High Affinity Binding of Epibatidine to Serotonin Type 3 Receptors

Abstract

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of ¹²⁵I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT₃Rs) block the remaining 25%. ¹²⁵I-Epibatidine binds with a high affinity to native 5-HT₃Rs of N1E-115 cells and to receptors composed of only 5-HT_3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT₃R-specific antagonist MDL72222, and the 5-HT₃R agonist chlorophenylbiguanide readily competed with ¹²⁵I-epibatidine binding to 5-HT₃Rs. Nicotine was a poor competitor for ¹²⁵I-epibatidine binding to 5-HT₃Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of ¹²⁵I-epibatidine binding to 5-HT₃Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT₃Rs in neuroblastoma cell lines and 5-HT_3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT₃Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT₃R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT₃Rs.

Epibatidine is an alkaloid originally isolated from Ecuadorian poison frog skin (1). Electrophysiological and ligand binding studies demonstrate that epibatidine is an agonist that binds with high affinity to a subset of nAChRs2 throughout the central and peripheral nervous systems (2–6). Epibatidine administration has toxic effects, such as seizures and hypertension (7–9). However, at similar doses, epibatidine has antinociceptive effects that are blocked by nAChR antagonists, such as mecamylamine, consistent with the analgesic activity of epibatidine being mediated through nAChRs (2, 10, 11). Administration of mecamylamine alone also has pronounced physiological effects that alter pain perception and blood pressure (7, 12, 13).

5-HT₃Rs and nAChRs are members of the same ionotropic neurotransmitter receptor family that also includes GABA_A and glycine receptors (14, 15). Within this neurotransmitter receptor family, 5-HT₃Rs are most similar to nAChRs, sharing up to 30% sequence homology (16, 17). Physiological studies indicate that 5-HT₃Rs and nAChRs regulate many of the same pathways (18–21). Co-expression of these two classes of receptors occurs in regions of the nervous system associated with pain processing, such as the dorsal horn of the spinal cord, nucleus of the solitary tract, raphe magnus nucleus, and nucleus dorsalis (22–25). Local or systemic administration of nAChR agonists reduces the pain response in rodents (2, 20, 26–30). 5-HT₃R ligands appear to be both pro- and antinociceptive, depending on the pain stimulus and route of administration (19, 31–35). 5-HT₃Rs and nAChRs also contribute to the modulation of blood pressure. Activation of nAChRs increases pressor and heart rate in normotensive and spontaneously hypertensive animals (18, 36–38). In spontaneously hypertensive rats, chronic inhibition of 5-HT₃Rs lowers blood pressure (39), whereas in normal rats, activation of 5-HT₃Rs decreases arterial pressure (40). Inhibition of 5-HT₃Rs likewise produced a significant decrease in blood pressure in obese rats prone to stress-induced hypertension (41). In addition to these effects, drug-induced and epilepsy-associated convulsive activity display an nAChR (42–44) and 5-HT₃R (45–47) sensitivity. These observations link 5-HT₃R function with responses associated with administration of some nAChR-specific ligands.

Experimental evidence indicates that select nAChR ligands cross-react with 5-HT₃Rs as either agonists or antagonists (48–52). In this study, using a combination of neuroblastoma cell lines, heterologous expression of 5-HT_3ARs, and rat brain preparations, we show that epibatidine binds with high affinity to 5-HT₃Rs. In addition, both epibatidine and mecamylamine function as antagonists when bound to 5-HT₃Rs. Thus, 5-HT₃Rs may participate in physiological effects previously thought to be nAChR-specific.

EXPERIMENTAL PROCEDURES

Cell Lines and Transfection—The human kidney epithelial cell line tsA201 (HEK cells) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Hyclone, Logan, UT). Hemagglutinin epitope-tagged mouse 5HT_3A was subcloned into the pcDNA3.1 vector using standard methodology. HEK cells were transiently transfected with the subunit cDNA constructs using a calcium phosphate protocol (53). In some experiments, for comparison with mouse 5-HT_3A receptors, HEK cells were transfected with cDNA encoding the human 5-HT_3A receptor subunit. Mouse neuroblastoma N1E-115 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C, 5% CO₂. Mouse neuroblastoma NB41A3 cells (ATCC) were maintained in F-12 medium supplemented with 15% horse serum, 5% fetal calf serum. Cells were plated onto 35-mm culture dishes (Fisher) for use in electrophysiological experiments. All medium was supplemented with penicillin (100 IU/ml) and streptomycin (100 μg/ml).

