Introduction

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Nicotinic acetylcholine receptors (nAChRs) are complexes of protein subunits that coassemble to form an ion channel that is gated through the binding of the neurotransmitter acetylcholine (ACh) to its ligand-binding site (Changeux et al., 1987). 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 to 8, 2 to 4 (Sargent, 1993; McGehee and Role, 1995). In heterologous expression systems, the neuronal subunits 2, 3, 4, and 6 lead to the assembly of functional nAChR in combination with either 2 or 4. They 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 7 and 8 subunits form part of a different group within the neuronal nAChR, because they can assemble into functional receptors in the absence of any other subunit and account for the -bungarotoxin-binding sites in the central nervous system (Couturier et al., 1990; Gerzanich et al., 1994).

The cloning of the 9 subunit added a peculiar member to the family of nAChRs (Elgoyhen et al., 1994). It is a distant member 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 39%. When expressed in Xenopus laevis oocytes, 9 forms a homomeric receptor-channel complex that is activated by ACh but not by nicotine; 9 also 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 9 receptor are strikingly similar to those described for the cholinergic receptor that mediates synaptic transmission between efferent cholinergic fibers and cochlear outer hair cells (Housley and Ashmore, 1991; Fuchs and Murrow, 1992; Elgoyhen et al., 1994; Erostegui et al., 1994). Moreover, the 9 subunit gene exhibits a unique and restricted expression pattern. Whereas 9 message has not been found in the central nervous system, it is present in the cochlear and vestibular hair cells (Elgoyhen et al., 1994; Hiel et al., 1996; Morley et al., 1998). This has led to the proposal that the 9 subunit is a component of the cholinergic receptor that is present at the base of the outer hair cells and therefore participates in the efferent modulation of the cochlear amplifier and the control of the dynamic range of hearing (Elgoyhen et al., 1994; Sewell, 1996).

The alkaloid strychnine, an established blocker of glycine-gated chloride channels, is one of the most potent antagonists described so far for both the recombinant 9 and the hair cell native receptors (Elgoyhen et al., 1994; Erostegui et al., 1994). Nicotinic AChRs as well as glycine receptors are members of a family of neurotransmitter-gated ion channels that also includes the type A -aminobutyric acid receptor (GABA_A) and the type 3 serotonin receptor (5-HT₃) (Karlin and Akabas, 1995). The subunits of these receptors have similar sequences and distributions of hydrophobic, membrane-spanning segments. Each subunit contains, in its ligand-binding, amino-terminal half, 2 cysteine residues separated by 13 other residues that are presumably disulfide-linked, thus giving this family the name of the Cys-loop receptors. Although at the level of detailed molecular mechanisms there do exist structural determinants that specify selectivity of ligand binding to each of these receptors, the potent strychnine block of the 9 nAChR indicates that some features are conserved between the 9 nACh and the glycine receptors.

The aim of the present work was to study, on the recombinant 9 receptor, the effect of selective drugs that interact with other members of the Cys-loop family. We report that the 9 nAChR shares several pharmacological properties with GABA_A, 5-HT₃, and glycine receptors.

	Experimental Procedures

Top Summary Introduction Experimental procedures Results Discussion References

Expression in X. laevis Oocytes and Electrophysiological Procedures. A full-length 9 rat cDNA constructed in the vector pGEMHE suitable for X. laevis oocyte expression studies was used as described previously (Elgoyhen et al., 1994). cRNA was synthesized using the mMessage mMachine T7 transcription kit (Ambion, Austin, TX), with plasmid linearized with NheI.

Materials. ACh chloride, GABA, strychnine HCl, ()-bicuculline methbromide, ICS-205,930 HCl, and ()-nicotine-di-d-tartrate were bought from Research Biochemicals (Natick, MA). Serotonin creatinine sulfate and glycine HCl were obtained from Sigma Chemical (St. Louis, MO). Drugs were dissolved in distilled water as 10 mM stocks and stored in aliquots at 20°C. BAPTA/AM-treated oocytes were incubated with the ester for 3 h before experiments. BAPTA/AM (Molecular Probes, Eugene, OR) was stored at 20°C as aliquots of a 100 mM solution in dimethyl sulfoxide. Aliquots were thawed and diluted 1000-fold into saline solution shortly before incubation of the oocytes.

