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NEUROPHARMACOLOGY

Molecular Determinants of Picrotoxin Inhibition of 5-Hydroxytryptamine Type 3 Receptors

Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas

Received November 6, 2004; accepted March 25, 2005.

	Abstract

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Previously, we reported that the GABA_A receptor antagonist picrotoxin also antagonizes serotonin (5-HT)₃ receptors and that its effects are subunit-dependent. Here, we sought to identify amino acids involved in picrotoxin inhibition of 5-HT₃ receptors. Mutation of serine to alanine at the transmembrane domain 2 (TM2) 2' position did not affect picrotoxin (PTX) sensitivity in murine 5-HT_3A receptors. However, mutation of the 6' TM2 threonine to phenylalanine dramatically reduced PTX sensitivity. Mutation of 6' asparagine to threonine in the 5-HT_3B subunit enhanced PTX sensitivity in heteromeric 5-HT_3A/3B receptors. Introduction of serine (native to the human 3B subunit) at the 6' position also increased PTX sensitivity, suggesting a species-specific effect. Mutation of the 7' leucine to threonine in 5-HT_3A receptors increased PTX sensitivity roughly 10-fold, comparable with that observed in GABA_A receptors, and also conferred distinct gating kinetics. The equivalent mutation in the 3B subunit (i.e., 7' valine to threonine) had no impact on PTX sensitivity in 5-HT_3A/3B receptors. Interestingly, [³H]ethynylbicycloorthobenzoate ([³H]EBOB), a high-affinity ligand to the convulsant site in GABA_A receptors, did not exhibit specific binding in 5-HT_3A receptors. The structurally related compound, tert-butylbicyclophosphorothionate (TBPS), which potently inhibits GABA_A receptors, did not inhibit 5-HT₃ currents. Our results indicate that the TM2 6' residue is a common determinant of PTX inhibition of both 5-HT₃ and GABA_A receptors and demonstrate a role of the 7' residue in PTX inhibition. However, lack of effects of EBOB and TBPS in 5-HT_3A receptors suggests that the functional domains in the two receptors are not equivalent and underscores the complexity of PTX modulation of LGICs.

The 5-HT_3A subunit was identified initially and shown to form functional homomeric receptors (Maricq et al., 1991). A second subunit (5-HT_3B) was subsequently cloned and characterized (Davies et al., 1999). The 5-HT_3B subunit is incapable of forming functional homomeric cell surface receptors because it is retained in the endoplasmic reticulum in the absence of the 3A subunit (Boyd et al., 2003). However, coexpression of 5-HT_3B with the 5-HT_3A subunit results in a heteromeric receptor with distinct biophysical properties (Davies et al., 1999). Recently, three additional putative 5-HT₃ subunits, 5-HT_{3C, 3D}, and _3E have been cloned (Niesler et al., 2003). The pharmacological and functional properties of these novel 5-HT₃-like subunits are currently unknown.

Like other members of this superfamily, the 5-HT₃ receptor is pentameric in nature, with the five subunits surrounding a central ion channel (Boess et al., 1995). The receptor shares structural features common to LGICs, such as a large extracellular N-terminal region, four transmembrane (TM) domains (TM1–TM4), and an intracellular loop between TM3 and TM4. Evidence suggests that the agonist binding site is located in the N-terminal region, whereas TM2 forms the pore (Akabas et al., 1994; Ortells and Lunt, 1995; Brejc et al., 2001).

The plant-derived alkaloid picrotoxin was originally shown to be a noncompetitive antagonist of GABA_A receptors (Takeuchi and Takeuchi, 1969). It was subsequently demonstrated that picrotoxin inhibits other anion-selective ligand-gated anion channels, including glycine and GABA_C receptors (Pribilla et al., 1992; Wang et al., 1995) and glutamate-gated Cl^- channels (Etter et al., 1999). Recently, we have shown that PTX also inhibits the cation-selective 5-HT_3A receptors in a noncompetitive, use-facilitated manner (Das et al., 2003). Additionally, the interaction of PTX with the 5-HT₃ receptor is subunit-dependent. Coexpression of the 5-HT_3B subunit with the 5-HT_3A subunit results in a substantial decrease in PTX sensitivity, making PTX the only antagonist that displays notable selectivity between homomeric and heteromeric 5-HT₃ receptors (Das and Dillon, 2003).