Rat Brain Membranes—Membranes were prepared as described previously (54). Briefly, adult rats were decapitated, and the entire brain was dissected and placed in ice-cold 50 mm NaPO₄, pH 7.4, 50 mm NaCl, 2 mm EDTA, and 2 mm EGTA plus protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 2 mm N-ethylmaleimide, chymostatin, pepstatin, 1-chloro-3-tosylamido-7-amino-2-heptanone, and leupeptin at 10 μg/ml). All chemicals were obtained from Sigma unless otherwise specified. The brain tissue was minced and homogenized in a Teflon-glass homogenizer. Homogenates were centrifuged at 100,000 × g for 1 h. Pellets were taken through one more cycle of homogenization and centrifugation. The resulting pellets were resuspended in 150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.4, 0.02% NaN₃ plus protease inhibitors and frozen at –80 °C until needed.

Ligand Binding Studies—Unlabeled epibatidine in phosphate-buffered saline was added to ¹²⁵I-epibatidine (2200 Ci/mmol) or [³H]epibatidine (47.3 Ci/mmol) (PerkinElmer Life Sciences) to a total epibatidine concentration of 1.7 and 10 μm, respectively. Equilibrium binding to whole cell membrane fractions was measured by incubating at room temperature in phosphate-buffered saline with increasing amounts of radiolabeled epibatidine on a table top rotator for 20 min. For competitive binding experiments, cells were preincubated with different serotonergic or nicotinic ligands at the specified concentrations, followed by the addition of 50 nm ¹²⁵I-epibatidine. Nonspecific binding was determined in the presence of 1 mm epibatidine, 100 μm MDL72222 or by performing binding on cells transfected with empty vector. Samples were washed three times rapidly with ice-cold phosphate-buffered saline in a Brandel apparatus. ¹²⁵I- and [³H]epibatidine counts were measured in a Wallac 1470 automatic γ counter and a Beckman LS6500 scintillation counter, respectively. Equilibrium binding constants were calculated based on the least squares equation, amount of ligand bound = B_max × [ligand]/K_D + [ligand]. Competition binding curves were generated with the least squared fit to the Hill equation, fraction of maximum = 1/(1 + IC₅₀/[ligand])ⁿ, where n represents the Hill coefficient. IC₅₀ values were converted to K_I values using the equation, K_I = IC₅₀/1 + ([epibatidine]/K_D). The K_D for epibatidine used to calculate K_I values for nicotine and MDL72222 in Fig. 1 was 8 pm (55) and 27 nm (see Fig. 2A), respectively.

FIGURE 1.

¹²⁵I-Epibatidine binds to 5-HT₃Rs in crude rat brain membranes. Samples were incubated with nicotine (A) or MDL72222 (B) prior to the addition of 50 nm ¹²⁵I-epibatidine. C, results from A and B in addition to experiments where nicotine + MDL72222 or nicotine + CPBG were added prior to 50 nm ¹²⁵I-epibatidine. The lines through the data represent the least squares fit to the Hill equation, fraction of maximum = 1/(1 + [ligand]/IC₅₀)ⁿ, where n represents the Hill coefficient. Each point represents the mean ± S.D. of three determinations. K_I values and Hill coefficients from the fit of the equation to the data are 2 ± 0.2 nm (n = 1.5) and 89 ± 5 nm (n = 0.7) for nicotine and MDL72222, respectively.

FIGURE 2.

Saturation and competition binding of serotonergic and nicotinic ligands to 5-HT₃Rs. A, representative saturation binding curves showing specific ¹²⁵I-epibatidine binding (K_D = 27 ± 6 nm) and [³H]epibatidine binding (K_D = 22 ± 5 nm) to N1E-115 cell membrane fractions. B, various nicotinic and serotonergic ligands compete for ¹²⁵I-epibatidine binding sites in N1E-115 cell membranes. C, saturation binding of ¹²⁵I-epibatidine (K_D = 14 ± 4 nm) to 5-HT_3ARs expressed heterologously in HEK cell membranes. D, competition binding experiments were performed as described in B on membrane fractions from HEK cells expressing the 5-HT_3AR. The graphs represent the least squares fit to the Hill equation, fraction of maximum = 1/(1 + [ligand]/IC₅₀)ⁿ, where n represents the Hill coefficient. K_I values calculated from IC₅₀ values and Hill coefficients are shown in Table 1. Each point represents the mean ± S.D. of three determinations.

