Introduction

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Neuronal nAChRs are ligand-gated cation channels composed of  and  subunits. Eight different  subunits (2-9) and three different  subunits (2-4) have been identified in vertebrate neuronal tissue, which allows for the possibility of multiple nAChR subtypes that display a range of functional and pharmacological characteristics. In rat brain, the two most abundant nAChR subtypes (as measured by the density of binding sites) are one composed of 4 and 2 subunits (Whiting et al., 1991; Flores et al., 1992) and another that may be composed of 7 subunits only (Schoepfer et al., 1990; Seguela et al., 1993). The relative abundance of the mRNA encoding nAChR subunits in brain is consistent with the prevalence of these two receptor subtypes (Wada et al., 1989; Seguela, et al., 1993). However, the CNS expresses mRNA for all of the nAChR subunits, and despite their lower abundance in brain overall, the receptors that contain 3 subunits may mediate important effects of acetylcholine and nicotine in specific regions of the brain (Mulle et al., 1991; Connolly et al., 1995; Clarke and Reuben, 1996; Albuquerque et al., 1997; Kulak et al., 1997), in the retina (McKay et al., 1994), and in the spinal cord (Flores et al., 1997).

In addition to their roles in the CNS, nAChRs comprised of 3 subunits in combination with either 2 or 4 subunits may play fundamental roles in peripheral neuronal tissues, including neurons of mammalian sympathetic ganglia (Covernton et al., 1994; Mandelzys et al., 1994), parasympathetic ganglia (Poth et al., 1997), sensory neurons such as the trigeminal ganglia (Flores et al., 1996), and adrenal chromaffin cells (Rogers et al., 1992; Campos-Caro, 1997). Nicotinic receptors containing 3 subunits also may predominate in chick ciliary ganglia (Boyd et al., 1988; Couturier et al., 1990), superior cervical ganglia (Couturier et al., 1990; Vernallis et al., 1993; Conroy and Berg, 1995), and dorsal root ganglion cells (Boyd et al., 1991).

Compared with the 4/2 nAChR in the CNS, receptors that contain 3 subunits seem to have much lower affinity for most nicotinic agonists; therefore, until recently, they could not be reliably measured or studied with the available radiolabeled ligands. This fundamental obstacle has hindered studies of the pharmacology and regulation of the binding sites of these receptor subtypes. EB, in contrast to most other nicotinic agonists, has high affinity for ganglionic-type receptors (Badio and Daly, 1994); in fact, [³H]EB binds with high affinity to several different defined nAChR subtypes in stably transfected cell lines (Xiao et al., 1996), as well as to the receptor or receptors in rat adrenal gland (Houghtling et al., 1995) and trigeminal ganglia (Flores et al., 1996). Furthermore, the iodinated analog of EB, [¹²⁵I]EB (formerly [¹²⁵I]IPH), labels receptors in the superior cervical ganglia (Dávila-García et al., 1997). These ganglionic tissues seem to contain nicotinic receptors composed predominantly of 3 subunits in combination with either 2 or 4 subunits, and in some cases, there may be an additional 5 subunit (Conroy et al., 1992; Conroy and Berg, 1995).

Another critical obstacle to the characterization of the pharmacology and regulation of nAChR subtypes containing 3 subunits is that the tissues in which they are found in high concentration, such as autonomic ganglia, adrenal gland, and specific nuclei within the brain, may contain more than one subtype of nicotinic receptor, making the assignment of characteristics to any one subtype difficult. Furthermore, these tissues provide only a very limited amount of tissue for study because of their relatively small size.

To study the pharmacological and functional characteristics of an important nAChR subtype that contains 3 subunits, we transfected HEK 293 cells with the genes encoding rat 3 and 4 nicotinic receptor subunits. A stable clonal cell line has been established, and it expresses this nAChR subtype at a very high density. Here, we report the characteristics of the 3/4 nAChR binding site labeled by [³H]EB and the activation of this receptor ligand-gated ion channel by nicotinic agonists.

	Experimental Procedures

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Materials and drugs. Tissue culture medium, serum, antibiotics, restriction endonucleases, modifying enzymes, and molecular size standards were obtained from GIBCO BRL (Gaithersburg, MD). [³H]EB and ⁸⁶RbCl were supplied by New England Nuclear Research Products (Boston, MA). [-³⁵S]dATPs, [-³²P]CTP, and [-³²P]ATP were obtained from Amersham (Arlington Heights, IL). DNA sequencing reagents were purchased from United States Biochemicals (Cleveland, OH). Electrophoresis reagents were purchased from BioRad Laboratories (Melville, NY). All other chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise stated. A-85380, (+)-anatoxin-A, DHE, and EB were from Research Biochemicals International (Natick, MA). ()-Nicotine, acetylcholine, carbachol, d-tubocurarine, and cytisine were from Sigma Chemical. Mecamylamine was from Merck Sharp & Dohme Research Lab (Rahway, NJ). Two plasmids, pPCA48E-3 and pZPC13, which carry the cDNA clones of rat neuronal nAChR subunit 3 and 4 genes, were described previously (Boulter et al., 1987; Duvoisin et al., 1989) and were generously provided by Dr. J. Boulter (Salk Institute, La Jolla, CA). HEK 293 cells (CRL 1573; American Type Culture Collection, Rockville, MD) were a gift from Y.-H. Wang and B.B. Wolfe (Georgetown University, Washington, D.C.).

