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Originally published In Press as doi:10.1074/jbc.M308974200 on September 11, 2003

J. Biol. Chem., Vol. 278, Issue 47, 46583-46589, November 21, 2003
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Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating*

Xiang-Qun Hu{ddagger}, Li Zhang, Randall R. Stewart, and Forrest F. Weight

From the Laboratory of Molecular and Cellular Neurobiology, NIAAA, National Institutes of Health, Bethesda, Maryland 20892-8115

Received for publication, August 13, 2003 , and in revised form, September 8, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT3A receptors and {alpha}7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wild-type and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT3 receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT3A receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the nervous system, serotonin type 3 (5-HT3)1 receptors can mediate fast excitatory synaptic transmission and modulate neurotransmitter release (1). To date two 5-HT3 receptor subunits have been identified: 5-HT3A and 5-HT3B (2, 3). The 5-HT3A receptor subunits can form functional channels homomerically (2), whereas the 5-HT3B receptor subunits are nonfunctional when expressed alone (3). However, the 5-HT3B receptor subunits can form heteromeric channels with the 5-HT3A receptor subunits, which results in modified biophysical characteristics compared with homomerically expressed 5-HT3A receptor subunits (3). 5-HT3A and 5-HT3B receptor subunits also have different distribution patterns in the nervous system. The 5-HT3A receptor subunits are expressed in both central and peripheral neurons, whereas the 5-HT3B receptor subunits are restricted to peripheral neurons (4). This suggests that homomeric 5-HT3A receptors play a dominant role in 5-HT3 receptor-mediated responses in the central nervous system.

5-HT3 receptors belong to a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine (nACh) receptors, glycine receptors, and {gamma}-aminobutyric acid type A receptors (5). The subunits in this superfamily are thought to assemble as pentamers with each subunit containing a large extracellular N-terminal domain, four transmembrane domains (TM1-TM4), a large intracellular loop between TM3 and TM4, and an extracellular C-terminal domain (Fig. 1A) (6). The agonist binding sites are thought to be located in the N-terminal domain at subunit-subunit interfaces, and the lining of the ion channel is believed to be formed by the second transmembrane (TM2) domain (6). The binding of agonist to the binding sites in the N-terminal domain presumably results in a conformational change of the channel protein, which is then transduced to the TM2 domain to result in channel opening.



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  		FIG. 1.
Putative 5-HT3A receptor topology and partial sequence alignment of 5-HT3A receptors and {alpha}7-nACh receptors. A, putative 5-HT3A receptor topology with four transmembrane domains (TM1-TM4), a large extracellular N-terminal domain, a large intracellular loop between TM3 and TM4, and a short extracellular C-terminal domain. C-C denotes the Cys loop. B, partial amino acid sequence alignment of the pre-TM1/TM1 of the cloned 5-HT3A receptors and {alpha}7-nACh receptors. Numbering corresponds to the mouse 5-HT3A receptor sequence. Note that the mouse 5-HT3A receptor arginine (R) 222, labeled with an asterisk, is conserved in all the 5-HT3A and {alpha}7-nACh receptors.

