Introduction

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Nicotinic acetylcholine receptors (nAChRs) are complexes of protein subunits that coassemble to form an ion channel gated through the binding of the neurotransmitter ACh to its ligand-binding site (Karlin, 2002). A diversity of subunits have been cloned in recent years. The nAChR at the neuromuscular junction mediates fast synaptic transmission and is thought to have a (1)₂1 stoichiometry (Galzi et al., 1991). Ten genes that encode neuronal nAChR subunits have been identified in the vertebrate central or peripheral nervous system: 2-8 and 2-4 (Le Novére and Changeux, 1999). In heterologous expression systems, the neuronal nAChRs may assemble from single -subunits, from multiple - and -subunits, and heteromeric nAChRs formed via pair-wise combinations of 2, 3, 4, or 6 with either the 2 or 4 subunits (for references, see Elgoyhen et al., 2001). Neuronal nAChRs preserve the structural motif of muscle nAChR, with a pentameric structure that includes two  and three  subunits (Anand et al., 1991; Cooper et al., 1991).

The cloning of the 9 and 10 subunits added two peculiar members to the family of nAChRs (Elgoyhen et al., 1994, 2001). They are distant members of the family; whereas neuronal nAChR  subunits and the muscle 1 subunit share sequence homologies ranging from 48 to 70%, the sequence identity between 9 and all known nAChR subunits is less than 40%. When expressed in Xenopus laevis oocytes, 9 and 10 subunits form a heteromeric receptor-channel complex that is activated by ACh but not by nicotine and displays a very distinct pharmacological profile that falls into neither the nicotinic nor the muscarinic subdivision of the pharmacological classification scheme of cholinergic receptors. However, the properties of the recombinant 910 receptor are strikingly similar to those described for the cholinergic receptor that mediates synaptic transmission between efferent cholinergic fibers and cochlear outer hair cells (Fuchs, 1996; Elgoyhen et al., 2001). Moreover, the 9 and 10 gene subunits exhibit a unique and restricted expression pattern. Whereas 9 and 10 message has not been found in the central nervous system, it is present in the cochlear and vestibular hair cells (Elgoyhen et al., 1994, 2001; Hiel et al., 1996; Morley et al., 1998). This has led to the proposal that efferent modulation of cochlear and vestibular hair cell function occurs, at least in part, via heteromeric nAChRs assembled from both 9 and 10 subunits (Elgoyhen et al., 2001).

Nicotinic AChRs are members of a family of neurotransmitter-gated ion channels that also includes, GABA_A, GABA_C, glycine, 5-HT₃ and some invertebrate anionic glutamate receptors (Le Novére and Changeux, 1999). The subunits of these receptors have similar sequences and distributions of hydrophobic, membrane-spanning segments. Each subunit contains in its ligand-binding N-terminal half two cysteine residues separated by 13 other residues that are presumably disulfide-linked, thus giving this family the name of the Cys-loop receptors. We have demonstrated (Rothlin et al., 1999) that homomeric 9 nAChRs share several pharmacological properties with GABA_A, glycine, and 5-HT₃ receptors. The aim of the present work was to perform an extensive characterization of the effects of 5-HT₃ receptor ligands on recombinant and native 910-containing nicotinic cholinergic receptors. Our results indicate that the recombinant 910 nAChR shares striking pharmacological properties with type 3 serotonin ligand-gated ion channels. Moreover, it demonstrates an in vivo correlate of these properties of the recombinant receptor, with those of the 910-containing nAChR of frog saccular hair cells.

	Materials and Methods

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Expression of Recombinant Receptors in X. laevis Oocytes. For expression studies, 4, 7, 9, 10, and 2 rat nAChR subunits were subcloned into a modified pGEMHE vector (Liman et al., 1992). Capped cRNAs were in vitro transcribed from linearized plasmid DNA templates using the mMessage mMachine T7 transcription kit (Ambion Corporation, Austin, TX). The maintenance of X. laevis and the preparation and cRNA injection of stage V and VI oocytes have been described in detail elsewhere (Katz et al., 2000). Typically, oocytes were injected with 50 nl of RNase-free water containing 0.01 to 1.0 ng of cRNA (at a 1:1 molar ratio when pair-wise combined) and maintained in Barth's solution at 17°C.