Electrophysiological Recording—The whole cell configuration of the patch clamp technique was used to record currents from cells expressing either recombinant 5-HT_3A receptors (HEK cells) or native 5-HT₃ receptors (N1E-115 and NB41A3 cells). The electrode solution contained 140 mm CsCl, 2 mm MgCl₂, 0.1 mm CaCl₂, 1.1 mm EGTA, and 10 mm HEPES (pH 7.4 with CsOH). The extracellular solution contained 140 mm NaCl, 2.8 mm KCl, 2 mm MgCl₂, 1 mm CaCl₂, 10 mm HEPES, and 10 mm glucose (pH 7.4 with NaOH). Cells were voltage-clamped at an electrode potential of –60 mV. 5-HT₃ receptors were activated by locally applying agonists to the cell by pressure (10 p.s.i.) ejection (Picospritzer II; General Valve Corp.) from modified patch pipettes. The recording chamber was continuously perfused with extracellular solution (5 ml/min). Experiments took place at 20–22 °C. Currents were monitored using an Axopatch 200B, low pass-filtered at 2 kHz, digitized at 10 KHz using a Digidata 1320A interface, and acquired using pCLAMP8 software (all from Axon Instruments) onto the hard drive of a personal computer for off-line analysis. The peak amplitudes of agonist-activated currents were measured using pCLAMP8 software. Concentration-response relationships were fitted with modified logistic functions to determine EC₅₀, IC₅₀, and Hill slope values, as described previously (56).

RESULTS

High Affinity Epibatidine Binding in Rat Brain to Sites Other than nAChRs—When relatively high concentrations of ¹²⁵I-epibatidine (50 nm) were used to measure binding to membranes prepared from rat brain homogenates, a component of the ¹²⁵I-epibatidine binding was insensitive to nicotine competition (Fig. 1A). Although nicotine blocked 75% of the binding to rat brain membranes, 25% of the binding remained intact even at nicotine concentrations as high as 1 mm. Nicotine blocked ¹²⁵I-epibatidine binding with a K_I of 2 nm, which was derived from the fit of the Hill equation to the data in Fig. 1A. The K_I value for nicotine is similar to other measurements using epibatidine binding to rat brain synaptosomal membranes (55) and rat cerebral cortex (57). These data indicate that 25% of the ¹²⁵I-epibatidine binding is to sites other than nAChRs. Since epibatidine has been shown to block serotonin-induced currents mediated by 5-HT3_ARs in oocytes (51), and nAChRs and 5-HT₃Rs share a high degree of sequence similarity (16), we tested the effects 5-HT₃R-specific ligands on ¹²⁵I-epibatidine binding to rat brain fractions. As shown in Fig. 1B, increasing concentrations of the 5-HT₃R-specific antagonist, MDL72222, blocked 25% of the ¹²⁵I-epibatidine binding in experiments parallel to those in Fig. 1A using the rat brain membranes. We obtained similar data using a 5-HT₃R-specific agonist, chlorophenylbiguanide (CPBG) (data not shown). The effects of nicotine and the 5-HT₃R-specific ligands were additive, as shown in Fig. 1C. Preincubation of the rat brain membranes with saturating concentrations of either nicotine and MDL72222 or nicotine and CPBG blocked all specific ¹²⁵I-epibatidine binding to the membrane. These results indicate that ¹²⁵I-epibatidine binds to two distinct subpopulations of receptors in rat brain: 1) a nicotine-sensitive subpopulation consisting of nAChRs and 2) a nicotine-insensitive subpopulation consisting of 5-HT₃Rs.