Construction of pKX3RC1 and pKX4RC1. To constitutively express the rat neuronal nAChR 3 subunit in mammalian cells, a 1.7-kb HindIII/EcoRI fragment was isolated from the pPCA48E-3 and subcloned into the eukaryotic expression vector pcDNA3 (InVitrogen, San Diego, CA). The resulting plasmid with the 3 coding sequence in the sense orientation was designated pKX3RC1. For generation of the eukaryotic expressable 4 gene, a 2-kb EcoRI/XbaI fragment was isolated from the pZPC13 and inserted into the pcDNA3 vector. The new construct with the 4 coding sequence in the sense orientation was denoted pKX4RC1. pKX3RC1 and pKX4RC1 were restriction mapped and sequenced to confirm correct ligation and orientation.

Cell culture and stable transfection. HEK 293 cells were maintained at 37° with 5% CO₂ in a humidified incubator. Growth medium for the HEK 293 cells was minimum essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin. For transfection experiments, plasmids were linearized by restriction digestion within their prokaryotic elements. Transfection was conducted according to the calcium phosphate method (Chen and Okayama, 1987). Briefly, exponentially growing HEK 293 cells were plated onto 100-mm dishes containing 10 ml of the growth medium 24 hr before transfection. For a single transfection experiment, 1 ml of transfection mixture was made. The mixture was composed of 10 µg of linearized DNA, 125 mM CaCl₂, 25 mM HEPES, 140 mM KCl, 6 mM glucose, and 0.75 mM Na₂HPO₄, pH 7.05. The mixture was added to the dish of cells in dropwise fashion. The cells were incubated with the transfection mixture for 16 hr in the incubator, after which the mixture was removed, and the cells were grown in fresh growth medium for 24 hr. The cells then were collected and plated at a range of densities onto six-well plates in the selection medium, which consisted of the growth medium containing 0.7 mg/ml geneticin (G418). The cells were grown in the selection medium for 3-4 weeks before G418-resistant clones were picked up by cloning cylinders. The stably transfected cells were maintained in the selection growth medium.

RNA isolation and RNase protection assay. Total cellular RNA was isolated from cells using RNA-STAT-60, an RNA isolation reagent (Tel Test B, Friendswood, TX). DNA templates for antisense riboprobes were prepared as described previously (Xiao et al., 1995). The size of the full-length probes and the expected protected fragments of the probes are for rat 2, 421 and 332 bases; rat 3, 308 and 231 bases; rat 4, 497 and 408 bases; rat 5, 450 and 388 bases; rat 2, 328 and 266 bases; rat 4, 258 and 174 bases; and human GAPDH, 224 and 154 bases, respectively. The probes were synthesized using T7 RNA polymerase (Ambion, Austin, TX) and [-³²P]CTP. Specific activities of [-³²P]CTP used for synthesizing the probes of rat nAChR subunit genes and the probe of GAPDH were 800 and 32 Ci/mmol, respectively. Approximately 50 µg of total RNA was hybridized with probes overnight at 42°. Nonprotected probes were digested with a mixture of RNase A and RNase T1, and the samples were processed using the RPA II kit (Ambion). The sizes of protected fragments were determined by electrophoresis on a 6% denaturing polyacrylamide gel.

Ligand binding studies. Cultured cells at >70% confluence were harvested in 50 mM Tris·HCl, pH 7.4, and homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). Homogenates were centrifuged at 35,000 × g for 10 min, and pellets were washed twice with fresh buffer. Membrane pellets were resuspended in fresh buffer, and aliquots equivalent to 60-200 µg of protein were used for binding assays. Ligand binding was measured as described previously (Houghtling et al., 1995) with minor modifications. Briefly, membrane preparations were incubated with [³H]EB for 4 hr at 24° in a final volume of 2.5 ml. Bound and free ligands were separated by vacuum filtration through Whatman GF/C filters treated with 0.5% polyethylenimine. The filter-retained radioactivity was determined by liquid scintillation counting. Total binding and nonspecific binding was determined in the absence and presence of ()-nicotine (300 µM), respectively. Specific binding was defined as the difference between total binding and nonspecific binding. Typically, total binding was measured in duplicate, and nonspecific binding was measured in singlet. In saturation binding experiments, receptor densities (B_max) and dissociation constants (K_d) were determined by nonlinear least-squares regression analyses (Accufit Saturation Two-Site Program; Beckman Instruments, Fullerton, CA). The one-site model was accepted unless the two-site model gave a statistically better fit of the data (p < 0.05 by F test). Hill coefficients (n_H) of specific binding curves were determined by linear regression analyses of those specific binding values that fell between 10% and 90% of B_max. The affinities of drugs at the receptors were determined from binding inhibition curves, in which a series of concentrations of each drug was incubated with a single concentration of [³H]EB. In some studies, the type of binding inhibition mechanism was investigated by including the drug in a [³H]EB binding saturation assay. Hill coefficients (n_H) and IC₅₀ values of binding inhibition curves were determined by linear regression analyses. The inhibition constants (K_i) were calculated according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