 
A number of studies have been carried out to determine the sequence elements involved in agonist binding and channel gating of 5-HT3A receptors (7–13). However, the mechanisms that transduce the binding of agonist to the opening of the channel are still poorly understood. Understanding the function of ligand-gated ion channels at the molecular level requires understanding how agonist binding to the receptor is converted to channel opening. Arginine (Arg) 222 is of particular interest because it is located at the distal end of the extracellular N-terminal domain, immediately adjacent to the first transmembrane (TM1) domain (Fig. 1A), and thus, it is between the presumed agonist binding sites in the N-terminal domain and TM2. Sequence alignments reveal that Arg-222 of the mouse 5-HT3A receptor is conserved in all of 5-HT3A receptors and {alpha}7-nACh receptors that have been cloned from various species (Fig. 1B). Moreover, some mutations at Arg-222 can alter the sensitivity of 5-HT3A receptors to agonists and produce channels that open spontaneously (14). In an attempt to understand the role of Arg-222 in the function of 5-HT3A receptors, we replaced the arginine residue with an alanine using site-directed mutagenesis. The wild-type and R222A 5-HT3A receptors were transiently expressed in human embryonic kidney (HEK) 293 cells, and the functional properties of the receptors were studied using the whole-cell patch clamp recording technique in combination with fast solution exchange. The results suggest that Arg-222 is involved in transducing the signal that couples agonist binding to channel opening in 5-HT3A receptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mutagenesis—Point mutation of the mouse 5-HT3A receptor was accomplished using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutation was verified by double strand DNA sequencing using an ABI Prism 377 automatic DNA sequencer (Applied Biosystems, Foster City, CA). The cDNAs were then subcloned into the vector pcDNA3.1 (Invitrogen) for expression in 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 maintained in a humidified incubator at 37 °C in 5% CO2. The HEK 293 cells were transiently transfected with the wild-type or R222A 5-HT3A receptor cDNA using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's instructions. Green fluorescent protein (pGreen Lantern, Invitrogen) was co-expressed with the 5-HT3A receptor subunits to permit selection of transfected cells under fluorescence optics.

Patch Clamp Recording—HEK 293 cells were recorded 1–3 days after transfection. Cells were continuously superfused with a solution containing 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 5mM glucose, and 10 mM HEPES (pH 7.4 with NaOH; ~340 mosmol with sucrose). Membrane current was recorded in the whole-cell configuration (15) using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) at 20–22 °C. Pipettes were pulled from borosilicate glass (TW-150F, World Precision Instruments, Sarasota, FL) using a two-stage puller (Flaming-Brown P-87; Sutter Instruments, Novato, CA) and had resistances of ~5 megaohms when filled with pipette solution containing 140 mM CsCl, 2 mM MgCl2, 10mM EGTA, 10 mM HEPES (pH 7.2 with CsOH; ~315 mosmol with sucrose). Cells were held at –60 mV unless otherwise indicated. Data were acquired using pClamp8.0 software (Axon). Currents were filtered at 2 kHz and digitized at 2–10 kHz. Agonists were applied with a piezoelectric device (PZ-150M; EXFO Burleigh Products Group Inc., Victor, NY) through two-barrel {theta} glass tubing (TGC150, Warner Instruments, Hamden, CT) that had been pulled to a tip diameter of ~200 µm. The piezoelectric device was driven by transistor-transistor logic pulses from pClamp 8.0 software. Voltage applied to the piezoelectric device produced a rapid lateral displacement of the {theta} tubing to move the interface between control and agonist solutions. Solution exchange rate was estimated using the potential change induced by switching from the control solution to a 140 mM N-methyl-D-glucamine test solution. The solution exchange time constants were ~0.3 ms for an open pipette tip and ~1.6 ms for whole-cell recording.