In the present report, we sought to identify the molecular determinants of PTX inhibition of 5-HT₃ receptors. Although the precise location of PTX binding in anion-selective channels is unknown, evidence indicates it acts at the cytoplasmic aspect of the highly conserved TM2 domain (Fig. 1) (Ffrench-Constant et al., 1993; Gurley et al., 1995; Buhr et al., 2001; Shan et al., 2001; Dibas et al., 2002). Because the mechanism of block by PTX appears comparable in anion-selective and 5-HT₃ receptors and because the TM2 domain of the 5-HT₃ receptor shares notable homology with the GABA_A receptor (Fig. 1), we focused on this region as the probable site of action of PTX in 5-HT₃ receptors. In the present study, we demonstrate that the 6' and 7' positions of TM2 are major determinants of PTX sensitivity in the 5-HT₃ receptor.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Map of the second transmembrane domain of members of the ligand-gated ion channel superfamily. Sequences for the GABAA 1 subunit, the glycine 1 subunit, and 5-HT3A and 5-HT3B subunits are shown. The 0' position represents the most cytoplasmic aspect of the channel, whereas the 19' position represents the extracellular aspect of the channel. The conserved 9' leucine is believed to form the channel gate. Residues at the 2' and 6' positions are strongly involved in picrotoxin-mediated inhibition, although other residues may also play a role (see text).

	Materials and Methods

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Cell Culture, Site-Directed Mutagenesis, and Expression of 5-HT₃ Receptor cDNA. Mouse 5-HT_3A (GenBank accession no. S41757) and 5-HT_3B subunits (AAF73284 [GenBank] kindly provided by Dr. D. Julius, USCF, San Francisco, CA and Dr. E. Kirkness, Institute of Genomic Research, Rockville, MD, respectively) were subcloned into pcDNA 3.1 (Invitrogen, Carlsbad, CA) for expression in HEK-293 cells. HEK-293 cells were maintained in medium containing minimum essential media, 10% fetal bovine serum, L-glutamine (200 mM), penicillin, and streptomycin (10,000 U/ml). Site-directed mutagenesis in the 5-HT_3A (A-S2'A, 2' serine to alanine; A-T6'F, 6'threonine to phenylalanine; and A-L7'T, 7' leucine to threonine) and 5-HT_3B (B-N6'S, 6' asparagine to serine; B-N6'T, 6' asparagine to threonine; and B-V7'T, 7' valine to threonine) subunits was performed using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA). For the double mutation in 5-HT_3B subunit (B-N6'T,V7'T), mutagenic primers were designed using B-N6'T as the template DNA. All mutations were confirmed by DNA sequencing (Texas Tech University, Lubbock, TX). Wild-type and mutant receptors were transfected in HEK-293 cells using the modified calcium phosphate transfection method (Chen and Okayama, 1987). For each transfection, approximately 4 to 10 µg of DNA was used. For cotransfection of receptor subunits, a 1:1 ratio of respective cDNA was used (unless otherwise stated). Cells were washed twice and were used for recording 12 to 48 h after transfection.

Radioligand Binding Assays. Saturation experiments were carried out using membranes harvested from a stable cell line expressing the 5-HT_3A receptor. Briefly, HEK-293 cells stably expressing the 5-HT_3A receptor were grown as described previously (Das et al., 2003) and harvested using the method described by Yagle et al. (2003). Membranes were suspended in sodium phosphate buffer (200 mM NaCl and 50 mM NaH₂PO₄·2H₂O, pH 7.4) such that the final concentration was approximately 2 mg/ml and stored at -80°C. Initial saturation experiments using tissue harvested from the stable cell line suggested high B_max values. Thus, relatively low amount of protein was used for subsequent assays. For experiments using [³H]GR65630, tissue was thawed and diluted in sodium phosphate buffer, and a fixed concentration of protein (5–12 µg) was added to varying concentrations of the radioligand (0.8–20 nM). Nonspecific binding was defined using 10 µM m-CPBG. Final volumes for all assays were 250 µl. Samples were incubated in borosilicate glass tubes for 30 min at 37°C, reaction was terminated by addition of ice-cold sodium phosphate buffer, and bound ligand was separated from free using a Millipore 12-well manifold vacuum filtration system (Millipore Corporation, Billerica, MA). Saturation experiments using [³H]EBOB were carried out using a similar protocol as described above. In this case, nonspecific binding was defined using 100 µM PTX, and samples were incubated at room temperature for 90 min. All assays were performed in triplicate, and a minimum of three experiments was carried out for each saturation assay. Radioactivity was measured using liquid scintillation (PerkinElmer Life and Analytical Sciences). Data analysis was performed using Origin 5.0 (Yagle et al., 2003).

