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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Characterization of Human Recombinant Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations α2β4, α3β4 and α4β4 Stably Expressed in HEK293 Cells

Kenneth A. Stauderman, L. Scott Mahaffy, Michael Akong, Gönül Veliçelebi, Laura E. Chavez-Noriega, James H. Crona, Edwin C. Johnson, Kathryn J. Elliott, Alison Gillespie, Richard T. Reid, Pamala Adams, Michael M. Harpold and Janis Corey-Naeve
Journal of Pharmacology and Experimental Therapeutics February 1998, 284 (2) 777-789;

Abstract

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human β4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human α2, α3 or α4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to α2 and β4, to α3 and β4 and to α4 and β4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean ± S.D. in pM) values of 42 ± 10, 230 ± 12 and 187 ± 29 and withBmax (fmol/mg protein) values of 1104 ± 338, 2010 ± 184 and 3683 ± 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (−)nicotine (Nic), ACh, cytisine (Cyt) and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: α2β4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; α3β4, Epi > DMPP = Cyt = Nic = Sub; α4β4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-β-erythroidine (DHβE) was highest at α3β4 compared with α2β4 or α4β4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human α2β4, α3β4 and α4β4 neuronal nicotinic receptors.

Neuronal nicotinic ACh receptors (nAChRs) are expressed in both the peripheral nervous system and the CNS, where they play an important role in the control of synaptic transmission (Gray et al., 1996; McGeheeet al., 1995; Role and Berg, 1996). These receptors are multisubunit complexes (Anand et al., 1991; Cooper et al., 1991), and a family of genes encoding 11 neuronal subunits has been identified (α2–α9, β2–β4; for reviews, see McGehee and Role, 1995; Sargent, 1993). The α7, α8 and α9 subunits are functional when expressed alone in Xenopus oocytes, whereas the α2, α3 and α4 subunits are functional only when expressed in pairwise combination with β2 or β4 subunits (McGehee and Role, 1995; Sargent, 1993). More recent data indicate that co-expression of α5 with heterologously expressed α4β2, α3β2 or α3β4 receptors can modify the pharmacological and biophysical properties of the corresponding pairwise combination (Wang et al., 1996;Ramirez-Latorre et al., 1996). Also, although functional roles for α6 and β3 subunits have not been firmly established, there now is evidence that α6 may form a functional receptor in combination with β4 (Gerzanich et al., 1997) and that the β3 subunit functions as a structural entity (Forsayeth and Kobrin, 1997).

The precise subunit composition of nAChRs in CNS neurons is a matter of considerable interest. Earlier studies suggested that the majority of immuno-isolated high-affinity binding sites for [3H]cytisine or [3H]nicotine in chick and rat brain contained the α4 and β2 subunits (Flores et al., 1991; Schoepfer et al., 1988; Whiting et al., 1987a; Whiting et al., 1987b). However, the existence of other receptor subunit combinations with either low affinity for [3H]nicotine and [3H]cytisine, or high-affinity sites in low abundance, is still likely. For example,in situ hybridization has revealed the presence of mRNA encoding β4 subunits in hippocampus, cortex, medial habenula, cerebellum and locus ceruleus (Dineley-Miller and Patrick, 1992). Although brains from transgenic mice lacking the β2 subunit lose a majority of their high-affinity binding sites for [3H]nicotine, nicotine-evoked electrophysiological responses were unaltered in neurons from the medial habenula (Picciottoet al., 1995). Additionally, the Type III current found in cultured rat hippocampal cells has been tentatively attributed to α3β4 receptors on the basis of a similar pharmacology observed for pairwise expression of these subunits in oocytes (Alkondon and Albuquerque, 1993). These data support the idea that certain nAChRs in the brain contain β4 subunits. Determining the functional properties of β4-containing receptors may therefore provide new insights into the role of nAChRs in the brain.

Studies examining the function of heterologously expressed nAChRs reveal a diverse pharmacological profile in receptors containing α2, α3 or α4 subunits in combination with β2 or β4 subunits (Chavez-Noriega et al., 1997; Gerzanich et al., 1995; Hussy et al., 1994; Luetje and Patrick, 1991). The pharmacological characteristics of specific nAChR subtypes also display species specificity. For example, nicotine acts as a full agonist on rat α3β2 receptors, whereas it is a partial agonist on chick and human α3β2 receptors (Chavez-Noriega et al., 1997; Hussyet al., 1994). Also, nicotine is more potent than DMPP on rat and chick α7 nAChRs (Gerzanich et al., 1993; Amaret al., 1993; Séguéla et al., 1993), whereas DMPP is more potent than nicotine on human α7 (Chavez-Noriegaet al., 1997; Peng et al., 1994). Therefore, in the discovery of nAChR-directed therapeutic agents for humans, it is preferable to use human nAChRs as primary targets.