Data Analysis—Average data are presented as means ± S.E. unless noted otherwise. Data analysis and curve fitting were performed with Origin 6.0 (Microcal Software, Northampton, MA), pClamp 8.0 (Axon), Statistica 5.5 (StatSoft Inc., Tulsa, OK), or GraphPad InStat 3.0 (GraphPad Software Inc., San Diego, CA) software. Concentration-response curves were fitted by the Hill equation, I/IMAX = 1/[1 + (EC50/[agonist])n], where I is the current amplitude activated by a given concentration of agonist ([agonist]), IMAX is the maximum response of the cell, n is the Hill slope, and EC50 is the concentration eliciting a half-maximal response. The time constants for activation, deactivation, and desensitization were determined with Levenberg-Marquardt algorithms.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Functional Characterization of the Wild-type and Mutant (R222A) 5-HT3A Receptors—The wild-type and R222A 5-HT3A receptors were transiently expressed in HEK 293 cells, and their responses to the full agonist, 5-HT, or the partial agonist, 2-Me-5-HT, were recorded in the whole-cell configuration with fast solution exchange. Typical responses activated by these agonists are shown in Fig. 2, A and B. At a holding potential of –60 mV, application of a maximally efficacious concentration of the full agonist, 5-HT (30 µM), induced a rapidly activating inward current in both the wild-type (Fig. 2A, left) and R222A (Fig. 2B, left) receptors. Application of a maximally efficacious concentration of the partial agonist, 2-Me-5-HT (100 µM), activated the wild-type receptor more slowly compared with the response activated by 30 µM 5-HT (Fig. 2A, right). By contrast, the R222A mutation resulted in a more rapidly activating response to 2-Me-5-HT (Fig. 2B, right). In addition, in the wild-type receptor the current activated by 100 µM 2-Me-5-HT was much smaller in amplitude than the current activated by 30 µM 5-HT (Fig. 2A), whereas in the R222A mutant receptor the current activated by 100 µM 2-Me-5-HT was similar in amplitude to that activated by 30 µM 5-HT (Fig. 2B). Concentration-response curves for agonists were constructed by normalizing the amplitude of current activated by a range of 5-HT or 2-Me-5-HT concentrations (I/IMAX for 5-HT and I/I30 µM 5-HT for 2-Me-5-HT), as shown in Fig. 2, C and D. The amplitude of currents activated by 5-HT or 2-Me-5-HT was concentration-dependent for both wild-type and R222A receptors. In addition, the concentration-response curves for both 5-HT and 2-Me-5-HT were shifted to the left by the R222A mutation (Fig. 2, C and D). In the R222A receptor the potency for 5-HT and 2-Me-5-HT was increased 5-fold and 12-fold, respectively (5-HT, EC50 = 0.5 ± 0.1 µM; 2-Me-5-HT, EC50 = 1.2 ± 0.2 µM), as compared with the wild-type receptor (5-HT, EC50 = 2.7 ± 0.4 µM; 2-Me-5-HT, EC50 = 14.4 ± 1.4 µM). The Hill slope of the concentration-response curve for 5-HT was not significantly changed by the R222A mutation (wild-type, 1.48 ± 0.08; R222A, 1.44 ± 0.06; p > 0.5), whereas the R222A mutation decreased the Hill slope for 2-Me-5-HT (wild-type, 2.48 ± 0.10; R222A, 1.20 ± 0.20; p < 0.05). In addition, the maximal response to 2-Me-5-HT was greatly increased by the R222A mutation, from 57.8 ± 2.3 to 103.3 ± 5.6% of the current activated by 30 µM 5-HT (p < 0.01).



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  		FIG. 2.
The R222A mutation increases the potency of agonists and converts the partial agonist, 2-Me-5-HT, into a full agonist. A and B, traces show current activated by 5-HT and 2-Me-5-HT in HEK 293 cells expressing wild-type (A) and R222A (B) receptors. C, concentration-response curves for 5-HT from WT ({circ}) and R222A (•) receptors. I and IMAX are the current at a given agonist concentration and the maximal current, respectively. The average IMAX values for 5-HT were 937 ± 114 pA for the WT receptors and 522 ± 44 pA for the R222A receptors. D, concentration-response curves for 2-Me-5-HT from WT ({circ}) and R222A (•) receptors. I and I30 µM 5-HT are the current at a given agonist concentration and the current activated by 30 MM 5-HT, respectively. The average IMAX values for 2-Me-5-HT were 544 ± 38 pA for the WT receptors and 560 ± 54 pA for the R222A receptors. The difference in the maximal currents evoked by 5-HT may reflect the expression efficiency of R222A receptors. Each data point represents mean ± S.E. from 6–9 cells.

 
Voltage Independence—To address whether the R222A mutation altered ion permeation, current-voltage (I-V) relationships were obtained by measuring the amplitude of current activated by either 30 µM 5-HT or 100 µM 2-Me-5-HT at holding potentials from –80 to +60 mV (Fig. 3, A and B). The current evoked by both 5-HT and 2-Me-5-HT reversed at ~5 mV, and the current-voltage relationship exhibited slight inward rectification for both agonists (Fig. 3, C and D). The R222A mutation had no significant effect on either the reversal potential for both 5-HT (wild-type, 4.90 ± 0.57; R222A, 4.49 ± 0.68; p > 0.5) and 2-Me-5-HT (wild type, 5.25 ± 0.0.62; R222A, 5.19 ± 0.0.67; p > 0.5) or the shape of the I-V relationship (Fig. 3, C and D).