⁸⁶Rb⁺efflux assay. The function of nAChRs expressed in the transfected cells was measured using a ⁸⁶Rb⁺ efflux assay as described by Lukas and Cullen (1988), with modifications. In brief, identical aliquots of cells in the selection growth medium were plated onto 24-well plates coated with poly-d-lysine. The plated cells were grown at 37° for 18-24 hr to reach 70-95% confluence. The cells then were incubated in growth medium (0.5-1 ml/well) containing ⁸⁶RbCl (2 µCi/ml) for 4 hr at 37°. The loading mixture was aspirated, and the cells were washed three times with 1 ml aliquots of buffer (15 mM HEPES, 140 mM NaCl, 2 mM KCl, 1 mM MgSO₄, 1.8 mM CaCl₂, 11 mM glucose, pH 7.4) for 30 sec, 5 min, and 30 sec, respectively. One milliliter of buffer, with or without drugs, was added to each well. After incubation for 2 min, the assay buffer was collected, and the amount of ⁸⁶Rb⁺ in the buffer was determined. Cells were lysed by the addition of 1 ml of 0.1 M NaOH to each well, and the lysate was collected for determination of the amount of ⁸⁶Rb⁺ that was in the cells at the end of the efflux assay. Radioactivity of the assay samples and lysates was measured by liquid scintillation counting. Total loading (cpm) was calculated as the sum of the assay sample and the lysate of each well. Values of total loading were 100,000-200,000 cpm/well. The amount of ⁸⁶Rb⁺ efflux was expressed as a percentage of ⁸⁶Rb⁺ loaded. Stimulated ⁸⁶Rb⁺ efflux was defined as the difference between efflux in presence and absence of nicotinic agonists. The EC₅₀, IC₅₀, and n_H values were estimated by linear regression analysis. Experiments with antagonists were done in two different ways. For obtaining an IC₅₀ value, inhibition curves were constructed in which different concentrations of an antagonist were included in the assay to inhibit efflux stimulated by 100 µM nicotine. For determination of the mechanism of antagonist blockade, concentration-response curves for activation by nicotine were constructed in the absence or presence of one or more concentrations of an antagonist.

Ca²⁺ and Na⁺ imaging assays. Effects of nicotinic drugs on changes in [Ca²⁺]_i or [Na⁺]_i were evaluated by Ca²⁺ and Na⁺ imaging assays. These assays were performed on an Attofluor Fluorescence Imaging System (Attofluor, Rockville, MD) with an Axiovert 135 fluorescence microscope (Zeiss, Germany). Cells were plated onto 25-mm glass coverslips (Fisher, Pittsburgh, PA) or 35-mm plastic tissue culture dishes (Nunc, Roskilde, Denmark), both precoated with poly-d-lysine. The plated cells were grown for 36-72 hr at 37°. Culture medium was removed, and cells were rinsed three times with Locke's buffer with the following composition: 140 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO₃, 1.3 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 10 mM HEPES, pH 7.4.

	Results

Top Summary Introduction Procedures Results Discussion References

Stable transfections of HEK 293 cells. Four sets of plasmid DNA were used for transfection experiments independently: (1) pcDNA3 (vector only), (2) pKX3RC1 (3 subunit gene), (3) pKX4RC1 (4 subunit gene), and (4) the combination of pKX3RC1 and pKX4RC1 (3 and 4 subunit genes). From each of the transfections, 36-72 stable, G418-resistant cell clones were isolated after cultivation in selection medium for 3-4 weeks. These clonal cell lines were then grown in selection medium for an additional 4 weeks.

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Multiple-probe RNase protection assay of expression of nAChR subunit genes in HEK 293 cells and transfected cell lines. RNase protection assay was carried out as described in Experimental Procedures. Total RNA from each cell line was hybridized with a mixture of antisense probes labeled with [32P] for six rat nAChR subunit genes (2, 3, 4, 5, 2, 4) and the human GAPDH gene. Nonprotected probes were digested with a mixture of RNase A and RNase T1. The sizes of protected RNA fragments were determined by electrophoresis on a 6% denaturing polyacrylamide gel. HEK 293, the parental cell line for transfection experiments; KXC1, the cell line that was transfected with the vector, pcDNA3; KX3R1, the cell line that was transfected with rat 3 subunit gene; KX4R1, the cell line that was transfected with rat 4 subunit gene; KX34R2, the cell line that was transfected with both rat 3 and 4 subunit genes. Labels, specific protected fragments and their sizes (bases). The GAPDH was used as an internal and loading control in the experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Specific binding of [3H]EB to membrane homogenates from HEK 293 cells, transfected cells, and rat forebrain (RFB). Binding assays were carried out as described in Experimental Procedures. The concentration of [3H]EB used in these experiments was ~3 nM. Total binding and nonspecific binding was determined in the absence and presence of 300 µM ()-nicotine, respectively. Specific binding was defined as the difference between total binding and nonspecific binding. See the legend for Fig. 1 for designations of cell lines. Values are the mean ± standard error from three to six independent experiments.