	Results

Top Summary Introduction Experimental procedures Results Discussion References

Interaction of GABAergic, Glycinergic, and Serotoninergic Drugs with the 9 nAChR. Voltage-clamped X. laevis oocytes injected with 9 cRNA responded to ACh with a fast peak current that rapidly decayed to a plateau level. Fig. 1 shows representative traces in the presence of 10 µM ACh, a concentration previously shown to correspond to the EC₅₀ of the agonist (Elgoyhen et al., 1994). As expected for an nAChR, neither GABA, glycine, nor serotonin evoked inward currents in 9-injected oocytes (Fig. 1). Moreover, neither GABA nor glycine modified responses to ACh, and traces obtained in the presence of these drugs did not differ from the control traces (Fig. 1, A and B). However, ACh-evoked currents were reduced by serotonin. As shown in Fig. 1, C and D, serotonin blocked both peak and plateau responses to ACh in a concentration-dependent manner with an IC₅₀ of 251 ± 30 µM.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of agonists of Cys-loop receptors on the 9 nAChR. Shown in A, B, and C are representative current responses to 10 µM ACh either alone or in the presence of GABA, glycine, or serotonin, respectively. D, inhibition curve performed by the coapplication of 10 µM ACh and increasing concentrations of serotonin. Only peak current values are plotted, expressed as the percentage of the peak control current evoked by ACh. The mean and S.E.M of five experiments are shown.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of antagonists of Cys-loop receptors on the 9 nAChR. A, representative traces to 10 µM ACh either alone or in the presence of increasing concentrations of bicuculline, strychnine or ICS-205,930. B, inhibition curves performed by the coapplication of 10 µM ACh and increasing concentrations of antagonists. Only peak current values are plotted, expressed as the percentage of the peak control current evoked by ACh. The mean and S.E.M of three to five experiments per group are shown.

Mechanism of Block. Serotonin interacts with the binding site of 5-HT₃ receptors and gates channel opening (Maricq et al., 1991). On the other hand, bicuculline, ICS-205,930, and strychnine are known to interact with GABA_A, 5-HT₃, and the glycine receptor-binding sites, respectively, and to block agonist-evoked responses in a competitive manner (Akaike et al., 1987; Maricq et al., 1991; Schmieden et al., 1992). To further characterize the mechanism underlying the blocking effects on the 9 nAChR, block by drugs was studied at increasing concentrations of the agonist. The concentrations of antagonists tested were the ones that corresponded to the IC₅₀ values derived from Figs. 1D and 2B. As shown in Fig. 3, 1 µM bicuculline, 20 nM strychnine, and 300 µM serotonin produced a parallel rightward shift of ACh-evoked currents. A significant increase of the ACh EC₅₀ values was observed, with no changes in agonist maximal responses and Hill coefficients (Table 1), which suggests a competitive type of block.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Displacements of ACh concentration-response curves. Concentration-response curves to ACh were performed either alone or in the presence of 1 µM bicuculline, 20 nM strychnine, or 300 µM serotonin. Peak current values were normalized and referred to the maximal peak response to ACh. Mean and S.E.M of six to eight experiments per group are shown.

Block of the 9 nAChR in BAPTA/AM-Treated Oocytes. In 9-injected oocytes, part of the ACh-evoked response is carried by a Ca⁺⁺-activated Cl current (Elgoyhen et al., 1994). To analyze whether the effect described is a direct block on the 9 receptor and not a nonspecific block of the oocyte Cl channel, the effect of drugs was studied in oocytes that had been treated with the fast Ca⁺⁺ chelator BAPTA/AM. The effectiveness of the treatment with BAPTA/AM was assessed as described in Experimental Procedures. Antagonists were applied at plateau responses achieved with two different ACh concentrations: a low, nonsaturating one (10 µM) and a saturating maximal concentration (300 µM) (Fig. 4). Responses to 10 µM ACh were blocked 82 ± 5% (n = 3), 40 ± 7% (n = 3), and 51 ± 8% (n = 3) in the presence of 1 µM bicuculline, 20 nM strychnine, and 300 µM serotonin, respectively. The blocking effect was drastically reduced or abolished when the ACh concentration was raised to 300 µM. This result suggests again that the block by the drugs tested is competitive and that the observed effect is a direct block on the 9 receptor.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Inhibition of ACh-evoked currents in BAPTA/AM-treated oocytes. BAPTA/AM-treated oocytes were incubated with the ester for 3 h before experiments and oocytes were voltage-clamped at 70 mV. Shown are representative traces of three experiments per group obtained at two different ACh concentrations: 10 µM in A and 300 µM in B. Either bicuculline, strychnine, or serotonin were applied at steady-state responses to ACh.