Electrophysiological Recordings. Whole-cell recordings were performed as described previously (Bell-Horner et al., 2000). Patch pipettes were pulled from thin-walled borosilicate glass using a horizontal micropipette puller (P-87/PC; Sutter Instrument Company, Novato, CA) and had a resistance of 1 to 3 M when filled with the following internal pipette solution: 140 mM CsCl, 10 mM EGTA, 4 mM Mg²⁺-ATP, and 10 mM HEPES-Na, pH 7.2. Coverslips containing the cultured cells were transferred to a small chamber (1 ml) on the stage of an inverted microscope (Olympus IMT-2; Olympus, Tokyo, Japan) and superfused continuously with the following external solution: 125 mM NaCl, 5.5 mM KCl, 1.5 mM CaCl₂, 0.8 mM MgCl₂, 20 mM HEPES-Na, and 10 mM glucose, pH 7.3. 5-HT-induced currents were obtained with an Axopatch 200A amplifier (Axon Instruments Inc., Union City, CA) equipped with a CV-4 headstage. Currents were low-pass filtered at 5 kHz, monitored simultaneously on a storage oscilloscope and a thermal head pen recorder (Gould TA240; Gould Instrument Systems Inc., Cleveland, OH), and stored on a computer using an online data acquisition system (pClamp 6.0, Axon Instruments Inc.). If a change in access resistance was observed during the recording period, the patch was aborted, and the data were not included in the analysis. All recordings were made at room temperature, and all cells were clamped at -60 mV.

Experimental Protocol. 5-HT (with or without PTX or TBPS) was dissolved in the external solution (above) and applied to the target cell through a Y tube positioned adjacent to the cell. With this system, solution exchange (measured as the 10–90% rise time of the junction potential at the open tip) averages 30 ms (Huang and Dillon, 1999). An equipotent concentration of 5-HT was used to gate the channel in most experiments evaluating the effects of antagonist. Once a stable 5-HT-gated current was established, the antagonist at varying concentrations was coapplied with 5-HT to the target cell.

Data Analysis. Both PTX (Dillon et al., 1995; Das et al., 2003) and TBPS (Van Renterghem et al., 1987; Dillon et al., 1995) are use-dependent blockers. Thus, for all experiments, the extent of inhibition by each agent was calculated as the current remaining at the end of the ligand application period (10–30 s), compared with the current amplitude at the same time point in the control (5-HT alone) recording. Inhibition-response relationships for PTX were fitted with the equation: I/I_max = 1/{1 + (IC₅₀/[PTX])ⁿ}, where I is the current amplitude normalized to control, IC₅₀ is the half-maximal blocking concentration, and n is the Hill coefficient. For inhibition curves, all Hill coefficients are understood to be negative. A minimum of four individual experiments was conducted for each paradigm. All data are presented as mean ± S.E.M.

	Results

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Figure 2 shows the concentration-response curves for 5-HT in wild-type and all mutant 5-HT_3A receptors. With the exception of the 5-HT_3A T6'F mutation (designated A-T6'F), only modest changes in EC₅₀ values were seen (Table 1), indicating the mutations were well tolerated and did not cause any gross alterations in channel structure. No detectable currents were recorded when the 5-HT_3A A-T6'F mutant was expressed alone. However, receptors incorporating this mutant were rescued when it was coexpressed with the wild-type 5-HT_3A subunit, and the resulting receptors had EC₅₀ and Hill coefficient values similar to wild type (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 2. 5-HT sensitivity in wild-type and mutant 5-HT3A receptors. Mutations in 2' and 7' positions in the 5-HT3A receptor did not appreciably affect 5-HT sensitivity, indicating that the gross structure of the mutant receptors was unaltered. The EC50 values and Hill coefficients are reported in Table 1. Values for the wild-type receptor are comparable with what we have reported previously (Das et al., 2003). Mutation of the 6' residue led to nonfunctional receptors that could be rescued upon coexpression with the wild-type 5-HT3A receptor. All data points are from a minimum of four to six cells.