Analysis of [³H]EB binding to 3/4 receptors in KX34R2 cell membrane homogenates. Specific binding of [³H]EB in cell membrane homogenates was saturable and represented >98% of the total binding throughout most of the concentration range used (Fig. 3A). The high density of nicotinic receptor binding sites in KX34R2 cells and the very low nonspecific binding of [³H]EB allowed binding to be analyzed over a wide range of concentrations. Over this range, [³H]EB binding fit a model for a single site with a Hill coefficient (n_H) close to 1, a K_d value of ~300 pM, and a site density of ~8900 fmol/mg of protein (Fig. 3B, see figure legend for calculated values).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Saturation binding of [3H]EB to KX34R2 membrane homogenates. Saturation binding assays were carried out as described in Experimental Procedures. Plots shown are a representative of four saturation binding experiments. A, Binding curves showing specific binding () and nonspecific binding (). Inset, semilogarithmic plot of the specific binding. B, Scatchard plot of the specific binding. Inset, Hill plot of the specific binding. Data from saturation binding experiments were analyzed by nonlinear least-squares regression (Accufit Saturation Two-Site Program) for curve-fitting and Kd and Bmax estimation. The Kd and Bmax values were 304 ±16 pM and 8942 ± 115 fmol/mg protein (mean ± standard error, four experiments), respectively. The Hill coefficient, estimated from Hill plots, was 1.06 ± 0.06 (mean ± standard error, four experiments).

Pharmacological characteristics of 3/4 receptor binding sites in KX34R2 cells. The pharmacological characteristics of the 3/4 nicotinic receptor binding sites in these cells were examined in binding competition assays in which drugs competed against 500 pM [³H]EB for binding sites in cell membranes (Fig. 4A, Table 1). EB was by far the most potent drug in competing for 3/4 receptor binding sites, with an affinity >2 orders of magnitude higher than that of any other drug tested. For example, among nicotinic agonist drugs, EB was >100 times more potent than anatoxin-A and A85380, >500 times more potent than cytisine, and >1000 times more potent than nicotine, acetylcholine, or carbachol (Fig. 4, Table 1). Among nicotinic antagonists, d-tubocurarine was nearly 10 times more potent than DHE in competing for 3/4 receptor binding sites (Fig. 4, Table 1). Neither mecamylamine nor hexamethonium at concentrations up to 1 mM competed effectively for [³H]EB binding sites.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Comparison of drug competition profiles of nAChRs in membrane homogenates from KX34R2 cells and rat forebrain. Binding assays were carried out as described in Experimental Procedures. Competition for [3H]EB binding sites in KX34R2 cell homogenates (A) and in rat forebrain membrane homogenates (B). Data shown are representative of five competition binding experiments. The concentration of [3H]EB used in competition experiment 5 was ~500 pM. See Table 1 for a summary and analyses of data from all experiments.

View this table: [in this window] [in a new window]   TABLE 1 Comparison of pharmacological properties of nAChR ligand binding in membrane homogenates from KX34R2 cell and rat forebrain Competition binding experiments were carried out as described in Experimental Procedures using 500 pM [3H]EB. Hill coefficients (nH) and IC50 values (not shown) of competition binding curves were estimated by Hill plot. The inhibition constants (Ki) were calculated according to the Cheng-Prusoff equation (1973). The Kd values of [3H]EB used in the calculation were 300 pM for KX34R2 cells and 50 pM for rat forebrain. Values shown are the mean ± standard error of three to five independent measurements.

Assessment of 3/4 receptor function in KX34R2 cells. To examine the function of the 3/4 nicotinic receptor in these cells, we measured ion flux stimulated by nicotinic agonists. Three types of ion flux were measured: efflux of ⁸⁶Rb⁺ from preloaded cells, increases in intracellular Ca²⁺ as measured by Fura-2/Ca²⁺ imaging, and increases in intracellular Na⁺ as measured by SBFI/Na⁺ imaging.