Isolation and Patch Clamp Recording from Saccular Hair Cells. Frog saccular hair cells were isolated enzymatically. The dissociation protocol, optimized for the purpose of observing reliable ACh responses, was described previously (Holt et al., 2001). Briefly, leopard frogs (Rana pipiens) were chilled, pithed,¹ and decapitated. Each side of the head was placed into a perilymph-like standard external solution composed of 105 mM NaCl, 2.5 mM KCl, 0.81 mM MgCl₂, 1.8 mM CaCl₂, 3.4 mM NaHCO₃, 0.5 mM NaH₂PO₄, 2.5 mM Na₂HPO₄, 1 mM ascorbate, 4 mM glucose, and 5 mM pyruvate. The otic capsule from each side was opened to gain access into the inner ear. The saccule was dissected, and the macula was excised from the saccule. The macula was then incubated in 0.05% trypsin in Hanks' balanced salt solution (Invitrogen, Carlsbad, CA) for 8 to 10 min, after 30 s of rinsing in a low-calcium dissociation solution (105 mM NaCl, 2.5 mM KCl, 0.81 mM MgCl₂, 0.1 mM CaCl₂, 3.4 mM NaHCO₃, 0.5 mM NaH₂PO₄, and 2.5 mM Na₂HPO4, 1 mM ascorbate, 4 mM glucose, and 5 mM pyruvate), which contained 10% fetal calf serum (Invitrogen). It was then washed in dissociation solution containing 500 µg/ml bovine serum albumin for 5 to 10 min. The macula was then transferred to the recording chamber, previously filled with external solution, and hair cells were gently separated from the epithelium with the help of a thin, hook-shaped glass wisp. The hair cells settled to the base of the recording chamber and adhered firmly to the bottom, which was precoated with 2 mg/ml concanavalin A. The bath was perfused with external solution at a rate of 1 ml/min. All solutions had an osmolality of 220 mOsm and a pH of 7.2.

Materials. ACh chloride or iodide was bought from Sigma Chemical Co. (St. Louis, MO). Serotonin hydrochloride, 1-(m-chlorophenyl)-biguanide hydrochloride, trimethyl serotonin iodide (5HTQ), 3-tropanyl-indole-3-carboxylate hydrochloride (tropisetron; ICS 205,930), and 3-tropanyl-3,5-dichlorobenzoate (MDL 72222) were obtained from RBI/Sigma (Natick, MA). Ondansetron hydrochloride was kindly donated by Raffo Laboratories (Buenos Aires, Argentina). Drugs were dissolved in distilled water as 10 mM stocks and stored aliquoted at 20°C. BAPTA-AM (Molecular Probes, Eugene, OR) was stored at 20°C as aliquots of a 100 mM solution in dimethyl sulfoxide, thawed, and diluted 1000-fold into saline solution shortly before incubation of the oocytes.

	Results

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Effect of Serotonin on ACh-Evoked Currents through Recombinant 910 nAChRs. Figure 1A shows representative responses to 10 µM ACh of X. laevis oocytes injected with 9 and 10 cRNAs. As expected for a nAChR, serotonin did not evoke currents through the 910 receptor. However, currents elicited by ACh were blocked by serotonin in a concentration-dependent manner (Fig. 1, A and B) with an IC₅₀ of 5.4 ± 0.6 µM (mean ± S.E.M.; n = 6). Block by serotonin was reversible, because initial control responses to ACh were recovered after washes of the oocytes with frog saline (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of serotonin on ACh-evoked currents through recombinant 910 nAChRs. A, representative traces to ACh either alone or in the presence of serotonin. B, inhibition curve performed by the coapplication of 10 µM ACh and increasing concentrations of serotonin. Oocytes were incubated with each concentration of serotonin for 2 min before the addition of ACh. Peak current values are plotted, expressed as the percentage of the peak control current evoked by ACh. The mean and S.E.M. of six experiments are shown.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Mechanism of blockage by serotonin of ACh-evoked responses. A, concentration-response curves to ACh performed either alone or in the presence of 10 µM serotonin. Peak current values were normalized and referred to the maximal peak response to ACh. The mean and S.E.M. of three to four experiments per group are shown. B, representative I-V curves obtained upon application of 2-s voltage ramps (120 to +50 mV) 10 s after the peak response to 10 µM ACh from a holding potential (Vhold) of 70 mV, either alone or in the presence of serotonin (n = 3 for each curve).