Lack of Effect of TM2 2' Serine on PTX Sensitivity in Homomeric 5-HT_3A Receptors. As shown in Fig. 3, PTX inhibited wild-type 5-HT_3A receptors with an IC₅₀ of 41.2 ± 5.6 µM. This IC₅₀ is comparable with the value we originally reported for PTX-mediated inhibition of these receptors (Das et al., 2003) and is 10-fold higher than picrotoxin's IC₅₀ in GABA_A receptors (Bell-Horner et al., 2000). The magnitude of inhibition was not different with cells recorded at -30 mV (current decreased to 38.5 ± 4.2% of control with 100 µM PTX, n = 4) and +30 mV (current reduced to 42.4 ± 7.3% of control with 100 µM PTX, n = 4), with no change in reversal potential, indicating picrotoxin's block is voltage-independent.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Effect of S2'A and T6'F mutations on picrotoxin sensitivity in 5-HT3A receptors. Typical traces showing effects of picrotoxin in wild type (A), A-S2'A receptors (B), or A-T6'F 5-HT3A receptors (C). Receptors were gated with an EC50 5-HT concentration. The PTX IC50 was not significantly shifted in receptors with the S2'A mutation as compared with the wild-type receptor but was drastically shifted in receptors expressing the T6'F mutation (Table 1). D, mean PTX inhibition curves for wild-type and mutant receptor configurations. n = 4 to 6 for each data point.

The TM2 6' Threonine Influences PTX Sensitivity in 5-HT_3A Receptors. In GABA_A and glycine receptors, mutation of the conserved 6' threonine to phenylalanine confers resistance to PTX (Gurley et al., 1995; Shan et al., 2001). Thus, we evaluated the role of this residue in conferring resistance to PTX in the 5-HT_3A receptor. Because we could not detect functional receptors when the A-T6'F mutant was expressed alone (no response to up to 100 µM 5-HT), we coexpressed it with wild-type cDNA in varying ratios. A 3:1 or greater ratio of A-T6'F to wild-type cDNA did not produce functional receptors. Cotransfection of these subunit cDNAs in 1:1 or 2:1 ratio produced functional receptors with comparable properties (i.e., EC₅₀ values and PTX sensitivity). The data presented here were obtained using a 2:1 ratio of A-T6'F:wild-type cDNA. The effects of PTX on the 5-HT_3A + A-T6'F receptor are shown in Fig. 3C. The PTX sensitivity was markedly reduced in receptors incorporating the A-T6'F mutation, compared with wild-type receptors (Fig. 3D; Table 1). Although PTX sensitivity was not completely abolished by the mutation, the nearly 50-fold shift in sensitivity to PTX strongly suggests that the 6' residue is a critical determinant of PTX sensitivity in the 5-HT_3A receptor.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Effect of L7'T mutation in the 5-HT3A receptor. In A-L7'T mutant receptors, PTX sensitivity was increased 10-fold compared with wild-type receptor (Table 1). B, traces in A are replotted from Fig. 3 for comparison. This mutation also conferred to the receptor distinct gating kinetics (see Fig. 5 below). Traces in A are replotted from Fig. 3 for comparison. C, mean PTX concentration-inhibition curve for the A-L7'T receptor compared with the wild-type receptor. n = 4 to 6 for each data point.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Effect of A-L7'T mutation on gating kinetics. A, mean activation kinetics in wild-type (solid line) and A-L7'T receptors (dotted line) in response to an EC50 5-HT concentration. Activation is reported as the 10 to 90% rise time. Mean results are given in Ab. B, deactivation was also enhanced in the A-L7'T receptors (dotted line) compared with wild-type receptors (solid line). Deactivation is reported as the 10 to 90% decay time. Mean deactivation times for the two receptors are given in Bb. Both activation and deactivation kinetics were significantly different in mutant compared with wild-type receptors (*, p < 0.01, Student's t test).

Evidence from substituted cysteine accessibility method studies in both anionic GABA_A and cationic 5-HT_3A receptors suggests that the amino acid residue at the 7' position may be exposed to the channel lumen (Xu et al., 1995; Reeves et al., 2001). In most GABA_A receptor subunits, a threonine exists at the 7' position. In contrast, a leucine residue is present at this position in the 5-HT_3A subunit. To evaluate the potential involvement of this residue in PTX-mediated inhibition, we mutated the 5-HT_3A TM2 7' leucine to threonine (A-L7'T). Although the A-L7'T mutation resulted in a very modest decrease in 5-HT sensitivity (Fig. 2), this mutation conferred a 10-fold increase in PTX sensitivity, compared with wild-type receptors (Fig. 4; Table 1). Interestingly, the sensitivity to PTX in A-L7'T receptors is comparable with that seen in GABA_A receptors (Xu et al., 1995; Bell-Horner et al., 2000). Thus, the TM2 7' position appears to play a role in determining picrotoxin sensitivity in homomeric 5-HT_3A receptors.