Stimulation of ⁸⁶Rb⁺ efflux. As shown in Fig. 5A, nicotine stimulated ⁸⁶Rb⁺ efflux from KX34R2 cells, which express both 3 and 4 subunits, but not from the parent HEK 293 cells or from HEK 293 cells transfected with either the vector only or the 3 or 4 subunit only. Both nicotine and acetylcholine stimulated ⁸⁶Rb⁺ efflux from KX34R2 cells in a concentration-dependent manner (Fig. 5B), with EC₅₀ values of 28 and 114 µM, respectively. Maximal ⁸⁶Rb⁺ efflux, 8-10 times greater than the basal efflux, occurred at a nicotine concentration of ~300 µM and an acetylcholine concentration of 2 mM. Concentrations of >1 mM nicotine and 10 mM acetylcholine tended to decrease ⁸⁶Rb⁺ efflux, possibly due to desensitization or agonist blockade of the channel (see Fig. 6B). The Hill coefficients for functional activation of the receptor by nicotine and acetylcholine were 1.6 and 1.5, respectively, and in both cases, these values were significantly different from 1 (p < 0.01). In addition to nicotine and acetylcholine, cytisine (30 µM), carbachol (300 µM), and EB (30 nM) all stimulated ⁸⁶Rb⁺ efflux from KX34R2 cells, and in each case, the efflux was nearly completely blocked by 10 µM mecamylamine (Fig. 5C).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effects of nicotinic agonists and antagonists on 86Rb+efflux from KX34R2 cells. 86Rb+ efflux was measured as described in Experimental Procedures. Cells were loaded with 86Rb+ and then exposed to buffer alone or buffer containing nicotinic drugs for 2 min. A, Effects of nicotine on 86Rb+ efflux from HEK 293 cells and transfected cells. Cells were exposed to buffer alone (basal efflux) or buffer containing 100 µM nicotine. The amount of 86Rb+ efflux was expressed as a percentage of 86Rb+ loaded. Data shown are the mean ± standard error from three separate measurements. B, Concentration-dependent activation of nAChR function in KX34R2 cells by nicotine and acetylcholine. The amount of 86Rb+ efflux was expressed as a percentage of 86Rb+ loaded. The stimulated 86Rb+ efflux was defined as the difference between efflux with and without nicotinic agonists (i.e., basal efflux, normally 4-8%, has been subtracted). Data shown are representative of five independent experiments. Parameters were estimated by Hill plot analyses. The EC50 values (mean ± standard error) for nicotine and acetylcholine were 28.2 ± 3.7 and 113.7 ± 24.2 µM, respectively. The Hill coefficients (mean ± standard error) for nicotine and acetylcholine were 1.59 ± 0.1 and 1.48 ± 0.04, respectively. These Hill coefficients are significantly different from 1 (p < 0.01). The efficacy of acetylcholine relative to nicotine was 98.8 ± 3% (mean ± standard error). C, Effects of nicotinic agonists on 86Rb+ efflux from KX34R2 cells. Cells were exposed to buffer alone (basal efflux) or buffer containing nicotinic agonists, with or without mecamylamine. The amount of 86Rb+ efflux was expressed as a percentage of 86Rb+ loaded. The concentrations of drugs used were 30 µM ()-nicotine (Nic), 10 µM mecamylamine (Mec), 30 µM cytisine (Cyt), 300 µM carbachol (Carb), and 30 nM (±)-EB. Data shown are the mean ± standard error from at least two independent measurements, each carried out in quadruplicate. D, Concentration-dependent inhibition by nicotinic antagonists of nicotine-stimulated 86Rb+ efflux from KX34R2 cells. Cells were exposed to buffer alone or buffer containing 100 µM nicotine with or without antagonists. Control equaled the amount of 86Rb+ efflux stimulated by 100 µM nicotine in the absence of antagonists. Parameters were estimated from Hill plots. The IC50 and Hill coefficients were, respectively, 1.2 ± 0.4 µM and 1.15 ± 0.1 (three experiments) for mecamylamine, 9.6 ± 1.4 µM and 0.78 ± 0.03 (four experiments) for d-tubocurarine, 100 ± 6 µM and 1.14 ± 0.16 (three experiments) for DHE, and 208 ± 27 µM and 1.20 ± 0.14 (five experiments) for hexamethonium (values are mean ± standard error).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of mecamylamine, hexamethonium, dihydro--erythroidine, and d-tubocurarine on the concentration-response relationship for nicotine-stimulated 86Rb+efflux from KX34R2 cells. 86Rb+ efflux was measured as described in Experimental Procedures. Cells were loaded with 86Rb+ and then exposed to buffer alone or buffer containing increasing concentrations of nicotine with or without antagonists for 2 min. The maximal stimulated 86Rb+ efflux was defined as the difference between maximal efflux in the presence of nicotine and basal efflux measured in buffer alone. Data shown are representative of five independent experiments. A, Effects of mecamylamine and hexamethonium. Control, concentration-response curve for activation by nicotine alone; Hex, concentration-response curve for activation by nicotine in the presence of 300 µM hexamethonium. Mec, concentration-response curve for activation by nic- otine in the presence of 2 µM mecamylamine. B, Effect of DHE. Control, concentration-response curve for activation by nicotine alone. DHE, concentration-response curve for activation by nicotine in the presence of 250 µM DHE. C, Effect of d-tubocurarine. Control, concentration-response curve for activation by nicotine alone. d-TC, concentration-response curve for activation by nicotine in the presence of d-tubocurarine at the indicated concentrations.

