 mM HEPES (pH was adjusted to 7.4 with NaOH and osmolarity was adjusted to 340 mOsmol with sucrose). Pipettes were pulled from borosilicate glass (TW-150F; WPI, Sarasota, FL) using a multistage puller (Flaming-Brown P-97; Sutter Instrument Company, Novato, CA), and they had resistances of 2 to 5 M when filled with pipette solution containing 140 mM CsCl, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES (pH was adjusted to 7.2 with CsOH, and osmolarity adjusted to 315 mOsmol with sucrose). Membrane current was recorded in the whole-cell configuration using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) at 20 to 22°C. Cells were held at –60 mV unless otherwise indicated. Data were acquired using pClamp 9.0 software (Molecular Devices). Currents were filtered at 2 kHz, and they were digitized at 5 to 10 kHz.

Agonists were applied with a piezoelectric device (PZ-150M; EXFO Burleigh Products Group Inc., Victor, NY) or a stepper motor-driven apparatus (Fast-Step; Warner Instruments, Hamden, CT) through a two-barrel theta glass tubing (TGC150; Warner Instruments, Hamden, CT) that had been pulled to a tip diameter of 200 µm. The cell was placed in front of the stream of control solution. The piezoelectric device or stepper motor-driven apparatus was driven by transistortransistor logic pulses from the pClamp 9.0 software (Molecular Devices), which produce a rapid lateral displacement (50 µm) of the tubing to move the interface between control and agonist solutions. Solution exchange rate for open pipette and whole-cell recording was estimated using the potential change induced by switching from the control solution to a 140 mM N-methyl-D-glucamine test solution at 0 mV in the absence of agonist; and the current rising phase was fit using an exponential function. The solution exchange time constants were 0.3 ms for an open pipette tip and 1.6 ms for whole-cell recording.

Data Analysis. Data analysis and curve fitting were performed with Origin 7.0 (OriginLab Corp., Northampton, MA), pClamp 9.0 (Molecular Devices), or InStat 3.0 (GraphPad Software Inc., San Diego, CA) software. Concentration-response data for dopamine were fit using the Hill equation I/I₃₀ µM 5-HT = 1/[1 + (EC₅₀/[agonist])^nh], where I is the current amplitude activated by a given concentration of agonist ([agonist]), I₃₀ µM 5-HT is the current amplitude of the cell in response to 30 µM 5-HT, n_h is the Hill coefficient, and EC₅₀ is the concentration eliciting a half-maximal response.

Parameters of channel activation and deactivation and desensitization were estimated by fitting appropriate current components using exponential functions of the general form A_ne^(-t/_n) + A_s, where A_n is the relative amplitude of the respective component, A_s is the steady-state current, n is the optimal number of exponential components, t is time, and _n is the respective time constant. Curve fitting was achieved in Clampfit 9.0 using the Levenberg-Marquardt algorithm. Additional components were accepted only if they significantly improved the fit, as determined by an F-test performed using the analysis software.

Activation rates were derived from exponential fitting of the rising phase of agonist activated current. Desensitization time constants were derived from exponential fits to the current decay starting just after the current peak and extending to the end of agonist application. Deactivation time constants were derived from exponential fits to the current decay after the removal of agonist. To facilitate direct comparison of desensitization with single versus multiple components, a weighted summation of time constants (a_nn) was used, where a_n is the fractional contribution of the respective component, _n is the respective time constant, and n is the optimal number of exponential components.

In some experiments, voltage ramps were applied to measure reversal potential and channel rectification. A ramp with a slew rate of 0.5 mV/ms was applied during the peak of current activated by 3 µM 5-HT and 10 mM 2,2,2-trichloroethanol (trichloroethanol). Current activated by a voltage ramp in the absence of agonist was subtracted from the ramp-activated current in the presence of agonist before plotting and analyzing these data.

Data are presented as mean ± S.E.M. Statistical significance was determined with the Student's t test or one-way analysis of variance. Differences were considered significant at p < 0.05.