Epibatidine Binding to Native and Heterologous 5-HT₃Rs—To further test whether there are ¹²⁵I-epibatidine binding sites on 5-HT₃Rs, we performed binding assays on preparations in which native and heterologously expressed 5-HT₃Rs were present in the absence of any significant nAChR expression. To study native 5-HT₃Rs, we used undifferentiated mouse neuroblastoma N1E-115 cells. These cells express endogenous 5-HT_3A and 5-HT_3B subunits (58) but little to no nicotinic receptors (59). Consistent with an absence of nAChR high affinity sites, we detected no specific binding to membranes from undifferentiated N1E-115 cells at ¹²⁵I-epibatidine concentrations of 0.5 nm. At higher concentrations, we observed an increasing level of specific, high affinity ¹²⁵I-epibatidine binding to the neuroblastoma cell 5-HT₃Rs with an estimated K_D of 27 ± 6 nm (Fig. 2A). We obtained similar results (an estimated K_D of 22 ± 5 nm) with [³H]epibatidine binding to neuroblastoma cell 5-HT₃Rs (Fig. 2A). Serotonin, MDL72222, and CPBG were highly potent inhibitors of ¹²⁵I-epibatidine binding to N1E-115 cell membranes as compared with nicotine in competition binding experiments (Fig. 2B). Surprisingly, mecamylamine, a noncompetitive antagonist of different nAChRs, blocked ¹²⁵I-epibatidine binding to 5-HT₃Rs. The relative potency in competing for ¹²⁵I-epibatidine binding sites in N1E-115 cell membranes was MDL72222 > epibatidine > 5-HT = CPBG > mecamylamine nicotine (Table 1).

TABLE 1.

Inhibition of ¹²⁵I-epibatidine binding to 5-HT₃Rs by various serotonergic and nicotinic ligands

	N1E-115 cells		Mouse 5-HT3ARs in HEK cells
	KI	Hill coefficient	KI	Hill coefficient
	m		m
Epibatidine	2.2 ± 0.5 × 10-6	0.66 ± 0.05	5.0 ± 1.0 × 10-7	0.38 ± 0.03
Mecamylamine	2.2 ± 0.6 × 10-5	0.53 ± 0.05	2.0 ± 0.2 × 10-6	0.93 ± 0.07
Serotonin	5.4 ± 1.2 × 10-6	0.38 ± 0.03	5.3 ± 1.4 × 10-6	0.53 ± 0.06
MDL72222	3.0 ± 0.8 × 10-7	0.44 ± 0.05	1.2 ± 0.2 × 10-6	0.45 ± 0.06
CPBG	5.6 ± 1.0 × 10-6	0.70 ± 0.10	9.7 ± 1.4 × 10-6	0.64 ± 0.05
Nicotine	2.0 ± 0.3 × 10-4	0.47 ± 0.05	2.7 ± 1.4 × 10-4	0.34 ± 0.06

5-HT₃Rs native to neuroblastoma cells have functional properties similar to those of homomeric 5-HT_3A receptors (60). To compare 5-HT₃Rs of neuroblastoma cells with those of known composition, we performed experiments on membranes from HEK cells expressing mouse 5-HT_3A subunits. As shown in Fig. 2C, 5-HT_3ARs bound ¹²⁵I-epibatidine with a K_D of 14 ± 4 nm, which is approximately half of the K_D observed for native receptors in N1E-115 cells. Competition binding experiments were also performed on membranes from HEK cells expressing 5-HT_3ARs. The relative potency in competing for ¹²⁵I-epibatidine binding sites from the HEK cell membranes was epibatidine > MDL72222 > 5-HT = mecamylamine > CPBG nicotine (Table 1).

The Action of Epibatidine on 5-HT₃R Function—Previous studies have shown that some nicotinic receptor agonists, including epibatidine, inhibit serotonin-induced currents in oocytes expressing 5-HT_3ARs (51, 52). Therefore, we investigated the effect of epibatidine on 5-HT₃R function in cells expressing native and recombinant 5-HT₃ receptors. N1E-115 neuroblastoma cells express endogenous functional 5-HT₃Rs (61). 5-HT (30 μm) activated inward currents recorded from N1E-115 cells voltage-clamped at –60 mV. Application of epibatidine to the recording chamber caused a concentration-dependent inhibition of 5-HT-evoked currents (Fig. 3A) with an IC₅₀ of 3.3 ± 0.3 μm (Table 2). Epibatidine had a similar potency (IC₅₀ = 4.7 ± 0.5 μm) as an inhibitor of 5-HT-evoked currents recorded from NB41A3 mouse neuroblastoma cells, which expresses native 5-HT₃Rs with properties similar to those of homomeric 5-HT_3ARs (60). We tested the effect of epibatidine on recombinant mouse 5-HT_3ARs expressed in HEK cells. Epibatidine inhibited 5-HT-evoked currents with an IC₅₀ of 4.1 ± 0.3 μm (Table 2). 5-HT elicited a concentration-dependent activation of current mediated by recombinant 5-HT_3ARs with an EC₅₀ of 5.7 ± 0.7 μm (Fig. 3B). In the presence of epibatidine (30 μm), this response is shifted to the right (EC₅₀ = 10.1 ± 0.3 μm) without altering the maximum current amplitude, indicating that epibatidine is a competitive antagonist at 5-HT₃Rs (Fig. 3B).