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  		FIG. 3.
The R222A mutation does not alter the current-voltage (I-V) relationship for 5-HT or 2-Me-5-HT. A and B, traces show current activated by 30 µM 5-HT (A) or 100 µM 2-Me-5-HT (B) at various holding potentials in cells expressing WT or R222A receptors. C and D, current-voltage relationships for 5-HT-(C) and 2-Me-5-HT-(D) activated current from WT ({circ}) and R222A (•) receptors. Each data point represents the mean ± S.E. from six cells.

 
Antagonist Profile—Because some Arg-222 mutants exhibited spontaneous channel opening when expressed in Xenopus oocytes (14), we examined whether the R222A mutation produced such activity in HEK 293 cells. Spontaneous opening of ligand-gated channels can be blocked with antagonists (14, 16). As shown in Fig. 4A, left, in cells expressing the wild-type receptors the application of 300 nM MDL 72222, a competitive 5-HT3 receptor antagonist, for 10 s in the absence of 5-HT did not alter the holding current. Similarly, in cells expressing the R222A receptors the application of 300 nM MDL 72222 for 10 s did not alter the holding current (Fig. 4A, right). We also tested the effectiveness of MDL 72222 in antagonizing 5-HT-activated currents. MDL 72222 at 300 nM completely blocked the current activated by 30 µM 5-HT in both the wild-type and the R222A receptors (Fig. 4B). Blockade of 5-HT-activated responses by MDL 72222 was readily reversed by washing (data not shown). Apomorphine has been reported to be an antagonist/very weak partial agonist in the 5-HT3 receptors (17). Fig. 4C, left, shows that in the wild-type receptors 30 µM apomorphine activated a very small current when compared with the current activated by 30 µM 5-HT. In addition, when co-applied with 5-HT in the wild-type receptors, 30 µM apomorphine inhibited the current activated by 30 µM 5-HT. On the other hand, 30 µM apomorphine activated a significant inward current in the R222A receptors (Fig. 4C, right). The bar graphs in Fig. 4D show the average action of apomorphine in both the wild-type and the R222A receptors. Apomorphine inhibited 5-HT-activated current by 82.1 ± 3.1% in the wild-type receptors (p < 0.01). In the wild-type receptors the average amplitude of the current activated by 30 µM apomorphine was less than 1% that activated by 30 µM 5-HT; however, in the R222A receptors the average amplitude of the current activated by 30 µM apomorphine was ~ 60% of the amplitude of the current activated by 30 µM 5-HT (wild type, 0.88 ± 0.35%; R222A, 60.8 ± 4.3%, p < 0.01).



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  		FIG. 4.
The R222A mutation does not alter the antagonist profile of MDL 72222, but it converts the antagonist/very weak partial agonist, apomorphine, to a potent agonist. A, traces show that the 5-HT3 antagonist, MDL 72222 (300 nM), does not alter the holding current in WT (left) or R222A (right) receptors. The bar indicates the time of MDL 72222 application. Similar responses were observed in at least 5–7 cells. B, traces show current activated by 30 µM 5-HT in the absence ({circ}) and presence of 300 nM MDL 72222 (•). MDL 72222 was pre-applied for 1.5 min before co-application with 5-HT. The bar indicates the time of 5-HT application. Similar responses were observed in at least 5–8 cells. C, traces show the current activated by 30 µM 5-HT or 30 µM apomorphine in WT (left) and R222A (right) receptors and inhibition of the current activated by 30 µM 5-HT by 30 µM apomorphine in WT receptors (left). The traces in the inset show the current activated by 30 µM apomorphine in WT receptors on an expanded amplitude scale. D, average action of apomorphine in WT (left) and R222A (right) receptors. Each bar represents the mean ± S.E. from 5–8 cells. **, p < 0.01.

