0022-3565/08/3243-10111018$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright 2008 by The American Society for Pharmacology and Experimental Therapeutics JPET 324:10111018, 2008
Vol. 324, No. 3 131011/3311495 Printed in U.S.A.
Arginine 246 of the Pretransmembrane Domain 1 Region Alters 2,2,2-Trichloroethanol Action in the 5-Hydroxytryptamine3A Receptor
Xiang-Qun Hu and Robert W. Peoples
Department of Biomedical Sciences, College of Health Sciences, Marquette University, Milwaukee, Wisconsin
Received August 30, 2007; accepted December 18, 2007
ABSTRACT 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-HT3A 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-HT3A 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.
Downloaded from jpet.aspetjournals.org by guest on June 8, 2011
The 5-hydroxytryptamine (5-HT) (serotonin) type 3 receptor is a member of the Cys-loop ligand-gated ion channel superfamily that also includes the nicotinic acetylcholine, GABAA, and glycine receptors (Karlin, 2002). Members of the Cys-loop ligand-gated ion channel superfamily are pentameric assemblies, with each subunit composed of four transmembrane domains, a large extracellular N terminus, a short extracellular C terminus, and loops connecting the transmembrane domains (TMs). It has been proposed that the agonist binding domains are formed by the extracellular N terminus at the interface between subunits, whereas the channel pore is lined by transmembrane domain 2 (Karlin, 2002). The activation of the Cys-loop ligand-gated ion channels involves the binding of the agonist to the receptor, the transduction of the binding signal to the channel gate, and the opening of the channel. The structural determinants in

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.
portions of loops 2, 7, and 9 and 8-9 linker of the extracellular N terminus (Absalom et al., 2003; Lee and Sine, 2005; Xiu et al., 2005; Sine and Engel, 2006; Gay and Yakel, 2007), preTM1 (Hu et al., 2003; Kash et al., 2003; Lee and Sine, 2005; Xiu et al., 2005; Keramidas et al., 2006; Mercado and Czajkowski, 2006), and transmembrane domain 2-3 loop (Grosman et al., 2000; Kash et al., 2003; Lummis et al., 2005; Xiu et al., 2005) have been implicated in relaying agonist binding to channel gating. It is interesting to note that those elements in the transduction pathway are also critical for allosteric modulation (Mihic et al., 1997; Krasowski et al., 1998b; Boileau and Czajkowski, 1999; Carlson et al., 2000; Chang et al., 2003; Hu and Lovinger, 2005; Jones-Davis et al., 2005). 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-HT3A subunit was first cloned in 1991 (Maricq et al., 1991). Since
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. 1011

Hu and Peoples

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-HT3A 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), GABAA (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-HT3A 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-HT3A receptor is a pivotal site for both coupling agonist binding to channel gating and mediating the action of trichloroethanol.

Materials and Methods

Mutagenesis. Point mutation of the mouse 5-HT3A receptor isoform 1 (NP_038589; a gift from Dr. D. Julius, University of California, San Francisco, CA) was accomplished using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The successful incorporation of mutations was verified by sequencing the clones using an ABI Prism 377 automated DNA sequencer (Applied Biosystems, Foster City, CA). The cDNAs were then subcloned into the vector pCDNA3.1 (Invitrogen, Carlsbad, CA) for expression in human embryonic kidney (HEK) 293 cells. 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 37C in 5% CO2. HEK 293 cells were transiently transfected with the wild-type or mutant 5-HT3A receptor cDNA using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturers instructions. Green fluorescent protein (pGreen Lantern; Invitrogen) was coexpressed with the 5-HT3A receptor subunits to permit selection of transfected cells under fluorescence optics. Each 35-mm dish was transfected with 3 g of cDNA encoding the wild-type or mutant receptors along with 1 g 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
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 M 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.