Effect of Serotonin Type 3 Receptor Agonists on the Recombinant 910 nAChR. To analyze whether the effect observed with serotonin could be extended to other serotoninergic agonists, the effect of 5HTQ and 1-(m-chlorophenyl)-biguanide, selective for 5-HT₃ receptors, was studied on 910-injected X. laevis oocytes. Contrary to that observed with serotonin, both 5-HT₃ agonists elicited inward currents through the 910 receptor (Fig. 3A). However, both compounds behaved as weak partial agonists, because maximal responses were only 10.8 ± 0.8% (n = 3) for 5HTQ and 3.1 ± 0.7% (n = 3) for 1-(m-chlorophenyl)-biguanide of the maximum obtained with ACh (Fig. 3B).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Effect of serotonin type 3 receptor agonists on the recombinant 910 nAChR. A, representative current responses to 5-HT3 receptor agonists. B, concentration-response curves to ACh, 5HTQ, and 1-(m-chlorophenyl)-biguanide. Peak current values were normalized and referred to the maximal peak response to ACh. The mean and S.E.M. of three experiments per group are shown.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of 1-(m-chlorophenyl)-biguanide on ACh-evoked currents. A, representative traces to ACh either alone or in the presence of 10 µM or 100 µM 1-(m-chlorophenyl)-biguanide. Oocytes were preincubated for 2 min with 1-(m-chlorophenyl)-biguanide before the addition of 10 µM ACh. B, responses to 10 µM ACh at different concentrations of 1-(m-chlorophenyl)-biguanide (n = 4-6); *, p < 0.05; **, p < 0.01, Tukey's test. C, representative I-V curves obtained upon application of 2-s voltage ramps (120 to +50 mV) 10 s after the peak response to 10 µM ACh from a holding potential (Vhold) of 70 mV, either alone or in the presence of 1-(m-chlorophenyl)-biguanide (n = 3 for each curve). D, concentration-response curves to ACh performed either alone or in the presence of 100 µM 1-(m-chlorophenyl)-biguanide. Peak current values were normalized and referred to the maximal peak response to ACh. The mean and S.E.M. of 10 experiments per group are shown.

Effect of Serotonin on ACh-Evoked Currents in Isolated Saccular Hair Cells. The effect of serotonin on a native hair cell cholinergic receptor preparation was analyzed on isolated frog vestibular hair cells. It has been described previously that hyperpolarization of the frog saccular hair cells in response to the exogenous application of ACh is caused by the activation of 9-containing nicotinic cholinergic receptors and the subsequent activation of the small-conductance, Ca²⁺-activated (SK) potassium current (Athas et al., 1997; Lioudyno et al., 2000; Holt et al., 2001). The 9-containing nAChR in frog saccular hair cells is pharmacologically indistinguishable from the 910 nAChR of the cochlear hair cells and the recombinant 910 nAChR, suggesting that both subunits are involved in the ACh-evoked response (Fuchs, 1996; Elgoyhen et al., 2001; Holt et al., 2001). As shown in the representative traces of Fig. 5A, the application of ACh to isolated saccular hair cells typically produces an outward current. The concentration of ACh that produces a half-maximal current response (EC₅₀) has been reported previously to be 18.4 µM (Holt et al., 2001).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effect of serotonin on ACh-evoked currents in isolated saccular hair cells. A, representative traces of ACh-evoked currents recorded from isolated frog saccular hair cells either alone or in the presence of serotonin (black traces). The membrane holding potential was stepped from 60 to 10 mV during the voltage-clamp protocol (gray, control traces). B, inhibition curve performed by the coapplication of 20 µM ACh and increasing concentrations of serotonin. Cells were incubated with each concentration of serotonin for 2 min before the addition of ACh. Current values are plotted, expressed as the percentage of the control current evoked by ACh. The data shown are the mean ± S.E.M. of four experiments.