Block of nicotine-stimulated ⁸⁶Rb⁺ efflux. The blockade of nicotine-stimulated ⁸⁶Rb⁺ efflux by several antagonists was investigated. As shown in Fig. 5D, despite the inability of mecamylamine to compete for the agonist binding site (Table 1), it blocked nicotine-stimulated ⁸⁶Rb⁺ efflux from KX34R2 cells quite potently, with an IC₅₀ value of 1 µM and a nearly complete blockade of efflux at a concentration of 10 µM. Similarly, d-tubocurarine, DHE, and hexamethonium blocked nicotine-stimulated ion efflux in a concentration-dependent manner, with IC₅₀ values of 10, 100, and 210 µM, respectively (Fig. 5D). In contrast to these drugs, -bungarotoxin (1.5 µM) had no effect on nicotine-stimulated ⁸⁶Rb⁺ efflux from KX34R2 cells (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effect of DHE and d-tubocurarine on the saturation binding of [3H]EB to KX34R2 membrane homogenates. Saturation binding assays were carried out as described in Experimental Procedures. A, Semilogarithmic plots showing specific binding of [3H]EB alone (control) and in the presence of 250 µM DHE or 30 µM d-tubocurarine. B, Lineweaver-Burk plots of the specific binding shown in A. Data from saturation binding experiments were analyzed by nonlinear least-squares regression (Accufit Saturation Two-Site Program) for curve-fitting and Kd and Bmax estimation. The apparent Kd and Bmax values were, respectively, 276 pM and 12,960 fmol/mg of protein for control, 1,076 pM and 12,700 fmol/mg of protein in the presence of 250 µM DHE, and 954 pM and 9,685 fmol/mg of protein in the presence of 30 µM d-tubocurarine.

Ca²⁺ ion imaging. As shown in Fig. 8A, nicotine stimulated an increase in [Ca²⁺]_i concentration in KX34R2 cells in a concentration-dependent manner. When the extracellular Ca²⁺ concentration was maintained at 1.3 mM, nicotine at concentrations of 10 and 100 µM increased [Ca²⁺]_i levels to 2-fold and 5-fold of base-line, respectively. When the extracellular Ca²⁺ was raised from 1.3 to 10 mM (Fig. 8B), the basal level of [Ca²⁺]_i increased slightly, but the increase in [Ca²⁺]_i stimulated by 10 µM nicotine was markedly enhanced to >10-fold of base-line. This nicotine stimulated increase in [Ca²⁺]_i was almost completely blocked by 10 µM mecamylamine (Fig. 8B).

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Effects of nicotine on [Ca2+]i in KX34R2 cells. Changes in [Ca2+]i were measured and calculated as described in Experimental Procedures. Cells were loaded with the Ca2+ fluorescent indicator Fura-2 and then exposed to buffer alone or buffer containing nicotine with or without mecamylamine. Traces, [Ca2+]i changes. Concentrations of nicotinic drugs during the period indicated (horizontal bars) are marked (above traces). Extracellular Ca2+ concentration and time scales are shown (under each trace). Nic, ()-nicotine. Mec, mecamylamine. Ca, extracellular Ca2+concentration. A, [Ca2+]i increase is stimulated by nicotine at 1.3 mM extracellular Ca2+. Data shown are representative of three independent experiments, and the trace shown is the average [Ca2+]i of 58 cells. B, [Ca2+]i increase is stimulated by nicotine and blocked by mecamylamine at an extracellular Ca2+ concentration of 10 mM. Data shown are representative of three independent experiments, and the trace shown is the average [Ca2+]i of 37 cells.

Na⁺ ion imaging. Nicotine also stimulated an increase in [Na⁺]_i, measured by SBFI/Na⁺ imaging, and this increase was blocked by mecamylamine (Fig. 9A). Unlike changes in [Ca²⁺]_i, however, the nicotine-stimulated increase in [Na⁺]_i was not affected by increasing the extracellular Ca²⁺ from 1.3 to 10 mM (Fig. 9B).

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Effects of nicotine on [Na+]i of KX34R2 cells. Relative changes in [Na+]i are shown in arbitrary units of fluorescence ratio F334/F380, as described in Experimental Procedures. Cells were loaded with the Na+ fluorescent indicator SBFI and then exposed to buffer containing nicotinic drugs. Traces, [Na+]i changes. Concentrations of nicotinic drugs during the period indicated (horizontal bars) are marked (above traces). Extracellular Ca2+concentration and time scales were shown (under each trace). Nic, ()-nicotine. Mec, mecamylamine. Ca, extracellular Ca2+concentration. A, [Na+]i increase is stimulated by nicotine and blocked by mecamylamine at 10 mM extracellular Ca2+. Data shown are representative of two independent experiments, and the trace shown is the average [Na+]i of 27 cells. B, [Na+]i increase is stimulated by nicotine at different extracellular Ca2+ concentrations. Data shown are representative of three independent experiments, and the trace shown is the average [Na+]i of 77 cells. Dotted horizontal lines, [Na+]i in cells that were permeabilized with 5 µM Gramicidine A when extracellular [Na+] was 154 mM (top line) and 0 mM (bottom line), respectively.