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 22C. 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 m. 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 m) of the
R246 in Alcohol Action at 5-HT3A Receptor
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 M 5-HT 1/[1 (EC50/[agonist])nh], where I is the current amplitude activated by a given concentration of agonist ([agonist]), I30 M 5-HT is the current amplitude of the cell in response to 30 M 5-HT, nh 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 Ane(t/n) As, where An is the relative amplitude of the respective component, As 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 (ann) was used, where an 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 M 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 Students t test or one-way analysis of variance. Differences were considered significant at p 0.05.

response curve to the left. The EC50 for the mutant receptor (55.8 10.3 M) was smaller than that for the wild-type receptor (140.0 4.5 M), 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 M 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 4fold 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

Results

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 M 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-

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 M 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.
components (fast component, 53.9 3.57.7 4.8%; slow component, 46.1 3.42.3 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 0.4 s; p 0.08). 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-HT3A receptor (Machu and Harris, 1994; Lovinger et al., 2000). Therefore, modulation of the 5-HT3A 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-HT3A 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. 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 M 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 M 5-HT. Pharmacological and Biophysical Properties of Trichloroethanol Currents. As shown in Fig. 5A, the current activated by trichloroethanol in the mutant receptor was completely blocked by 300 nM tropanyl 3,5-dichlorobenzoate (MDL 72222), a competitive 5-HT3 receptor antagonist (Fozard, 1984). Figure 5B shows a voltage ramp from 80 to 60 mV at the peak of current activated by 3 M 5-HT in the wild-type and mutant receptors and by 10 mM trichloroethanol in the mutant receptor. The shape of the current-voltage relationship was similar for both trichloroethanol and 5-HT; and the current reversed at 1.6 0.5 mV for trichloroethanol in the mutant receptor, at 1.8 0.4 mV for 5-HT in the mutant receptor, and at 1.5 0.4 mV for 5-HT in the wildtype receptor (analysis of variance; p 0.8), respectively.

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.
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 M 5-HT for each cell. Each data point represents mean S.E.M. from 8 to 25 cells.
Predesensitizing the R246A Receptor with Trichloroethanol Abolishes 5-HT Current. The response to 30 M 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 M 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 M 5-HT immediately after trichloroethanol preincubation failed to activate a detectable current in the mutant receptor (Fig. 6B). Kinetics of Trichloroethanol-Activated Current in the R246A Receptor. We also examined the kinetics of trichloroethanol-activated current in the mutant receptor. Figure 7A depicts representative traces of 10 mM trichloroethanol-activated current in the R246A receptor. The onset and offset of the current seem to be fast, and both could be fitted with a monoexponential function. Trichloroethanolactivated current decayed exponentially in the continuous presence of trichloroethanol after reaching peak. Desensitization time course was best fitted by a monoexponential function. The average time constants for activation, deactivation and desensitization are 140, 200, and 3400 ms, respectively (Fig. 7B).

Discussion

Dopamine can function as a partial agonist at the 5-HT3 receptor (van Hooft and Vijverberg, 1996; Hu and Lovinger, 2005). The R246A mutation dramatically increased the potency and relative efficacy of dopamine. This observation confirms the notion that the R246 residue plays an important role in channel gating of the 5-HT3A receptor (Hu et al., 2003). Trichloroethanol, an allosteric modulator at the 5-HT3 receptor (Lovinger and Zhou, 1993; Lovinger et al., 2000; Hu et al., 2006), barely activated the wild-type 5-HT3A receptor;
however, it was converted into an agonist by the R246A mutation. The direct activation of the R246A receptor by trichloroethanol provides further evidence to support a role of the R246 residue in channel gating. 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 2 GABAA 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-HT3A 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-HT3A 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-HT3A (Zhang et al., 2002) and 2(T262W) GABAA (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 agonistbound 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.,