Block of the 9 nAChR in Ba⁺⁺ Frog Saline. To preclude the possibility of activation of a remaining Cl current in BAPTA/AM-treated oocytes, a set of experiments was performed in frog saline in which Ca⁺⁺ was replaced by Ba⁺⁺. As described for BAPTA/AM-treated oocytes, antagonists were applied at plateau responses achieved with two different ACh concentrations: a low, nonsaturating one (10 µM) and a saturating maximal concentration (300 µM) (Fig. 5). Responses to 10 µM ACh were blocked 92 ± 6% (n = 3), 69 ± 12% (n = 3), and 68 ± 6% (n = 4) in the presence of 1 µM bicuculline, 20 nM strychnine, and 300 µM serotonin, respectively. The blocking effect was drastically reduced or abolished when the ACh concentration was raised to 300 µM. This result yet again indicates that the effect of the drugs tested is a direct block on the 9 receptor.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Inhibition of ACh-evoked currents in Ba++ frog saline. Oocytes were voltage-clamped at 90 mV. Shown are representative traces of three to four experiments per group obtained at two different ACh concentrations: 10 µM in A and 300 µM in B. Either bicuculline, strychnine, or serotonin were applied at steady-state responses to ACh.

	Discussion

Top Summary Introduction Experimental procedures Results Discussion References

The present study contributes to the pharmacological characterization of the newly cloned 9 nAChR and indicates that this receptor shares striking properties with other members of the Cys-loop family of receptors. Thus, the recombinant 9 nAChR is blocked by GABA_A, 5-HT₃, and glycine receptor antagonists.

The IC₅₀ values found for bicuculline and strychnine block of 9, 0.8 µM and 0.02 µM, respectively, are similar to those reported for GABA_A (0.9 µM; Sigel et al., 1992) and glycine receptors (0.05 µM; Schmieden et al., 1992) expressed in X. laevis oocytes. In addition, the nanomolar potency of ICS-205,930 to block ACh-evoked currents in 9-injected oocytes is in the same order of magnitude as that required for both recombinant (Maricq et al., 1991) and native 5-HT₃ receptors present in the guinea pig submucosal plexus and rabbit heart (Vanner and Suprenant, 1990; Turconi et al., 1991). Moreover, among all of the nicotinic antagonists tested on 9-injected oocytes, only -bungarotoxin and -bungarotoxin have high blocking potencies that are comparable to those of strychnine and ICS-205,930 (Elgoyhen et al., 1994; Johnson et al., 1995). Nicotinic drugs such as d-tubocurarine, mecamylamine, and dihydro--erythroidine have IC₅₀ values in the micromolar range (Elgoyhen et al., 1994; unpublished observations). Taken together, these results indicate that, based on its pharmacological properties, the 9 subunit is an unusual member of the nAChR family. It is activated by ACh (although not by nicotine; Elgoyhen et al., 1994); therefore, it should be considered to be in the cholinergic family of ionotropic receptors. However, the profile of block by antagonists does not allow the inclusion of the 9 subunit in any specific Cys-loop subfamily of receptors. The present observations are in accordance with the finding that the comparison of sequence similarities and gene structure indicates that 9 is the most distant member within the nAChR family. Some amino acid residues that are conserved along all members of the gene family have a nonconservative substitution in the 9 primary structure, which might contribute to the unique properties of this receptor (Elgoyhen et al., 1994).