 
Activation Kinetics—To evaluate the kinetics of 5-HT3A receptor activation, 5-HT or 2-Me-5-HT was applied at different concentrations for a sufficient duration to allow the receptor-agonist interaction to reach equilibrium. Fig. 5, A and B, shows normalized responses to the application of 2.7 and 30 µM 5-HT (Fig. 5A) and 14.4 and 100 µM 2-Me-5-HT (Fig. 5B), respectively, from cells expressing the wild-type or the R222A receptors. The records show that high agonist concentrations elicited a faster activation than low agonist concentrations for both the wild-type and the R222A receptors. In addition, the rate of receptor activation was agonist-dependent for both the wild-type and the R222A receptors; fast for 5-HT and slower for 2-Me-5-HT. The R222A mutation significantly increased the rate of receptor activation for both 5-HT and 2-Me-5-HT. The activation of 5-HT3A receptors could be fitted with a single exponential function. The time constants for 2.7 and 30 µM 5-HT were 124.0 ± 7.4 and 16.2 ± 0.65 ms, respectively, in the wild-type receptors and 19.3 ± 1.4 and 8.7 ± 0.5 ms, respectively, in the R222A receptors. The time constants for 14.4 and 100 µM 2-Me-5-HT were 753.1 ± 43.4 and 288.7 ± 12.4 ms, respectively, in the wild-type receptors and 19.6 ± 1.4 and 13.0 ± 1.0 ms, respectively, in the R222A receptors. Fig. 5, C and D, plots the activation rate (inverse of the activation time constant, {tau}activation) as a function of 5-HT (Fig. 5C) and 2-Me-5-HT (Fig. 5D) concentration for the wild-type and the R222A receptors. Although the concentration at which the activation rate was half-maximal was 3.7 µM 5-HT for the R222A receptor, 25 µM 5-HT was required to achieve the same activation rate for the wild-type receptor. Similarly, 23 µM 2-Me-5-HT produced the half-maximal activation rate in the R222A receptor, but extrapolation suggests that 1800 µM 2-Me-5-HT would be needed to produce the same activation rate for the wild-type receptor.



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  		FIG. 5.
The R222A mutation accelerates the activation rate of 5-HT3A receptors in response to 5-HT and 2-Me-5-HT. A and B, traces show receptor activation elicited by 2.7 µM 5-HT (EC50 at WT) and 30 µM 5-HT (A) and 14.4 µM 2-Me-5-HT (EC50 at WT) and 100 µM 2-Me-5-HT (B) in WT and R222A receptors. Agonist-activated responses were normalized to peak current for comparison. The bar indicates the time of agonist application. C and D, plots of the activation rate (inverse of {tau}activation) versus 5-HT concentration (C) and 2-Me-5-HT concentration (D) in WT ({circ}) and R222A (•) receptors. Each data point represents mean ± S.E. from 10–24 cells.

 
Gating—Activation of ligand-gated ion channels involves agonist binding and conformational changes that lead to gating. Previous studies on ligand-gated ion channels suggest that at low agonist concentrations, agonist binding is the rate-limiting step; however, when the agonist concentration is high, gating becomes the rate-limiting step, and the activation rate approximates the channel opening rate (18). In view of this, we examined the activation of both the wild-type and the R222A receptors with saturating concentrations of 5-HT. The increase in activation rate with increasing agonist concentrations appeared to reach a plateau at ~300 µM 5-HT for both the wild-type (Fig. 6A) and R222A (Fig. 6B) receptors, since the activation rate did not appear to be accelerated by increasing 5-HT concentration above 300 µM (up to 3 mM). On the other hand, the activation rate was faster in R222A receptors than in wild-type receptors (Fig. 6C). In the wild-type receptors, for 5-HT concentrations >=300 µM, average activation rates (Fig. 6D) were not significantly different (analysis of variance, p > 0.5). Similarly, in the R222A receptors, average activation rates (Fig. 6D) were not significantly different for 5-HT concentration >=300 µM (analysis of variance, p > 0.3). However, the R222A mutation significantly accelerated the activation rate for 300 µM (p < 0.01), 1 mM (p < 0.001), and 3 mM 5-HT (p < 0.001) compared with the wild-type receptors (Fig. 6D).