Effect of Serotonin Type 3 Receptor Antagonists on ACh-Evoked Currents through Recombinant 910 nAChRs. Tropisetron, an established blocker of 5-HT₃ receptors (Vanner and Sruprenant, 1990; Maricq et al., 1991), is one of the most potent antagonists described so far for the 910 nAChR (Fig. 6) (Elgoyhen et al., 2001). High sensitivity of 910 to 5-HT₃ antagonists seems to be a pharmacological property of this receptor and is not a peculiarity restricted to tropisetron. Thus, ACh-evoked currents in 910-injected oocytes were also blocked by the 5-HT₃ antagonists ondansetron and MDL 72222 (Fig. 6, A and B). In all cases, the effect was concentration-dependent, with a rank order of potency of tropisetron (IC₅₀, 70.1 ± 0.9 nM, n = 8) > ondansetron (IC₅₀, 0.6 ± 0.1 µM, n = 6) = MDL 72222 (IC₅₀, 0.7 ± 0.1 µM, n = 4). Block by antagonists was reversible, because initial control responses to ACh were recovered after washes of the oocytes with frog saline (not shown). Moreover, antagonists did not elicit per se responses in oocytes expressing 910 receptors. On the other hand, ketanserin (3 µM), an antagonist of 5-HT2A and 5-HT2C metabotropic serotonin receptors, did not significantly block ACh-evoked currents (n = 3 oocytes).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of serotonin type 3 receptor antagonists on ACh-evoked currents through recombinant 910 nAChRs. A, representative traces to 10 µM ACh, either alone or in the presence of tropisetron, ondansetron, or MDL 72222. B, inhibition curves performed by the coapplication of 10 µM ACh and increasing concentrations of the antagonists. Oocytes were incubated with each concentration of antagonists for 2 min before the addition of ACh. Peak current values are plotted, expressed as the percentage of the peak control current evoked by ACh. C, concentration-response curves to ACh performed either alone or in the presence of 0.3 µM tropisetron. Peak current values were normalized and referred to the maximal peak response to ACh. The mean and S.E.M. of four to ten experiments per group are shown in B and C.

Effect of Serotonin Type 3 Receptor Antagonists on ACh-Evoked Currents in Isolated Saccular Hair Cells. The native vestibular and cochlear hair cell cholinergic receptor and the recombinant 910 nAChR share similar pharmacological properties (for references, see Elgoyhen et al., 2001). To analyze whether this also holds true in the case of 5-HT₃ receptor antagonists, the effect of these compounds was studied on a native hair cell cholinergic receptor preparation, such as the isolated frog saccular hair cells.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of serotonin type 3 receptor antagonists on ACh-evoked currents in isolated saccular hair cells. A, representative traces to 20 µM ACh, either alone or in the presence of tropisetron, ondansetron, or MDL 72222 (black traces). The cells were held at 60 mV and the command potential was stepped from 60 to 10 mV during the voltage-clamp protocol (gray, control traces). B, inhibition curves performed by the coapplication of 20 µM ACh and increasing concentrations of the antagonists. Cells were incubated with each concentration of antagonist for 2 min before the addition of ACh. Peak current values are plotted, expressed as the percentage of the peak control current evoked by ACh. The mean ± S.E.M of four to five experiments per antagonist concentration are shown.

Effect of Tropisetron on 42 and 7 nAChRs. To analyze whether the block by tropisetron is selective for the 910 nAChR or is a general feature common to other nAChRs, the effect of this compound was studied on recombinant 42 and 7 nAChRs. Although 42 nAChRs were blocked by tropisetron (Fig. 8A), the IC₅₀ value (5.9 ± 0.6 µM, n = 5) derived from the concentration-response curves, was 2 orders of magnitude higher than that required to block 910 nAChRs. On the other hand, tropisetron elicited inward currents in 7-injected oocytes, albeit with a maximal response that reached only 15.4 ± 1.1% of the maximum obtained with nicotine (Fig. 8B).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effect of tropisetron on 42 and 7 nAChRs. A, inhibition curve performed by the coapplication of 10 µM ACh and increasing concentrations of tropisetron to 42-injected oocytes. Peak current values are plotted, expressed as the percentage of the peak control current evoked by ACh. B, concentration-response curves to tropisetron and nicotine in 7-injected oocytes. Peak current values were normalized and referred to the maximal peak response to nicotine. The mean and S.E.M. of three to five experiments per group are shown.