FIGURE 3.

Epibatidine inhibits 5-HT-evoked currents. A, epibatidine (0.3–30 μm) inhibited currents activated by 5-HT recorded from NIE-115 cells voltage-clamped at –60 mV in the whole cell patch clamp configuration. On each cell tested, 5-HT (30 μm) was pressure-applied locally for a time sufficient to evoke ∼10% maximal current. The IC₅₀ for epibatidine is 3.3 ± 0.7 μm with a slope of 1.5 ± 0.2, determined from the logistic fit to the data points (n = 5). This value is similar to that determined from similar experiments utilizing either NB41A3 cells (another neuroblastoma cell line) or HEK cells expressing recombinant mouse 5-HT_3ARs (Table 2). B, the 5-HT concentration-response relationship, recorded from HEK cells expressing recombinant mouse 5-HT_3ARs, was shifted to the right by epibatidine (30μm) without reducing the maximum 5-HT-evoked current amplitude. Data were normalized to the maximum amplitudes of currents activated by 5-HT (30 μm) in each cell in the absence of epibatidine. The EC₅₀ and slope values for the 5-HT concentration-response relationship, determined from the logistic fit to the data obtained in the absence and presence of epibatidine, are 5.7 ± 0.7 μm, 1.8 ± 0.3 (n = 8) and 10.1 ± 0.3 μm, 1.8 ± 0.1 (n = 7).

TABLE 2.

Inhibition by epibatidine of 5-HT-evoked currents

	IC50	Hill coefficient
	μm
5-HT3Rs in neuroblastoma cells
N1E-115	3.3 ± 0.3	1.5 ± 0.2
NB41A3	4.7 ± 0.5	1.1 ± 0.1
Ectopic 5-HT3ARs in HEK cells
Mouse 5-HT3A	4.1 ± 0.3	1.6 ± 0.1
Human 5-HT3A	7.6 ± 0.7	1.1 ± 0.1

d-Tubocurarine, a plant alkaloid that inhibits nAChR function, also inhibits 5-HT₃Rs (50). The potency of d-tubocurarine as an antagonist of 5-HT₃R-mediated currents is highly species-dependent. Human 5-HT_3ARs are >1000 times less sensitive to inhibition by d-tubocurarine than are rodent 5-HT_3ARs. Therefore, we examined whether the antagonist potency of epibatidine also varies between mouse and human 5-HT_3ARs. 5-HT (30 μm)-activated currents recorded from HEK cells expressing recombinant human 5-HT_3ARs were inhibited by epibatidine with an IC₅₀ of 7.6 ± 0.7 μm (n = 5), a value that is only slightly higher than the IC₅₀ of epibatidine as an inhibitor of mouse 5-HT_3ARs (Table 2).

Since mecamylamine displaces binding of epibatidine to 5-HT₃Rs (Fig. 2B and Table 1), we examined whether the nAChR antagonist affects 5-HT-activated currents mediated by recombinant 5-HT_3ARs. Mecamylamine (10 μm) caused a 30 ± 4% (n = 4) inhibition of 5-HT-activated currents mediated by human 5-HT_3ARs.

DISCUSSION

In this study, we identify high affinity ¹²⁵I-epibatidine binding sites in the brain with the pharmacological characteristics of 5-HT₃Rs. These sites are distinctly serotonergic, since the 5-HT₃R agonist (CPBG) and antagonist (MDL72222) effectively block ¹²⁵I-epibatidine binding, whereas nicotine is ineffective at blocking binding. Nicotine is also a poor competitor against ¹²⁵I-epibatidine binding to 5-HT_3AR homomers expressed heterologously in HEK cells and endogenous 5-HT₃Rs expressed by N1E-115 mouse neuroblastoma cells. In these cells, epibatidine blocks serotonin-induced currents in a competitive manner with IC₅₀ values similar to that observed for the inhibition of recombinant 5-HT₃R-mediated currents in oocytes (51). Thus, in addition to being an agonist at nAChRs, we conclude that epibatidine is a competitive antagonist of 5-HT₃Rs.