	Results

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R246 Mutation Increases the Relative Efficacy of the Partial Agonist Dopamine. Both the wild-type and R246 mutant receptors were transiently expressed in HEK 293 cells; and the effect of the R246 mutation on the current activated by dopamine, a 5-HT₃ receptor partial agonist, was examined with whole-cell patch-clamp recording. Representative responses activated by 5-HT and dopamine are shown in Fig. 1B. At maximally efficacious concentrations, 3 mM dopamine was less effective than 30 µM 5-HT in activating the wild-type receptor, confirming the partial agonism of dopamine at the 5-HT₃ receptor. However, dopamine was as effective as 5-HT in activating the mutant receptor. Application of dopamine activated concentration-dependent responses in both the wild-type and mutant receptors (Fig. 1C). The R246A mutation shifted the dopamine concentration-response curve to the left. The EC₅₀ for the mutant receptor (55.8 ± 10.3 µM) was smaller than that for the wild-type receptor (140.0 ± 4.5 µM), suggesting an increased agonist potency by the R246A mutation. The Hill coefficient for the wild-type and mutant receptor was 1.9 ± 0.1 and 1.1 ± 0.1, respectively. In addition, the R246A mutation increased the maximal response activated by dopamine from 29.8 ± 1.4 to 91.6 ± 1.4% of the response activated by 30 µM 5-HT. Dopamine at 3 mM elicited a much faster-activating inward current in the mutant receptor than in the wild-type receptor (Fig. 1D); and the R246A mutation resulted in a 25-fold increase in activation rate of the receptor in response to dopamine (Fig. 1E).

R246A Mutation Essentially Eliminates Trichloroethanol Effect on Desensitization. Desensitization is a common feature of ligand-gated ion channels. As shown in Fig. 2A, 5-HT-activated current decayed during prolonged agonist application in the wild-type receptor, and coapplication of trichloroethanol with 5-HT slowed down the current decay. Consistent with previous findings (Hu et al., 2003), desensitization was faster in the R246A mutant receptor than in the wild-type receptor (Fig. 2A). Coapplication of trichloroethanol and 5-HT seemed to slightly decrease desensitization in the mutant receptor. Desensitization time course was best fit with a monoexponential function in the wild-type receptor, and the rate of desensitization was 4-fold slower in the presence of trichloroethanol (Fig. 2B). However, desensitization in the mutant receptor, displayed a biexponential function. It seems that the R246A mutation largely abolished the trichloroethanol effect on receptor desensitization. For example, trichloroethanol did not alter the time constant for the fast component of desensitization, whereas it slightly slowed down the time constant for the slow component of desensitization (Fig. 2B). In addition, trichloroethanol did not alter the proportion of the fast and slow components (fast component, 53.9 ± 3.1 57.7 ± 4.8%; slow component, 46.1 ± 3.1 42.3 ± 4.8%; p = 0.5). Furthermore, the overall desensitization measured as the weighted desensitization time constant was not significantly altered by trichloroethanol in the R246A receptor (1.8 ± 0.2 2.2 ± 0.4 s; p > 0.08).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Effect of the R246A mutation on modulation of 5-HT3A receptor desensitization by trichloroethanol (TcEt). A, traces show desensitization of WT (left) and R246A (right) receptors in response to 30 µM 5-HT in the absence or presence of 1 mM TcEt. Currents were normalized to peak current, and they are superimposed for comparison. Bar indicates the time of agonist application. Traces in the absence of TcEt are in black, whereas those in the presence of TcEt are in gray. B, average desensitization kinetics in the WT and R246A receptors in the absence or presence of 1 mM TcEt. Each bar represents the mean ± S.E.M. from 5 to 12 cells.