Binding of [³H]EB to cell surface receptors. The KX34R2 cells produce a very high density of 3/4 receptors, and the ligand binding studies indicate that these receptors have characteristics of a single homogenous population. However, ligand binding measurements in membrane homogenates do not distinguish between receptors on the cell surface membrane and receptors on intracellular membranes. Therefore, to estimate the fraction of the total 3/4 receptor population that is located on the cell surface of KX34R2 cells, [³H]EB binding was measured in intact cells attached to 24-well tissue culture plates, and nonspecific binding was determined with either nicotine (which easily crosses cell membranes) or carbachol (which does not readily cross cell membranes). As shown in Fig. 10, nicotine blocked >95% of the [³H]EB binding sites in intact cells, whereas in contrast, the nonpermeant carbachol blocked a maximum of 40% of the [³H]EB binding sites even at a concentration 25,000 times higher than its affinity constant (K_i) for the receptor, as determined in membrane binding assays (see Table 1). Thus, we estimate that 40% of the total number of 3/4 receptors measured in KX34R2 cell homogenates are located on the cell surface.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Binding of [3H]EB to intact KX34R2 cells. Intact cell binding assays were carried out as described in Experimental Procedures. Total binding of [3H]EB (3 nM) was determined in the absence of any competing ligand. Nic, ()-nicotine. Carb, carbachol. Values are the mean ± standard error from six independent experiments.

	Discussion

Top Summary Introduction Procedures Results Discussion References

The KX34R2 cells described here stably express 3/4 nAChRs that bind [³H]EB with high affinity and function to gate cations through their channels in response to nicotinic agonists. In contrast, cells expressing the mRNA for either the 3 or 4 subunit alone do not express [³H]EB binding sites or cation channels that respond to nicotine. Both [³H]EB saturation binding and drug competition studies indicate that KX34R2 cells express a single class of nicotinic receptor binding sites; similarly, the studies of the receptor function are consistent with a single class of receptors. These cells produce a very high density of 3/4 receptors, and binding studies in intact cells indicate that ~40% of the total number of receptors are located on the cell surface. Only the receptors on the cell surface would be expected to mediate functional responses, whereas receptors on intracellular membranes might be in various stages of their receptor cycle, either before insertion into the cell surface membrane or after removal from it. Furthermore, we cannot exclude the possibility that these cells express receptors with more than one stoichiometric combination of 3 and 4 subunits but very similar pharmacological and functional properties. Nevertheless, these cells provide a model system in which the pharmacology, function, and regulation of the 3/4 nAChR can be examined in detail.

Drug binding competition studies indicated that the affinity of every agonist examined is lower at the 3/4 receptor binding site in these cells than at the 4/2 nicotinic receptor site in rat forebrain, with affinity ratios ranging from 7 for EB to >750 for A85380. One consequence of the lower affinity of agonists for 3/4 receptors is that with the exception of [³H]EB, radioligands such as [³H]cytisine, [³H]nicotine, and [³H]acetylcholine, which have been very useful for labeling the 4/2 nAChR, the predominant receptor subtype in brain, probably would not be useful for labeling 3/4 receptors. This is because their lower affinities would require ligand concentrations (200-880 nM) at which nonspecific binding would obscure specific binding. This probably explains why these radioligands have not been useful for labeling nicotinic receptors in autonomic ganglia or adrenal gland, where a receptor subtype containing 3 subunits seems to predominate. Although the affinity of EB also is lower at 3/4 receptors than at 4/2 receptors in brain, its affinity at these 3/4 receptors (300 pM) is still very high, making it an excellent radioligand for these receptors.

The affinities of d-tubocurarine and DHE, the two antagonists examined that competed for the binding site, like those of the agonists, are lower at the 3/4 receptor binding site than at the 4/2 binding site in brain. In fact, DHE displays the largest difference in affinity for these two receptor subtypes of any drug examined. Thus, DHE, with a receptor binding site affinity ratio of >7500, and A85380, with an affinity ratio of >750, could be useful drugs for distinguishing between these two subtypes of nicotinic receptors in native tissues. In addition to the absolute difference in affinity of DHE, a second way to distinguish between these two receptor subtypes could be to take advantage of the difference in the rank orders of affinities of d-tubocurarine and DHE. Thus, at 3/4 receptor binding sites, d-tubocurarine has >9 times higher affinity than DHE, whereas in contrast, at 4/2 binding sites, DHE has >75 times higher affinity than d-tubocurarine.

Consistent with a previous report (Houghtling et al., 1995), both d-tubocurarine and DHE show low Hill coefficients in competing for brain [³H]EB binding sites, which are predominantly 4/2 receptors. Similarly, d-tubocurarine shows a low Hill coefficient in competing for 3/4 receptors in these transfected cells. These low Hill coefficients suggest the interesting possibility that some antagonists can distinguish between two states of the same receptor, for example, as the receptor conformation changed from a resting state to a desensitized state (with high affinity for agonists) during prolonged incubation with an agonist, as in the receptor binding assay.

The 3/4 nAChR function and its pharmacology were assessed by measuring agonist-stimulated ⁸⁶Rb⁺ efflux. Nicotine and acetylcholine both stimulated ⁸⁶Rb⁺ efflux in a concentration-dependent manner, with EC₅₀ values of 28 and 114 µM, respectively. The difference between the affinity of the receptor for these agonists as assessed by this measurement of receptor function compared with binding studies (Table 1) presumably reflects the shift of the receptor to a state with high affinity for agonists under equilibrium binding conditions. In the cases of nicotine and acetylcholine, the EC₅₀ values for functional activation were 59- and 129-fold higher, respectively, than the K_i values measured in binding studies.