	Discussion

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The present study contributes to the pharmacological characterization of the 910 nAChR and indicates that this receptor shares striking properties with type 3 serotonin ligand-gated ion channels. Moreover, it demonstrates an in vivo correlate of these properties of the recombinant receptor, with those of the 910-containing nAChR of frog saccular hair cells. Finally, it poses the possibility that the biogenic amine serotonin might act as a neuromodulator of the cholinergic efferent transmission in the vestibular apparatus and in the organ of Corti.

The IC₅₀ values found for tropisetron, ondansetron, and MDL 72222 to block ACh-evoked currents on the 910 and on the native hair cell receptor are in the same order of magnitude as those required for both recombinant (Maricq et al., 1991; Dubin et al., 1999) and native 5-HT₃ receptors present in the guinea pig submucosal plexus and the rabbit heart (Vanner and Sruprenant, 1990; Turconi et al., 1991). Moreover, among all the compounds tested on the 910 nAChR (Elgoyhen et al., 2001; Sgard et al., 2002), the 5-HT₃ antagonist tropisetron/as well as the glycinergic antagonist strychnine and the nicotinic antagonist -bungarotoxin, have the highest blocking potencies. Thus, the heteromeric 910 receptor has peculiar pharmacological properties within the nAChR family, and resembles what has been described previously for the homomeric 9 recombinant receptor, which preserves pharmacological properties that are characteristic of other members of the Cys-loop family of ligand-gated ion channels (Rothlin et al., 1999). Moreover, this finding is in accordance with the fact that 9 shares with 10 the highest amino acid sequence identity, being more distantly related to other members of the nAChR family (Elgoyhen et al., 2001).

The binding of 5-HT₃ antagonists such as tropisetron to 5-HT₃ receptors is known to require several tryptophan residues in the extracellular amino terminus, probably involved in cation- interactions with the positive amine in the tropane of these antagonists (Venkataraman et al., 1999; Yan et al., 1999; Spier and Lummis, 2000). Interestingly, in the 9 and 10 nAChR subunits, most residues aligned with the critical tryptophans are conserved. Thus the high-affinity site of interaction of tropisetron with the 910 nAChR could be extracellular and resemble its site on the 5-HT₃ receptor. This is supported by the finding that blockage of the 910 receptor by tropisetron is surmounted at high concentrations of ACh, compatible with a competitive type of block. Competitive antagonists of members of the Cys-loop family of ligand-gated ion channels are most probably binding with an extracellular site of the receptor (Karlin, 2002). Moreover, tropisetron turns to a high-affinity agonist of an 910 receptor bearing a leucine-to-threonine mutation at position L9' within transmembrane region II (P. V. Plazas, E. Katz, and A. B. Elgoyhen, unpublished observations), further reinforcing the hypothesis of an extracellular amino-terminal binding site for this compound.

Within the nAChR family, high-affinity (nanomolar) block by the 5HT₃ antagonist tropisetron seems to be a distinctive feature of 9 (Rothlin et al., 1999) and 910 receptors. As derived from Fig. 8, micromolar concentrations of this antagonist are required to block 42 receptors expressed in X. laevis oocytes. Moreover, affinity constants for tropisetron on 42 and 11 derived from binding assays are all in the micromolar range (Macor et al., 2001). On the other hand, although tropisetron binds with high affinity to 7 nAChRs (Macor et al., 2001), it behaves as a weak partial agonist of this receptor subtype (Fig. 8) (Macor et al., 2001).

From a pharmacological standpoint, ondansetron and tropisetron are widely prescribed drugs for chemotherapy-induced emesis (Hesketh, 2000). A myriad of signaling pathways lead from the periphery to the emetic center, including inputs from the vestibular apparatus, of special importance during motion sickness (Pasricha, 2001). Because of its high affinity for the 910 receptor, tropisetron will block the 910-containing vestibular cholinergic receptor at the concentrations used in therapeutics. This effect might (or might not) contribute to its antiemetic action, a question that will only be elucidated when a better understanding of the function of the efferent system to the vestibular apparatus is gained.