R246A Mutation Reduces Trichloroethanol Modulation of Dopamine Current. In addition to decreasing desensitization kinetics, alcohol has also been found to increase the peak current activated by dopamine and low concentrations of 5-HT at the 5-HT_3A receptor (Machu and Harris, 1994; Lovinger et al., 2000). Therefore, modulation of the 5-HT_3A receptor by trichloroethanol was further examined using dopamine to activate the receptor. In addition, the effect of ethanol on dopamine-activated current was also studied based on the structural similarity between trichloroethanol and ethanol. In this set of experiments, three concentrations of dopamine (EC₃₅, EC₆₅, and EC₁₀₀) were used for both the wild-type and mutant receptors. The activation of the 5-HT_3A receptor by dopamine seemed much faster in the mutant receptor at each corresponding concentration. Coapplication of either trichloroethanol or ethanol with dopamine enhanced peak current in the wild-type receptor, and trichloroethanol seemed to be more potent than ethanol. The potentiation by both compounds was dopamine concentration-dependent, with greater enhancement seen at lower concentrations. However, the potentiation of dopamine-activated current by trichloroethanol and ethanol was less obvious in the mutant receptor (Fig. 3A). Averaged data revealed that dopamine-activated current amplitude was enhanced by 2100, 700, and 300% with trichloroethanol and by 130, 70, and 60% with ethanol at EC₃₅, EC₆₅, and EC₁₀₀ of dopamine, respectively, in the wild-type receptor (Fig. 3B). However, ethanol only enhanced dopamine-activated current amplitude by 20% at EC₃₅ and EC₆₅ of dopamine; and it failed to potentiate EC₁₀₀ dopamine-activated current in the mutant receptor. Furthermore, the potentiation by trichloroethanol was dramatically reduced by the R246A mutation; trichloroethanol increased dopamine-activated current amplitude by only 100, 80, and 15% at EC₃₅, EC₆₅, and EC₁₀₀ of dopamine in the mutant receptor, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effect of R246A mutation on modulation of DA-activated current by TcEt and ethanol (EtOH). A, traces show modulation of current amplitude activated by EC35 (left), EC65 (middle), and EC100 (right) DA with 100 mM EtOH and 1 mM TcEt in the WT (top) and R246A (bottom) receptors. B, averaged data show alteration of DA-activated responses by 100 mM EtOH (top) and 1 mM TcEt (bottom) in the WT and R246A receptors. Each bar represents the mean ± S.E.M. from 7 to 19 cells.

R246A Mutation Converts Trichloroethanol to a Partial Agonist. Trichloroethanol at 10 mM in the absence of agonist barely activated detectable current in the wild-type receptor. However, 10 mM trichloroethanol alone was able to activate the mutant receptor (Fig. 4A). The maximal response activated by trichloroethanol was less than 1% of the response activated by 30 µM 5-HT in the wild-type receptor. In contrast, trichloroethanol activated the mutant receptor at concentrations 1 mM in a concentration-dependent manner (Fig. 4B). The trichloroethanol EC₅₀ and Hill coefficient values were 4.2 ± 0.44 and 3.5 ± 0.33 mM, respectively, in the mutant receptor. In addition, the R246A mutation significantly increased the maximal response activated by trichloroethanol to 10% of the response activated by 30 µM 5-HT.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Effect of the R246A mutation on direct activation of the 5-HT3A receptor by TcEt. A, traces show currents activated by 5-HT and TcEt in HEK 293 cells expressing wild-type (top) and R246A (bottom) receptors. B, concentration-response curves for TcEt in the WT and R246A receptors. Data were normalized to peak current activated by 30 µM 5-HT for each cell. Each data point represents mean ± S.E.M. from 8 to 25 cells.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Properties of TcEt-activated currents in the R246A receptor. A, traces show current activated by 10 mM TcEt in the absence (left) and presence of 300 nM MDL 72222 (right). The bar indicates the application time for TcEt or MDL 72222. Similar responses were observed in four cells. B, traces show voltage ramps obtained at the peak of current activated by 3 µM 5-HT in the WT and R246A receptors and 10 mM TcEt in the R246A receptor (left). Current-voltage relationships for 5-HT in the WT and R246A receptors and TcEt in the R246A receptor (right). Currents activated by the voltage protocol in the absence of 5-HT and TcEt have been subtracted. Similar results were obtained from 12 to 17 cells.