The blockage of the 9 nAChR by serotonin resembles what has been described for other nAChRs. The function of native and recombinant nAChRs can be modified by this neurotransmitter (García-Colunga and Miledi, 1995; Palma et al., 1996). Thus, 7 nAChRs expressed in X. laevis oocytes are blocked by micromolar concentrations of serotonin (Palma et al., 1996). In contrast to that found for the 9 nAChR, the block of the 7 receptor is noncompetitive, which suggests different underlying modes of action. Sensitivity to bicuculline has been reported for nAChRs present in isolated pig pituitary intermediate lobe cells and cultured embryonic rat skeletal muscle (Zhang and Feltz, 1991; Liu et al., 1994). However, the IC₅₀ values reported in those preparations are 1 to 2 log units higher than those found for bicuculline block of both 9 and GABA_A (present results; Sigel et al., 1992). Moreover, although 7 nAChRs are also blocked by strychnine (Gerzanich et al., 1994), the potency of this antagonist on 7 receptors is 2 orders of magnitude lower than that reported for both 9 (present observations) and glycine receptors (Schmieden et al., 1992). Therefore, our results indicate that among the nAChR gene family, it is only 9 that most closely resembles other members of the Cys-loop family.

Members of the Cys-loop family of receptors include both cationic, 5-HT₃, and nACh, as well as anionic, glycine, and GABA_A receptors. They all have a high degree of amino acid sequence similarity and some highly characteristic sequence motifs, both in the binding, extracellular, amino-terminal domain, and in the four hydrophobic, putative transmembrane regions (Karlin and Akabas, 1995). They all share a common evolutionary ancestor, and within the cationic branch, the homo-oligomeric receptors are probably the most primitive of all receptors because they conserve the closest similarity with the hypothetical ancestor (Le Novere and Changeux, 1995; Ortells and Lunt, 1995). When expressed in X. laevis oocytes, the 9 nAChR forms homo-oligomeric receptors, as well as continuing to conserve pharmacological properties typical of each of the subfamilies, which suggests that it is a primitive member of the Cys-loop family and that it had a very early evolutionary split. In support of this hypothesis is the observation that nAChRs in organisms that appeared before mammals in evolution, such as the nematode Ascaris summ, the marine snail Aplysia sp. and the insect Schistocerca sp., are sensitive to both strychnine and bicuculline block (Ono and Salvaterra, 1981; Marshall et al., 1990; Walker et al., 1992).

The simplest interpretation of the competitive type of block as suggested here for bicuculline, strychnine, and serotonin on the 9 nAChR is that these compounds share at least part of the binding pocket with the agonist, in such a way that occupancy of the site is mutually exclusive. Within the Cys-loop family of receptors, the amino-terminal extracellular domain is known to form the binding site. In the most thoroughly characterized member of this class of receptors, the nAChR, several residues in the extracellular domain of the  subunit have been identified as forming part of the agonist- and antagonist-binding sites using photoaffinity labeling and site-directed mutagenesis (Galzi et al., 1991; Karlin and Akabas, 1995). The fact that the 9 nAChR shares pharmacological properties with GABA_A, 5-HT₃, and strychnine receptors indicates that it must conserve in its primary structure some residues that are common to each of these receptors and that are responsible for agonist and antagonist binding. Asp-148, Tyr-161, and Tyr-202, known as determinants in the strychnine-binding site of the glycine receptor, are conserved in the 9 nAChR (Vandenberg et al., 1992a, 1993; Elgoyhen et al., 1994). Thus, the binding sites for antagonists on the glycine receptor and the 9 nAChR would be conserved and would form a similar tertiary structure, leading to a common mechanism of antagonism in these receptors. Glycine and GABA are simple molecules, and the number of specific interactions that they can achieve with their respective receptors is limited. Removal of one such interaction would be expected to result in a dramatic reduction in the affinity of agonists, but not antagonists, for the receptor (Vandenberg et al., 1992b). This might explain the fact that although both bicuculline and strychnine block the 9 nAChR, neither GABA nor glycine modify ACh-evoked currents or elicit responses in 9-injected oocytes. Thr-204, shown to be important for glycine-binding but not strychnine-binding to its receptor, is not conserved in the 9 nAChR (Vandenberg et al., 1992b; Elgoyhen et al., 1994).

Although not typical of what has been described for an nAChR, the bicuculline and strychnine block of the recombinant 9 nAChR resembles what has been shown for the native cholinergic receptor present in outer hair cells. Thus, nanomolar concentrations of strychnine and micromolar concentrations of bicuculline block ACh-evoked currents in both isolated guinea pig outer hair cells (Erostegui et al., 1994) and 9 injected oocytes. As suggested previously (Elgoyhen et al., 1994), these findings add further data to support the hypothesis that the 9 nAChR is a component of the cholinergic receptor present at the base of the outer hair cells, responsible for the efferent modulation of the cochlear amplifier.