The TM2 7' Residue Modulates Gating Kinetics in 5-HT_3A Receptors. In addition to altering PTX sensitivity, the A-L7'T mutation dramatically altered channel kinetics. Activation, desensitization, and deactivation were all enhanced compared with wild-type 5-HT_3A receptors. Figure 5 illustrates the changes in activation and deactivation conferred by this mutation. The 10 to 90% rise time in the presence of an EC₅₀ 5-HT concentration was 3.5 ± 0.76 and 0.37 ± 0.08 s in wild-type and mutant receptors, respectively (p < 0.01). Deactivation time was similarly enhanced in the mutant (12.1 ± 0.91 s in wild-type compared with 2.28 ± 0.54 s in the mutant A-L7'T receptors, p < 0.01). Also, in contrast to wild-type 5-HT_3A receptors that showed little or no desensitization when gated with an EC₅₀ concentration of 5-HT (current amplitude decreased only 1.7 ± 0.8% at end of 5-HT application period), the A-L7'T receptors exhibited significant desensitization (current amplitude decreased 13.5 ± 2.9% at end of 5-HT application period, p < 0.01 compared with wild-type receptors). Thus, mutation of 7'L to T in the 5-HT_3A receptor also profoundly altered gating kinetics.

The 3B Subunit TM2 6' Position Is Responsible for Reduced PTX Sensitivity in Heteromeric Receptors. We recently reported that heteromeric receptors formed by murine 5-HT_3A and 5-HT_3B subunits are notably less sensitive to PTX than homomeric 5-HT_3A receptors (Das and Dillon, 2003). Following our observation that the 6'T residue in the 5-HT_3A subunit was critical in PTX-mediated inhibition, we sought to examine if the 6' residue in the 5-HT_3B subunit [asparagine (N) instead of threonine; Fig. 1] was responsible for the reduced sensitivity to PTX in the heteromeric receptors. Serotonin sensitivity in heteromeric receptors expressing the B-N6'T mutation was modestly increased compared with that observed in wild-type receptors (Fig. 6; Table 1). Consistent with our previous report, sensitivity to PTX was significantly decreased in heteromeric receptors (Fig. 7). As expected, PTX sensitivity was significantly enhanced in 5-HT_3A + B-(N6'T) receptors (Fig. 7), confirming that the 6'N in the 5-HT_3B subunit is in part responsible for the reduced sensitivity of heteromeric receptors to PTX. However, PTX sensitivity was not fully converted to that seen in homomeric receptors, suggesting other residues contribute to the reduced PTX sensitivity in these receptors.

View larger version (26K): [in this window] [in a new window]   Fig. 6. 5-HT sensitivity in wild-type and mutant 5-HT3A/3B receptors. Agonist sensitivity was increased approximately 2-fold on average in heteromeric receptors with mutations in the 5-HT3B subunit (Table 1). Values for the wild-type receptor are similar to what we have reported previously (Das and Dillon, 2003).

View larger version (27K): [in this window] [in a new window]   Fig. 7. PTX sensitivity is partially restored in heteromeric receptors with a converse mutation at the 6' position. A, wild-type heteromeric 5-HT3A/3B receptors show reduced sensitivity to PTX, as reported previously (Das and Dillon, 2003). B, heteromeric receptors in which the B subunit 6' asparagine has been mutated to threonine (B-N6'T) displayed an increased sensitivity to PTX, although this was not fully converted to the sensitivity observed in homomeric 5-HT3A receptors. C, mutation of the 6' asparagine to serine, which is present in the human 5-HT3B subunit, also conferred enhanced sensitivity to PTX. D, mean PTX concentration-inhibition curves in wild-type and mutant heteromeric 5-HT3 receptors. PTX IC50 and Hill coefficient values for receptors expressing these mutations are reported in Table 1. Effects of PTX on homomeric receptors (solid squares) is replotted here and in Fig. 8 for comparison.