Predesensitizing the R246A Receptor with Trichloroethanol Abolishes 5-HT Current. The response to 30 µM 5-HT (in the absence of trichloroethanol) immediately after preincubation with 10 mM trichloroethanol for 20 s was not altered in the wild-type receptor (Fig. 6A). There was a delayed rebound current after termination of 5-HT application, which accounted for an increase in response by 8.9 ± 1.7%. Preincubation with 10 mM trichloroethanol activated current that was 11.5 ± 1.5% of 30 µM 5-HT-activated response in the mutant receptor, and this current gradually decayed back to the baseline during the 20-s preincubation. Application of 30 µM 5-HT immediately after trichloroethanol preincubation failed to activate a detectable current in the mutant receptor (Fig. 6B).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effect of preincubation with TcEt on 5-HT-activated current. A, traces show currents activated by 30 µM 5-HT with or without 10 mM TcEt preincubation in the WT and R246A receptors. The arrow indicates the application of 5-HT for 250 ms, and the bar indicates the application time for TcEt. B, averaged data show effects of TcEt preincubation on current amplitude activated by 30 µM 5-HT in the WT and R246A receptors. Each bar represents mean ± S.E.M. from 8 to 13 cells.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Kinetics of TcEt-activated current. A, exponential fitting of the activation, deactivation, and desensitization of 10 mM TcEt-activated current. The bar indicates the time of agonist application, and the arrow indicates the time point of removal of TcEt. B, averaged data show kinetics of TcEt-activated current in the R246A receptor. Each bar represents mean ± S.E.M. from 13 to 16 cells.

	Discussion

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A cluster of positively charged residues is present in the preTM1 region of the Cys-loop ligand-gated ion channels. It has been demonstrated that there is an electrostatic interaction between K215 in the preTM1 and D146 in the loop 7 of the β2 GABA_A receptor subunit (Kash et al., 2004) and between R209 in the preTM1 and E45 in loop 2 of the nicotinic acetylcholine receptor subunit (Lee and Sine, 2005), implicating that charge interactions play a potential role in coupling agonist binding to channel gating. A previous study also implies that the R246 residue of the 5-HT_3A receptor could participate in forming salt bridges (Zhang et al., 2002). However, it has also been argued that the overall, rather than specific, charge interactions at the gating interfaces govern the coupling mechanism (Xiu et al., 2005). It is possible that the participation of the R246 residue in electrostatic interactions may stabilize the 5-HT_3A receptor in the closed state, and that the R246A mutation disrupts this interaction leading to facilitated transduction and channel activation. The increased relative efficacy for dopamine and trichloroethanol could well be explained by this action. The possibility of a reduction in repulsion of the charged dopamine molecule leading to an enhanced efficacy cannot be excluded, because dopamine mainly exists as cationic form at pH 7.4 (Barlow, 1976).

It is interesting to note that the R246A mutation dramatically increased the agonism of trichloroethanol. Direct activation by ethanol has been observed in some R246 mutant 5-HT_3A (Zhang et al., 2002) and 2(T262W) GABA_A (Ueno et al., 2000) receptors expressed in Xenopus oocytes. Observations such as similar reversal potentials for trichloroethanol and 5-HT currents, blockade of trichloroethanol current by the competitive 5-HT₃ receptor antagonist MDL 72222, and abolition 5-HT current by desensitizing the receptor with trichloroethanol suggest that trichloroethanol directly activates the R246A receptor. The activation kinetics of trichloroethanol current in the mutant receptor is much slower, whereas the deactivation kinetics of trichloroethanol current in the mutant receptor is much faster than that of 5-HT current in both the wild-type and mutant receptors (Hu et al., 2003). However, the deactivation kinetics of trichloroethanol current is similar to that of the low-affinity agonist dopamine current in the wild-type receptor (Hu and Lovinger, 2005). In addition, the desensitization of the trichloroethanol current is similar to that of 5-HT current in the wild-type receptor (Hu et al., 2003). Therefore, trichloroethanol seems to bind to the R246A receptor with low affinity to activate the receptor. The exact location of the trichloroethanol binding sites for direct activation is not clear. Those sites may overlap with the agonist/competitive antagonist sites on the receptor. Alternatively, trichloroethanol could function as an allosteric agonist by binding to an allosteric site(s).