Among the antagonists tested, mecamylamine, with an IC₅₀ value of 1 µM, was the most potent in blocking nicotine-stimulated ⁸⁶Rb⁺ efflux in KX34R2 cells. It was ~10 times more potent than d-tubocurarine, 100 times more potent than DHE, and 200 times more potent than hexamethonium. Both mecamylamine and hexamethonium blocked receptor function by a noncompetitive mechanism, suggesting that they block the receptor ion channel. This would explain their effective block of receptor function in the face of their near-total inability to compete for the agonist binding site of the 3/4 receptor.

Tubocurarine, on the other hand, presents a more interesting case. In the binding assay, it competes for the 3/4 receptor agonist recognition site with a K_i value of 23 µM (Table 1), but at similar concentrations, its blockade of receptor function seems to be entirely by a noncompetitive mechanism. In fact, there was no indication that d-tubocurarine competes for the agonist binding site in the ⁸⁶Rb⁺ efflux assay; that is, the concentration-response curves for nicotine were not shifted to the right in the presence of any concentration of tubocurarine tested (Fig. 6C). One explanation for this apparent contradiction between the binding assay and the ⁸⁶Rb⁺ efflux assay is that in the receptor's functional state, as measured in the ⁸⁶Rb⁺ efflux assay, tubocurarine has virtually no affinity for the agonist recognition site of the receptor but nevertheless very effectively blocks receptor function noncompetitively by blocking the ion channel. However, when the receptor conformation shifts to a desensitized state with its higher affinity for nicotinic agonists, as occurs in the binding assay, its affinity for tubocurarine also increases, enabling this antagonist to then compete with micromolar affinity for the agonist recognition site. This explanation also can account for the observation that tubocurarine competes for the agonist recognition sites of both the 3/4 receptor in these cells and the 4/2 receptor in rat brain with a low Hill coefficient because this could reflect the different affinities of the drug for the different conformations of the receptors. In fact, the [³H]EB saturation binding studies shown in Fig. 7 suggest that in the presence of 30 µM tubocurarine, ~25% of the receptors may remain in a conformation with an affinity for [³H]EB too low to be measured in a typical binding assay.

In contrast to these other antagonists, DHE blocked receptor function in a competitive manner. Its affinity constant for the functional state of the receptor, calculated from its IC₅₀ value in blocking nicotine-stimulated ⁸⁶Rb⁺ efflux (after correction for the nicotine concentration), is ~20 µM, which is ~10 times lower than the K_i value derived from the ligand binding competition assays (Table 1). In other words, DHE is ~10 times more potent in blocking receptor function than would have been predicted from the binding assay. This suggests that DHE has higher affinity for the functional conformation of the receptor than for its conformation in the desensitized state, which would occur during the binding assay.

Activation of the 3/4 receptor in KX34R2 cells resulted in an increase in both [Ca²⁺]_i and [Na⁺]_i. The nicotine-stimulated increase in [Ca²⁺]_i was markedly enhanced when the concentration gradient was increased by raising the extracellular Ca²⁺ concentration from 1.3 to 10 mM. The higher extracellular Ca²⁺ concentration, however, seemed to have no effect on the nicotine-stimulated increase in [Na⁺]_i. These results suggest that extracellular Ca²⁺ can actually pass through the 3/4 receptor channel.

The 3/4 nAChR has been expressed previously in HEK 293 cells, both transiently (Wong et al., 1995) and stably in conjunction with a voltage-gated Ca²⁺ channel (Stetzer et al., 1996). Although pharmacological comparisons of these previously expressed 3/4 nAChRs and those in the KX34R2 cells are very limited, there seems to be agreement that acetylcholine, nicotine, and cytisine are full agonists, confirming studies in frog oocytes (Luetje and Patrick, 1991; Covernton et al., 1994; Papke and Heinemann, 1994). However, nicotine and acetylcholine seemed to be somewhat more potent in stimulating the 3/4 receptors in these KX34R2 cells than was reported in the other transfected HEK 293 cells.

In conclusion, KX34R2 cells provide a model system in which to study 3/4 receptors, a subtype that may mediate neurotransmission in autonomic ganglia and brain. These cells have maintained a high level of receptor expression and function for >300 generations and thus have allowed a detailed examination of the pharmacology of the receptor. The pharmacological studies reported here, for both drug binding and functional activation, should help in studying the distribution, physiological role, and pharmacology of 3/4 nAChRs in brain and peripheral tissues. In addition, this cell line provides a stable and convenient source of highly functional channels for patch-clamp experiments to study in detail the electrophysiological properties of 3/4 nAChRs (Zhang et al., 1997). Furthermore, the binding site of the receptor and its function in these cells are altered by exposure to nicotinic drugs (Meyer et al., 1997); thus, these stably transfected cells should prove useful in studying the chronic and acute effects of nicotinic drugs on the density, cellular distribution, and function of 3/4 nAChRs.