The 7' Residue in the 3B Subunit Is a Minimal Determinant of PTX Sensitivity in Heteromeric Receptors. Based on our above finding that the 7' position of the 3A subunit affects sensitivity to PTX in homomeric receptors, we assessed whether this position in the 3B subunit could similarly alter PTX-mediated inhibition in heteromeric receptors. Receptors expressing the wild-type 3A subunit along with the 3B subunit 7' valine to threonine (B-V7'T) mutant had slightly enhanced 5-HT sensitivity compared with wild-type heteromeric receptors (Fig. 6). Figure 8, A and C, illustrates that PTX sensitivity in these receptors was not significantly increased. To further evaluate a possible contribution of the 3B subunit 7' position to PTX sensitivity, we generated a 5-HT_3B double mutant (B-N6'T, V7'T). As shown in Fig. 8, B and C, heteromeric receptors expressing the double mutant did not display any additional sensitivity to PTX than receptors expressing only the 6' mutation. Thus, in heteromeric receptors, the 7' position of the 5-HT_3B subunit does not appear to play a significant role in modulating the effects of PTX.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Effect of the TM2 7' position in the 5-HT3B subunit on PTX sensitivity in heteromeric 5-HT3A/3B receptors. A, effect of PTX in cells expressing 3A + B-(V7'T) subunits. No significant increase in PTX sensitivity was observed in these receptors compared with wild-type 5-HT3A/3B receptors. B, typical recordings showing effects of PTX in cells expressing the B-N6'T, V7'T double mutant along with the wild-type 3A subunit. No additional gain in sensitivity was observed in these receptors when compared with heteromeric receptors expressing the B-N6'T mutation alone. C, mean PTX concentration-inhibition curves in cells expressing wild-type and mutant 5-HT3A/3B heteromeric receptors.

The Caged Convulsants EBOB and TBPS Do Not Interact with 5-HT_3A Receptor. [³H]EBOB binds with high affinity to the convulsant site in GABA_A receptors (Huang and Casida, 1996; Yagle et al., 2003). Although the exact binding site for [³H]EBOB has not been defined, it is believed to overlap the binding site for PTX since PTX and related convulsant drugs competitively displace [³H]EBOB (Cole et al., 1995). Based on our above findings, we hypothesized that [³H]EBOB would also bind to 5-HT_3A receptors. To test this possibility, we performed [³H]EBOB saturation binding studies using membranes harvested from a stable cell line expressing the 5-HT_3A receptor. As expected, [³H]GR65630, a ligand for the 5-HT₃ receptor agonist binding site, bound to these receptors with high affinity (K_d = 8 ± 1.7 nM, B_max = 23.8 ± 1.6 pmol/mg protein). In contrast, we detected no interaction of [³H]EBOB with 5-HT_3A receptors because exposure of the membranes to up to 180 nM of [³H]EBOB did not yield significant specific binding (Fig. 9A). We subsequently tested whether TBPS, which is structurally related to EBOB and also presumably binds to the PTX site in GABA_A receptors (Van Renterghem et al., 1987; Dillon et al., 1995), could interact with 5-HT_3A receptors. As shown in Fig. 9B, application of up to 100 µM TBPS had no significant effect on 5-HT currents. In contrast, a concentration of TBPS 10-fold lower completely abolished steady-state GABA-activated currents (Fig. 9C). Thus, although the interaction of PTX with 5-HT_3A receptors is similar to that observed in GABA_A receptors, the caged convulsants EBOB and TBPS do not similarly interact with the two receptors.

View larger version (17K): [in this window] [in a new window]   Fig. 9. The noncompetitive GABAA receptor antagonists EBOB and TBPS do not interact with 5-HT3A receptors. A, saturation curve for analysis of [3H]EBOB binding in 5-HT3A receptors. The plot shown is representative of one experiment performed in triplicate. Filled triangles represent total binding, open squares represent nonspecific binding, and open circles represent specific binding. A similar lack of specific binding of [3H]EBOB to 5-HT3A receptors was observed in three additional experiments. B, TBPS, a high-affinity noncompetitive antagonist of GABAA receptors, was tested for its ability to block 5-HT3A receptors. As shown, 100 µM TBPS had no significant effect of 5-HT-gated current. C, in contrast, steady-state GABA currents recorded from HEK293 cells expressing 122 GABAA receptors were completely abolished by a 10-fold lower concentration of TBPS.

	Discussion

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For decades, picrotoxin was considered to be a fairly selective antagonist of GABA_A receptors. The discovery that PTX also inhibits glycine receptors and other anion-selective ligand-gated ion channels is more recent (Pribilla et al., 1992; Etter et al., 1999; Shan et al., 2001). Following our report that PTX also inhibits the cation-selective 5-HT₃ receptor (Das et al., 2003) and that it does so in a subunit-dependent manner (Das and Dillon, 2003), the focus of the present investigation was to determine the molecular basis for PTX inhibition of 5-HT₃ receptors.