The affinity of epibatidine for 5-HT₃R sites is approximately 3 orders of magnitude lower than for nAChR subtypes that bind epibatidine with high affinity. Epibatidine binds to these nAChRs with K_D values in the 10–100 pm range (57, 62, 63). This variability might arise from ligand depletion during binding studies that can result in underestimation of the affinity of epibatidine for nAChRs. In studies that avoided ligand depletion, epibatidine binds to a single population of brain nAChRs with K_D values in the 1–8 pm range (4, 55). Because of its extremely high affinity for nAChRs, virtually all studies measuring equilibrium binding have used epibatidine concentrations less than 1 nm, which would result in little, if any, binding to 5-HT₃Rs. Thus, the failure to observe epibatidine binding to 5-HT₃Rs in earlier studies is most likely due to the relatively low concentrations of radiolabeled epibatidine used to measure binding to nAChRs. When epibatidine concentrations much higher than 1 nm were used in radiolabeled epibatidine binding studies, a second population of binding sites with lower affinity for epibatidine was observed (4, 64, 65). Based on the works of Marks et al. (65), using nAChR subunit null mice, at least some of the lower affinity epibatidine sites consist of nAChRs. However, data presented in this study suggest that a portion of the lower affinity sites may also consist of 5-HT₃Rs.

Our results are consistent with earlier studies that have established significant pharmacological crossover between 5-HT₃Rs and nAChRs. For example, the nAChR antagonist, d-tubocurarine, and 5-HT₃R-specific antagonists, such as MDL72222 and GR65630, bind to 5-HT₃Rs with comparable affinities. Furthermore, d-tubocurarine blocks 5-HT₃R-mediated currents (49, 50). Similar actions are observed for the nAChR channel blockers, chlorpromazine and QX222 (48). Other studies also show that tropisetron, which is a selective 5-HT₃R antagonist, binds with high affinity and is a potent partial agonist at the α7 nAChR (66), whereas the α7 agonist, PSAB-OFP, acts as a potent agonist at 5-HT₃Rs (67). Relevant to our results, the nAChR agonists, anabaseine and epibatidine, are shown to block serotonin-activated currents in oocytes expressing 5-HT_3ARs with IC₅₀ values of 14 and 8 μm, respectively (51, 52). Similarly, we find that epibatidine effectively blocks rodent 5-HT₃R-mediated currents in a competitive manner with IC₅₀ values of 3.3 and 4.1 μm in neuroblastoma cells and HEK cells, respectively.

The mechanisms underlying the physiological effects of epibatidine have not been fully characterized. The concentrations of epibatidine required for analgesia are in the micromolar range (27, 29, 30, 68, 69) and also produce adverse symptoms, including hypothermia, hypertension, ataxia, and seizures (8, 70). These concentrations are similar to the concentrations that we found antagonize 5-HT₃Rs and are orders of magnitude higher than what is needed to saturate the high affinity binding site on nAChRs but in line with the epibatidine concentrations that activate nAChRs. As an agonist, epibatidine (10–100 nm) increases neurotransmitter release from hippocampal slice preparations (70, 71) and current responses in hippocampal and retinal neurons (72, 73). Using heterologous expression of α4β2 nAChRs, Buisson et al. found a similar dose dependence for epibatidine as an nAChR agonist (74). Further evidence that agonist activity of epibatidine underlies its physiological role is that the nicotinic antagonist mecamylamine blocks both the functional activity and antinociceptive effects of epibatidine (2, 10, 11). However, the fact that mecamylamine has pronociceptive properties (12) and our data showing that mecamylamine competes with the epibatidine binding to 5-HT₃Rs (Table 1) indicate that the previous findings with mecamylamine are also consistent with these ligands interacting with 5-HT₃R sites. Our findings that epibatidine binds with high affinity to and antagonizes 5-HT₃Rs raise questions about where epibatidine elicits its physiological actions and indicate that 5-HT₃Rs should be considered as a potential target.