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 M 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.
Fig. 6. Effect of preincubation with TcEt on 5-HT-activated current. A, traces show currents activated by 30 M 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 M 5-HT in the WT and R246A receptors. Each bar represents mean S.E.M. from 8 to 13 cells.
2000). Those results suggest that gating efficacy of the 5-HT3A 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-HT3A receptor by trichloroethanol and ethanol, suggesting that the R246 residue plays a role in the actions of alcohol at the 5-HT3A
receptor. It has been suggested that the S267 residue of the 1 glycine receptor subunit and S270 residue of the GABAA 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 GABAA receptor, of the 5-HT3A 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 GABAA 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 GABAA receptor (Scheller and Forman, 2002) and R246 and L293 residues in the 5-HT3A 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 GABAA 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 GABAA 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 GABAA receptor (Rusch et al., 2004). However, several studies dem onstrated that the positive modulation and direct activation of the GABAA receptor by propofol and pentobarbital are mediated by binding to different sites (Krasowski et al.,

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.
1998b; Dalziel et al., 1999; Chang et al., 2003). Our data have demonstrated that trichloroethanol has multiple actions on the function of the 5-HT3A receptor. The R246A mutation ablated the potentiation of 5-HT3A 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-HT3A 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.

References

Absalom NL, Lewis TM, Kaplan W, Pierce KD, and Schofield PR (2003) Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor. J Biol Chem 278:5015150157. Barlow RB (1976) The effects of pH on the activity of coryneine and related phenolic quaternary ammonium salts on the frog rectus preparation. Br J Pharmacol 57:517519. Barnes NM and Sharp T (1999) A review of central 5-HT receptors and their function. Neuropharmacology 38:10831152. Boileau AJ and Czajkowski C (1999) Identification of transduction elements for benzodiazepine modulation of the GABAA receptor: three residues are required for allosteric coupling. J Neurosci 19:1021310220. Carlson BX, Engblom AC, Kristiansen U, Schousboe A, and Olsen RW (2000) A single glycine residue at the entrance to the first membrane-spanning domain of the -aminobutyric acid type A receptor 2 subunit affects allosteric sensitivity to GABA and anesthetics. Mol Pharmacol 57:474 484. Chang CS, Olcese R, and Olsen RW (2003) A single M1 residue in the beta2 subunit alters channel gating of GABAA receptor in anesthetic modulation and direct activation. J Biol Chem 278:42821 42828. 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. Dalziel JE, Cox GB, Gage PW, and Birnir B (1999) Mutant human 11(T262Q) GABAA receptors are directly activated but not modulated by pentobarbital. Eur J Pharmacol 385:283286. 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 397:359 363. Dubin AE, Huvar R, DAndrea MR, Pyati J, Zhu JY, Joy KC, Wilson SJ, Galindo JE, Glass CA, Luo 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. Fozard JR (1984) MDL 72222: a potent and highly selective antagonist at neuronal 5-hydroxytryptamine receptors. Naunyn Schmiedebergs Arch Pharmacol 326:36 44. Gay EA and Yakel JL (2007) Gating of nicotinic ACh receptors; new insights into structural transitions triggered by agonist binding that induce channel opening. J Physiol 584:727733. Grosman C, Salamone FN, Sine SM, and Auerbach A (2000) The extracellular linker