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  		FIG. 6.
The R222A mutation accelerates opening rate of 5-HT3A receptors. A and B, normalized and superimposed receptor activation in response to supermaximal concentrations of 5-HT (100, 300, 1000, and 3000 µM) in WT (A) and R222A (B) receptors. Activation rate appears similar when 5-HT concentration is greater than 300 µM in both WT and R222A receptors. The bar indicates the time of 5-HT application. C, superimposed traces from A and B show the difference of activation rate for WT and R222A receptors in response to 1000 µM 5-HT, with a faster time scale. D, average activation rate at supermaximal concentrations of 5-HT in WT ({square}) and R222A ({blacksquare}) receptors. Each bar represents mean ± S.E. from 7–8 cells. **, p < 0.01.

 
Deactivation Kinetics—Deactivation is the process of agonist unbinding and channel closing, and it is generally considered as an index of agonist affinity (19, 20). To determine whether the R222A mutation alters the rate of deactivation, we examined the current deactivation kinetics after rapid removal of the agonist. The records in Fig. 7, A and B, illustrate the current deactivation after rapid removal of 1 mM 5-HT (Fig. 7A) and 100 µM 2-Me-5-HT (Fig. 7B) in cells expressing the wild-type and R222A 5-HT3A receptors. 5-HT was removed after a 2-ms application, whereas 2-Me-5-HT was removed before desensitization was observed. When the agonist was rapidly washed-out, the currents decayed back to base line as a mono-exponential function for both the wild-type and the R222A receptors. The R222A mutation did not alter the deactivation rate (inverse of deactivation time constant, {tau}deactivation) for either 5-HT (wild type, 0.34 ± 0.03 s–1; R222A, 0.34 ± 0.03 s–1; p > 0.5) or 2-Me-5-HT (wild type, 0.57 ± 0.02 s–1; R222A, 0.58 ± 0.02 s–1; p > 0.5). However, the deactivation rate was agonist-dependent, and on average, 2-Me-5-HT had a significantly faster deactivation rate than 5-HT (p < 0.01) for both the wild-type and the R222A receptors, Fig. 7C).



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  		FIG. 7.
The deactivation rate of 5-HT3A receptors is not altered by the R222A mutation. A and B, traces show deactivation after termination of the application of 1 mM 5-HT (A) or 100 µM 2-Me-5-HT (B) from cells expressing WT (left) or R222A (right) receptors. The agonist-activated responses are normalized to peak current for comparison. The arrow indicates the start of agonist washout. 5-HT was applied for 2 ms and was then quickly removed, whereas 2-Me-5-HT washout was started before desensitization (current decay) was observed. C, average deactivation kinetics for 5-HT (left) and 2-Me-5-HT (right) from the WT ({square}) and R222A ({blacksquare}) receptors. Each bar represents mean ± S.E. from 13–24 cells.

 
Desensitization Kinetics—In the continued presence of agonist, the activated current exhibited a marked decrease in amplitude after reaching a peak, indicating receptor desensitization. Examples of the desensitization resulting from the application of 30 µM 5-HT and 100 µM 2-Me-5-HT are shown in Fig. 8, A and B, respectively. For the wild-type 5-HT3A receptors, the desensitization of the current was relatively slow and well described by a mono-exponential function. In addition, the desensitization time constant for 2-Me-5-HT in the wild-type receptors was significantly slower than for 5-HT (p < 0.04). For the R222A receptors, a bi-exponential function was required to adequately fit the desensitization of the current activated by 5-HT or 2-Me-5-HT. Both the fast and slow desensitization components of the R222A receptors were similar for 5-HT and 2-Me-5-HT (p > 0.50). The fast component contributed ~60% of the total current decay for both agonists (Fig. 8, C and D). The slow component of the decay in the R222A receptor for either 5-HT or 2-Me-5-HT was faster than the current decay in the wild-type receptor (p < 0.01).