The observation that 910 and the native saccular hair cell receptors were blocked by serotonin at low micromolar concentrations and by an apparent noncompetitive mechanism was unexpected. Block of the closely related homomeric 9 receptor by serotonin seems to be competitive and requires high micromolar to millimolar concentrations (IC₅₀, 251 µM; Rothlin et al., 1999), thus suggesting that serotonin probably binds to different sites within the 9 and the 910 receptor proteins. Moreover, the fact that block of ACh-evoked currents through 910 was dependent upon variations of the membrane holding potential, being evident only at hyperpolarized potentials, might suggest that serotonin is acting at a site within the channel lumen. An interaction of serotonin with the ion channel lumen has been reported for other nAChRs (Arias, 1998). However, although serotonin also blocks neuronal 7, 34, 44, 24, and 32 (Nakazawa et al., 1995; Palma et al., 1996), the apparent affinity for these receptor subtypes is 1 or 2 orders of magnitude lower than that reported here for the 910 nAChR. Therefore, within the nAChR family of receptors, the antagonist specificity of serotonin is highest for the 910 receptor.

Serotonin differs from other 5-HT₃ receptor agonists such as 5HTQ and 1-(m-chlorophenyl)-biguanide in that the latter behave as weak partial agonists of the recombinant 910 receptor. However, as observed in Fig. 4, the effect of 1-(m-chlorophenyl)-biguanide is more complex than just a partial agonism, because both potentiation and inhibition of ACh-evoked responses were observed, probably indicating more than one site of interaction of the compound with the receptor. As reported for the effect of atropine on 44 nicotinic receptors and curare-like drugs on neuronal nicotinic receptors containing the 4 subunit, where both potentiation and blockage of ACh-evoked responses are observed (Cachelin and Rust, 1994; Zwart and Vivjerberg, 1997), potentiation might result from the interaction with the agonist binding site of the receptor, where simultaneous occupation of the two binding sites, one agonist recognition site with ACh and the other agonist recognition site with 1-(m-chlorophenyl)-biguanide, leads to subsequent ion channel activation. On the other hand, blockage was noncompetitive and independent of the membrane holding potential, thus suggesting an interaction with a regulatory site independent of the ion channel pore. As for all experiments in which more than one receptor subunit is expressed, one cannot preclude the possibility of assembly of different receptors subtypes with different stoichiometries to account for the different effects of 1-(m-chlorophenyl)-biguanide. Further studies that go beyond the scope of the present work will be required to fully explain the complex effects of 1-(m-chlorophenyl)-biguanide on the 910 receptor.

Does the interaction of serotonin with the 910 recombinant and the saccular hair cell cholinergic receptor underlie a physiological implication? The function of a serotonergic innervation to the inner ear is unknown. Intracochlear injection of serotonin reduces the compound action potential of the auditory nerve (Bobbin and Thompson, 1978). Recent evidence suggests the presence of serotonin and its metabolite 5-hydroxyindole-3-acetic acid in the mammalian cochlea and vestibule (Gil-Loyzaga et al., 1997a,b, 2000) and subunit A of 5-HT₃ receptors has been identified by in situ hybridization in the cochlear and vestibular sensory epithelium of the developing rat (Johnson and Heinemann, 1995). In the case of the organ of Corti, serotonergic fibers reach the cochlea accompanying the olivocochlear efferent system. At the periphery, these fibers form glomerulus-like structures within the inner spiral bundle and could directly contact inner hair cells. They reach the Corti's tunnel and then follow a spiral distribution, branching at least on the first row of outer hair cells (Gil-Loyzaga et al., 2000). The fact that serotonergic fibers are present at the base of the outer hair cells, together with the finding that low micromolar concentrations of serotonin block ACh-evoked currents through the 910 receptor, opens the possibility that serotonin might function as a neuromodulator of the efferent cholinergic innervation to the inner ear.