Under a saturating concentration of agonist, the response is largely determined by the interplay between agonist-bound closed and open states of the receptor (Colquhoun, 1998). We showed that trichloroethanol and ethanol enhanced the response activated by a saturating concentration of dopamine in the wild-type receptor, which is consistent with previous observations in NCB-20 cells (Lovinger et al., 2000). Those results suggest that gating efficacy of the 5-HT_3A receptor is enhanced by both trichloroethanol and ethanol. Either an increase in open time or a decrease in closed time or both could contribute to the increase in gating efficacy. The decreased desensitization may be secondary, at least in part, to the prolonged stay of the receptor in the open state. Because the enhancement by trichloroethanol and ethanol was more obvious at lower concentrations of dopamine, the possibility that trichloroethanol and ethanol increase the affinity of dopamine also cannot be ruled out.

It is interesting to note that the R246A mutation considerably reduced the allosteric modulation of the 5-HT_3A receptor by trichloroethanol and ethanol, suggesting that the R246 residue plays a role in the actions of alcohol at the 5-HT_3A receptor. It has been suggested that the S267 residue of the 1 glycine receptor subunit and S270 residue of the GABA_A receptor subunits participate in forming the binding pocket for alcohol and anesthetics (Mascia et al., 2000). We have recently demonstrated that the L293 residue, an amino acid at the equivalent position of the S267 residue in the glycine receptor and S270 residue in the GABA_A receptor, of the 5-HT_3A receptor is critical for alcohol modulation (Hu et al., 2006). However, little relationship could be established between the physicochemical properties of the substituted amino acids at this position and alcohol actions. Indeed, whether the S267 residue of the glycine receptor or S270 residue of the GABA_A receptor is part of the alcohol/anesthetic binding site has been questioned (Carlson et al., 2000; Chang et al., 2003). It has been established that the S270 residue in the GABA_A receptor (Scheller and Forman, 2002) and R246 and L293 residues in the 5-HT_3A receptor (Hu et al., 2003, 2006) are critical residues for channel gating. Those residues may be part of the common transducing elements for both agonists and allosteric modulators such as ethanol and trichloroethanol, so that the increase in gating efficacy due to mutations diminishes additional enhancement provided by alcohol binding. Therefore, the abolition of alcohol modulation with mutations at these residues could be secondary to the alteration in the gating process. Similar results have been observed in the GABA_A receptor. Mutagenesis studies have implicated several structural elements for allosteric modulation, such as preTM1, TM2, and TM2-TM3 loop of the 2 subunit for benzodiazepines and anesthetics actions at the GABA_A receptor. It is interesting to note that the residues at those locations are also critical for channel gating. Therefore, those elements have been defined as transducing sites for the allosteric modulators (Boileau and Czajkowski, 1999; Carlson et al., 2000; Chang et al., 2003).

A single binding site for etomidate has been proposed for its allosteric modulation and direct activation at the GABA_A receptor (Rüsch et al., 2004). However, several studies demonstrated that the positive modulation and direct activation of the GABA_A receptor by propofol and pentobarbital are mediated by binding to different sites (Krasowski et al., 1998b; Dalziel et al., 1999; Chang et al., 2003). Our data have demonstrated that trichloroethanol has multiple actions on the function of the 5-HT_3A receptor. The R246A mutation ablated the potentiation of 5-HT_3A receptor-mediated current and markedly enhanced the agonism of trichloroethanol, indicating that potentiation and direct activation by trichloroethanol seem to be distinct processes, and they probably involve at least two discrete binding sites.

In conclusion, we have confirmed that the R246 residue in the preTM1 region of the 5-HT_3A receptor is a critical site for coupling agonist binding to channel gating. In addition, our study reveals that the R246 residue is crucial for allosteric modulation by alcohols. Our findings are consistent with the idea that residues located at the membrane interface are the transducing sites for both agonist and allosteric modulators.

	Footnotes

 
This work was supported by institutional funds from Marquette University and by a grant from the National Institutes of Health (to R.W.P.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.131011.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); TM, transmembrane domain; preTM, pretransmembrane domain; HEK, human embryonic kidney; MDL 72222, tropanyl 3,5-dichlorobenzoate; TcEt, trichloroethanol; DA, dopamine; WT, wild type; EtOH, ethanol.

Address correspondence to: Dr. Robert W. Peoples, Department of Biomedical Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881. E-mail: robert.peoples{at}marquette.edu

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