Minimal Role of the TM2 2' Residue in PTX Sensitivity of 5-HT₃ Receptors. In the homomeric Drosophila GABA receptor, a naturally occurring mutation at the 2' position (A2'S) confers resistance to PTX (Ffrench-Constant et al., 1993). Mutations at this position also modify PTX sensitivity in mammalian GABA_A receptors (Xu et al., 1995), GABA_C receptors (Wang et al., 1995), and glutamate-gated Cl^- channels (Etter et al., 1999). Because the presence of serine or threonine instead of alanine at the 2' position in the Drosophila GABA receptor and glutamate-gated Cl^- channels, respectively, causes PTX resistance, we hypothesized that the reverse mutation in the homomeric 5-HT_3A receptor (S2'A) might lead to an increase in sensitivity to PTX. However, the picrotoxin sensitivity in 5-HT_3A receptors expressing the S2'A mutant was not significantly different from wild-type 5-HT_3A receptors. Our results are analogous to those of Shan et al. (2001), who found that substitution of the native 2' residue in glycine 1 or subunits (glycine and proline, respectively) with alanine did not increase PTX sensitivity in homomeric 1 or heteromeric 1 receptors. Thus, the role of the 2' position on picrotoxin sensitivity is receptor-dependent; in 5-HT₃ receptors, like in glycine receptors (Shan et al., 2001), it appears to play a minimal role.

The TM2 6' Residue Is a Critical Determinant of PTX Sensitivity in Both 5-HT_3A and 5-HT_3A/3B Receptors. The presence of phenylalanine at the TM2 6' position has been shown to confer PTX resistance in GABA_A (Gurley et al., 1995), GABA_C (Zhang et al., 1995), and glycine receptors (Shan et al., 2001). Our finding that 5-HT_3A receptors that express a T6'F mutation are significantly less sensitive to PTX demonstrates the 6' position is also a critical determinant of PTX sensitivity in cation-selective LGICs. In addition, our ability to convert picrotoxin-resistant heteromeric 5-HT_3A/3B receptors to picrotoxin-sensitive by mutation of the native 3B 6' residue (asparagine) confirms its involvement in PTX-mediated inhibition of 5-HT₃ receptors.

The precise role the 6' residue plays in PTX sensitivity is not clear. Based on molecular modeling, Zhorov and Bregestovski (2000) hypothesized that picrotoxin's electronegative hydrophilic domain is stabilized in the channel of GABA_A and glycine receptors by hydrogen bonding with the native 6' threonine residues; the inability of phenylalanine to hydrogen bond destabilizes PTX binding. The experimental results of Shan et al. (2001) support this hypothesis. However, our results demonstrate that asparagine, which can act as a hydrogen bond donor, confers resistance when present at the 6' position of the 5-HT_3B subunit. Thus, either aparagine's NH₂ group is not properly oriented to act as a hydrogen bond donor for the picrotoxin molecule, or other factors at this 6' position are also critical in conferring sensitivity to PTX.

The TM2 7' Position Differentially Affects PTX Sensitivity in 5-HT_3A and 5-HT_3A/3B Receptors. All , , and subunits of the GABA_A receptor have a threonine at the 7' position of TM2 (Tyndale et al., 1995), whereas a leucine exists at this position in the 5-HT_3A subunit. Based on the fact that the 7' residue is exposed to the lumen of the channel in both GABA_A (Xu et al., 1995) and 5-HT_3A receptors (Reeves et al., 2001) and the fact that it is adjacent to the 6' residue, we hypothesized that the 7' position may be responsible for the 5- to 10-fold lower PTX sensitivity of 5-HT_3A receptors than GABA_A receptors (Bell-Horner et al., 2000). Mutation of the TM2 7' leucine to threonine resulted in a 10-fold increase in sensitivity to PTX in the 5-HT_3A receptor. This is the first report of a role of the 7' residue in PTX inhibition of a member of the LGIC superfamily. Interestingly, this residue in the 3B subunit does not appear to play a role in PTX sensitivity in 5-HT_3A/3B receptors because mutation of this residue to T did not enhance sensitivity to PTX when expressed alone or in combination with the 6' threonine mutation (i.e., B-N6'T, V7'T). The basis for the differential effect of the 7' mutation in the two subunits is not known. Although the 7' residue is known to project toward the channel lumen in homomeric 5-HT_3A receptors (Reeves et al., 2001), the orientation of the 7' position of the 3B subunit in heteromeric 5-HT_3A/3B receptors has not been determined. Subunit-dependent effects of equivalent TM2 residues near the 7' position have been reported in both GABA_A (Chang and Weiss, 1999) and glycine (Shan et al., 2001) receptors. However, the similarity of effect of 6' mutations in the two subunits would seem to indicate the orientation of residues at this position is not dramatically different. Thus, differences at the level of 7' would likely be subtle.