of muscle acetylcholine receptor channels is a gating control element. J Gen Physiol 116:327340. Hapfelmeier G, Tredt C, Haseneder R, Zieglgansberger W, Eisensamer B, Rupprecht R, and Rammes G (2003) Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor. Biophys J 84:1720 1733. Hu XQ and Lovinger DM (2005) Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2. J Physiol 568:381396. Hu XQ, Hayrapetyan V, Gadhiya JJ, Rhubottom HE, Lovinger DM, and Machu TK (2006) Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions. Br J Pharmacol 148:88 101. Hu XQ, Zhang L, Stewart RR, and Weight FF (2003) Arginine 222 in the pretransmembrane domain 1 of 5-HT3A receptors links agonist binding to channel gating. J Biol Chem 278:46583 46589. Jones-Davis DM, Song L, Gallagher MJ, and Macdonald RL (2005) Structural determinants of benzodiazepine allosteric regulation of GABAA receptor currents. J Neurosci 25:8056 8065. Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3:102114. Kash TL, Dizon MJ, Trudell JR, and Harrison NL (2004) Charged residues in the beta2 subunit involved in GABAA receptor activation. J Biol Chem 279:4887 4893. Kash TL, Jenkins A, Kelley JC, Trudell JR, and Harrison NL (2003) Coupling of agonist binding to channel gating in the GABAA receptor. Nature 421:272275. Keramidas A, Kash TL, and Harrison NL (2006) The pre-M1 segment of the 1 subunit is a transduction element in the activation of the GABAA receptor. J Physiol 575:1122. Krasowski MD, Finn SE, Ye Q, and Harrison NL (1998a) Trichloroethanol modulation of recombinant GABAA, glycine and GABA 1 receptors. J Pharmacol Exp Ther 284:934 942. Krasowski MD, Koltchine VV, Rick CE, Ye Q, Finn SE, and Harrison NL (1998b) Propofol and other intravenous anesthetics have sites of action on the -aminobutyric acid type A receptor distinct from that for isoflurane. Mol Pharmacol 53:530 538. Lee WY and Sine SM (2005) Principal pathway coupling agonist binding to channel gating in nicotinic receptors. Nature 438:243247. Lovinger DM and Zhou Q (1993) Trichloroethanol potentiation of 5-hydroxytryptamine3 receptor-mediated ion current in nodose ganglion neurons from the adult rat. J Pharmacol Exp Ther 265:771776. Lovinger DM, Sung KW, and Zhou Q (2000) Ethanol and trichloroethanol alter gating of 5-HT3 receptor-channels in NCB-20 neuroblastoma cells. Neuropharmacology 39:561570. Lummis SC, Beene DL, Lee LW, Lester HA, Broadhurst RW, and Dougherty DA (2005) Cis-trans isomerization at a proline opens the pore of a neurotransmittergated ion channel. Nature 438:248 252. 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 5HT3 receptor, a serotonin-gated ion channel. Science 254:432 437. Mascia MP, Trudell JR, and Harris RA (2000) Specific binding sites for alcohols and anesthetics on ligand-gated ion channels. Proc Natl Acad Sci U S A 97:93059310. Mercado J and Czajkowski C (2006) Charged residues in the 1 and 2 pre-M1 regions involved in GABAA receptor activation. J Neurosci 26:20312040. 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 389:385389. Niesler B, Frank B, Kapeller J, and Rappold GA (2003) Cloning, physical mapping and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3C, HTR3D and HTR3E. Gene 310:101111. Rusch D, Zhong H, and Forman SA (2004) Gating allosterism at a single class of

Xiu X, Hanek AP, Wang J, Lester HA, and Dougherty DA (2005) A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors. J Biol Chem 280:41655 41666. 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.
etomidate sites on 122L GABAA receptors accounts for both direct activation and agonist modulation. J Biol Chem 279:2098220992. Scheller M and Forman SA (2002) Coupled and uncoupled gating and desensitization effects by pore domain mutations in GABAA receptors. J Neurosci 22:8411 8421. Sine SM and Engel AG (2006) Recent advances in Cys-loop receptor structure and function. Nature 440:448 455. Ueno S, Lin A, Nikolaeva N, Trudell JR, Mihic SJ, Harris RA, and Harrison NL (2000) Tryptophan scanning mutagenesis in TM2 of the GABAA receptor alpha subunit: effects on channel gating and regulation by ethanol. Br J Pharmacol 131:296 302. van Hooft JA and Vijverberg HP (1996) Selection of distinct conformational states of the 5-HT3 receptor by full and partial agonists. Br J Pharmacol 117:839 846.
Address correspondence to: Dr. Robert W. Peoples, Department of Biomedical Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 532011881. E-mail: robert.peoples@marquette.edu