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  		FIG. 8.
The R222A mutation accelerates the desensitization rate of 5-HT3A receptors. A and B, traces show desensitization of WT and R222A receptors to a 10-s application of 30 µM 5-HT (A) or 100 µM 2-Me-5-HT (B). The agonist-activated currents are normalized to peak current and superimposed for comparison. The bar indicates the time of agonist application. The desensitization is best fit with one exponential function in the WT receptors, whereas two exponential functions best fit the decay of current in the R222A receptors. C and D, average desensitization kinetics for 5-HT (C) and 2-Me-5-HT (D) in WT and R222A receptors. Each bar represents the mean ± S.E. from 8–10 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we investigated the role of amino acid residue Arg-222 in the function of 5-HT3A receptors. We found that the R222A mutation increased the apparent potency of both the agonist, 5-HT, and the partial agonist, 2-Me-5-HT. The R222A mutation also increased the efficacy of the partial agonist, 2-Me-5-HT, and converted the antagonist/very weak partial agonist, apomorphine, to a potent agonist. In addition, the R222A mutation accelerated the rate of 5-HT3A receptor activation and desensitization, but it did not alter the rate of 5-HT3A receptor deactivation.

Ligand-gated ion channels exist in at least three intercon-vertible states (resting, open, and desensitized), and the function of these channels is thought to be determined by the transitions among these states (6). Activation of ligand-gated ion channels involves the binding of agonist to the receptor, which results in a conformational change of the protein that opens the channel. In the present study, how the R222A mutation affects 5-HT3A receptor activation was examined.

Because deactivation is defined as the process of the unbinding of agonist from the open state of the receptor and the channel returning to the closed resting state, it is expected that receptors with a higher affinity for agonist in the open state would decrease the probability of agonist unbinding from the receptor. Thus, agonist affinity could be assessed from agonist unbinding rate (19, 20). If the R222A mutation increases agonist affinity, the deactivation rate of the receptor would be expected to be slower. We found, however, that the R222A mutation did not change the rate of deactivation, because currents for both wild-type and R222A receptors decayed with similar kinetics upon removal of agonist. The observation that the mutation did not alter the deactivation rate for either 5-HT or 2-Me-5-HT is, thus, inconsistent with an increase in agonist affinity for R222A receptors. This is consistent with the observation in a single-oocyte binding assay (14). However, we cannot exclude the possibility from this study that Arg-222 is part of the agonist binding pocket.

2-Me-5-HT is a partial agonist at mouse wild-type 5-HT3A receptors (21). Because the relative efficacy of an agonist is dependent in part upon the gating process (22), the conversion of the partial agonist, 2-Me-5-HT, into a full agonist suggests the possibility of an enhanced gating efficacy in the R222A receptors. The increase in efficacy by R222A mutation is not unique to 2-Me-5-HT, because this mutation also converted the extremely weak 5-HT3 receptor partial agonist, apomorphine, to a potent agonist. In addition, the R222A mutation was found to enhance the rate of 5-HT3A receptor activation at saturating concentrations of 5-HT, which is also consistent with a facilitation of the gating process. For the ligand-gated ion channels, it has been found that a mutation that facilitates gating may only cause a shift to the left of the agonist concentration-response curve for a full agonist (22). The observations that the R222A mutation accelerated activation of 5-HT3A receptors by 5-HT and increased the apparent potency of 5-HT suggest that the efficacy of 5-HT to gate the channel is also enhanced by the mutation. Alterations in apparent potency and efficacy of agonists by mutations have been observed for other ligand-gated ion channels. For example, point mutations in {alpha}7-nACh receptors at Leu-247 (23) and {alpha}-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors at Ala-636 (24) converted an antagonist into an agonist and increased the apparent potency of agonists. On the other hand mutations in the NR1 subunit of N-methyl-D-aspartate receptors at Asp-732 (25) and the {alpha}2 subunit of nACh receptors at Asp-200 (26, 27) decreased apparent agonist potency and converted partial agonists into competitive antagonists.

The distance between the agonist binding sites and the channel pore of the nACh receptor is estimated to be ~30 Å (28). The agonist binding sites of the 5-HT3A receptor are thought to be comparable with those of the nACh receptor (29). Because the agonist binding sites are at a distance from the channel gating mechanism, the binding signal must involve a transduction mechanism to open the channel. The binding of agonist to the receptor is thought to provide the driving force to open the channel (30, 31). It is proposed for nACh receptors that the binding of agonist triggers a localized disturbance at the binding sites that transmits to transmembrane domains of the receptor through a small rotation of the N-terminal domain (32). Our observations on the role of Arg-222 in the gating of 5-HT3A receptors suggest that this amino acid residue may be involved in the transduction of the signal from agonist binding to channel gating.