Because it has been postulated that picrotoxin acts by stabilizing a desensitized state in other receptors (Newland and Cull-Candy, 1992; Dillon et al., 1995), we considered the possibility that the enhanced effect of PTX in A-L7'T receptors may be secondary to the altered kinetics present in these receptors. This does not appear to be the case, however, because we have recently made the reverse mutation (T7'L) in homomeric glycine 1 receptors. As expected, this mutation in glycine receptors caused opposite effects on kinetics than the L7'T mutation caused in 5-HT_3A receptors. However, PTX sensitivity was in fact slightly enhanced, not decreased as would be expected if its effects were secondary to alterations in kinetics of the channel (E. B. Gonzales and G. H. Dillon, unpublished data). Thus, the effects of the 7' residue on PTX sensitivity do not appear to be due to alterations in channel kinetics.

Is the Binding Site for PTX the Same in 5-HT₃ Receptors and GABA_A Receptors? The present report demonstrates that the 6' TM2 residue, like in the GABA_A receptor, is a critical determinant of PTX action in 5-HT₃ receptors. However, the differential contribution in the two receptors of the 2' residue to PTX sensitivity indicates the binding domains are not equivalent. Our finding that the picrotoxin site ligands [³H]EBOB and TBPS had insignificant effects on the 5-HT₃ receptor demonstrates an additional difference of the convulsant site in the two receptors. The lack of effect of these ligands on 5-HT₃ receptors was initially somewhat unexpected. However, given that [³H]EBOB binding is strongly influenced by the 2' residue (Cole et al., 1995), its lack of affinity for the 5-HT₃ receptors is not unreasonable. With regard to TBPS, the 3' leucine of the GABA_A receptor subunit may contribute to a high-affinity binding site for this convulsant (Jursky et al., 2000). Thus, in the GABA_A receptor, radioligand binding studies, site-directed mutagenesis, and substituted cysteine accessibility method studies are suggestive of a PTX binding site at or near the cytoplasmic aspect of the ion channel, encompassing the 2' to 3' vicinity. [³H]EBOB, which displaces PTX binding in the GABA_A receptors, did not specifically bind to the 5-HT_3A receptors. In addition, TBPS, a GABA_A receptor antagonist that presumably binds at the 3' region in GABA_A receptors (Jursky et al., 2000), did not inhibit whole-cell currents induced by 5-HT in the 5-HT_3A receptor. Taken together, our data demonstrate the 6' to 7' region is the more likely site of PTX action than the 2' to 3' region in 5-HT₃ receptors. An allosteric effect of the mutations we have made on PTX sensitivity cannot be ruled out, however. It is possible, for instance, that mutations at the 6' to 7' region are inducing conformational changes in receptor structure at a distant site that are influencing the ability of PTX to bind at that site. Indeed, there is evidence in anion-selective receptors that picrotoxin may interact at the extracellular aspect (15'–17') of TM2 (Perret et al., 1999; Dibas et al., 2002).

The TM2 7' Position Is a Determinant of Channel Gating Kinetics. In addition to influencing PTX sensitivity, substitution of the native 7' leucine with threonine also notably altered kinetics in 5-HT_3A receptors. Activation, desensitization, and deactivation kinetics were all increased significantly compared with wild-type receptors. The TM2 7' residue is likely a determinant of gating kinetics in the LGIC superfamily in general because, as noted above, the reverse mutation in glycine 1 receptors (T7'L) had opposite effects on channel kinetics (E. B. Gonzales and G. H. Dillon, unpublished data). The involvement of the 7' position in gating kinetics is not surprising given that neighboring residues, including the 9' (Chang and Weiss, 1999) and 6' (Xu et al., 1995; Shan et al., 2001) positions, are known to influence channel gating. A better understanding of the physicochemical traits of 7' residues necessary for rapid channel kinetics will require additional investigation.

	Acknowledgements

 
We thank David Julius for providing 5-HT_3A cDNA, Ewen Kirkness for providing 5-HT_3B cDNA, and Cathy Bell-Horner for technical assistance.

	Footnotes

 
This work was supported by National Institutes of Health Grant ES07904 (to G.H.D.).

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

doi:10.1124/jpet.104.080325.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; LGIC, ligand-gated ion channel; TM, transmembrane; PTX, picrotoxin; m-CPBG, meta-chlorophenylbiguanide; EBOB, ethynylbicycloorthobenzoate; TBPS, tert-butylbicyclophosphorothionate; [³H]GR65630, [³H]3-(5-methyl-1H-imidazol-4-yl)-1-methyl-1H-indol-3-yl)-1-propanone.

Address correspondence to: Dr. Glenn H. Dillon, Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. E-mail: gdillon{at}hsc.unt.edu

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