The pre-TM1 region and TM2-TM3 loop of several ligandgated ion channels have been found to be critical in the coupling of agonist binding to channel gating. Certain residues of the TM2-TM3 loop in nACh receptors (33, 34), glycine receptors (35), and {gamma}-aminobutyric acid type A receptors (36) have been found to participate in the transduction of agonist binding to channel gating. For N-methyl-D-aspartate receptors, the pre-TM1 segment was found to link agonist binding to channel gating and to affect entry into open and desensitized states (37). In addition, a recent study suggests that an interaction of residues in TM1 of nACh receptors, which are near Arg-222 in 5-HT3A receptors, and TM2 contributes to the gating process (38). Our data suggest that Arg-222, located in pre-TM1 of 5-HT3A receptors, may also play a crucial role in the transduction mechanism coupling agonist binding to channel opening and entry into the desensitized state. In this context, Arg-222 may serve as a constraint to gating of the 5-HT3A receptors by maintaining the wild-type receptors in the resting closed state. The R222A mutation may release this constraint and reduce the energy barrier for channel opening, increasing apparent agonist potency and partial agonist efficacy as a result of enhanced coupling between agonist binding and gating.

In the present study, it appears that the R222A mutation reduced the energy barrier for agonist to open the channel. In this regard, it should be noted that arginine and alanine residues differ in their size, polarity, and hydrophobicity. Facilitated coupling resulting from the R222A mutation might be due to the decreased side-chain size, the increased hydrophobicity, or the reduced charge. Any of these changes may reduce the energy barrier for channel opening and, thus, facilitate the transition from a closed to an open state. Mutations at Val-385 of the {alpha} subunit of muscle type nACh receptors revealed that both volume and stereochemistry contribute to channel gating but not to binding affinity (39). The observation that some 5-HT3A receptor mutants, such as R222F and R222I, exhibits spontaneous channel opening (14) is consistent with the notion that Arg-222 mutations can reduce the energy barrier for channel opening.

It has been proposed that the closed resting state is the most stable conformation in the absence of agonist, whereas the closed desensitized state is the most stable conformation in the presence of agonist (6). Desensitization is a widespread phenomenon among ligand-gated ion channels (40). Our observation that the R222A receptors exhibited a significantly faster desensitization than the wild-type receptors suggests that although the R222A mutation facilitated gating of the receptor, it also increased the proportion of receptors entering into the desensitized state from the open state. It has been proposed for the nACh receptor that gating and desensitization are energetically coupled events (41). Our observation that an increase in gating efficacy is associated with an enhanced desensitization of 5-HT3A receptors is consistent with such a notion and suggests that desensitization of 5-HT3A receptors is positively coupled to gating.

In summary, we have found that the R222A mutation can enhance the apparent potency of agonists and the efficacy of partial agonists in 5-HT3A receptors. Kinetic analysis indicates that these alterations are associated with faster activation and desensitization of this receptor channel. The unique position of Arg-222 may allow it to couple the binding of agonist to the opening of the channel, since the R222A mutation appears to reduce the energy barrier for gating. A reduced energy barrier may speed up activation, which in turn may increase the apparent potency of agonists and the efficacy of partial agonists. Thus, our data suggest that Arg-222 has a functional role in the signal transduction mechanism of 5-HT3A receptors.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Laboratory of Molecular and Cellular Neurobiology, NIAAA, National Institutes of Health, Park Bldg., Rm. 150, Bethesda, MD 20892-8115. Tel.: 301-443-8163; Fax: 301-480-6882; E-mail: xhu{at}mail.nih.gov.

1 The abbreviations used are: 5-HT3, serotonin type 3; nACh, acetylcholine; TM, transmembrane domain; 2-Me-5-HT, 2-methyl-5-hydroxytryptamine; HEK cells, human embryonic kidney cells; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Amir Ghazanfari and Julia Healey for technical assistance, Dr. David Julius for providing mouse 5-HT3A receptor cDNA, and Drs. David Lovinger and Robert Peoples for reviewing the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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