The pharmacological properties of recombinant human nAChRs are now beginning to emerge. Reports have appeared describing the characteristics of human α7 (Peng et al., 1994), as well as some limited information on α3β2 and α3β4 receptors (Gerzanich et al., 1995), expressed in Xenopusoocytes. A full characterization of the pharmacological properties of human α2β4, α3β4, α4β4, α2β2, α3β2, α4β2 and α7, expressed in Xenopus oocytes, was recently completed (Chavez-Noriega et al., 1997). To date, however, only α7 and α4β2 human recombinant receptors have been characterized after stable expression in mammalian cells (Gopalakrishnan et al., 1995; Buisson et al., 1996; Gopalakrishnan et al., 1996), which is a system more adaptable to drug discovery by high-throughput screening. We describe here the pharmacological characterization of human recombinant α2β4, α3β4 and α4β4 receptors stably expressed in HEK293 cells.

Materials and Methods

Cell culture and stable transfection of HEK293 cells.

HEK293 cells (American Type Tissue Collection, Rockville, MD) were grown in Dulbecco’s modified Eagle medium supplemented with 6% bovine calf serum, 50 units/ml penicillin and 50 μg/ml streptomycin in a humidified atmosphere containing 6% CO2. The isolation and characterization of cDNAs encoding human α2, α3, α4 and β4 nAChR subunits were reported previously (Elliott et al., 1996). In order to optimize expression levels, the cDNAs encoding the α2 and α4 subunits were modified by replacing the 5′ untranslated region with an efficient ribosomal binding site, 5′-GCCACC-3′ (Kozak, 1987). The A4B4 cell line (previously referred to as 10C4-6; Staudermanet al., 1995) was developed using expression constructs in which the cDNAs encoding the human α4 and β4 subunits were subcloned into the vector pCMV-T7, a modified version of pCMVβ (Clontech, Palo Alto, CA) containing a T7 promoter site. The plasmid pSV2neo (Clontech), which carries the neomycin-resistance gene, was cotransfected with the α4 and β4 plasmids in construction of this cell line. The expression constructs used to develop the A2B4 cell line (previously referred to as 13B5-12; Stauderman et al., 1995) were generated by subcloning cDNAs containing the entire coding sequences of human α2 and β4 into the pcDNA3 plasmid (Invitrogen, San Diego, CA), which carries the neomycin-resistance gene. The same β4 construct was used to develop the A3B4.2 cell line, in combination with human α3 cDNA subcloned into the vector pZeoCMV, a modified version of pcDNA3 (Invitrogen) that carries the Zeocin-resistance gene from pZeoSV (Invitrogen).

Cells were transfected using LipofectAMINE (Gibco BRL, Gaithersburg, MD) according to the manufacturer’s instructions. All transfections were maintained for 2 weeks in selection medium composed of growth medium containing 500 μg/ml G418 (Gibco BRL) (A2B4 and A4B4 cell lines) or 100 μg/ml G418 and 40 μg/ml Zeocin (Invitrogen) (A3B4.2 cell line). Antibiotic-resistant colonies were isolated and maintained in the presence of the selection medium. Screening for cell colonies expressing functional receptors was performed by a fluo-3-based assay (see below) measuring agonist-stimulated changes in [Ca++]i. Colonies demonstrating functional receptors were subcloned by limiting dilution, and the resulting subclones were screened by the fluo-3 assay. HEK Neo/Zeo, a negative control cell line, was developed by cotransfection of HEK293 cells with pcDNA3 and pZeoSV. Cells that survived a 2-week selection in 100 μg/ml G418 and 40 μg/ml Zeocin demonstrated no agonist-stimulated changes in [Ca++]i as measured in the fluo-3 assay (data not shown).

RNA isolation and northern blot analysis.

Total cellular RNA was isolated from stably transfected cells using the RNeasy Total RNA Kit (QIAGEN, Inc., Chatsworth, CA). Then 5 μg of each RNA sample was fractionated by electrophoresis on a 1% agarose gel containing 1 M formaldehyde and transferred to Zeta-Probe (BioRad, Hercules, CA) by downward alkaline blotting (Chomczynski, 1992). Ribosomal RNAs were visualized by staining the membrane with methylene blue. Blots were sandwiched between two sheets of Whatman 3MM paper and hybridized in 250 mM sodium phosphate buffer, pH 7.2, 250 mM NaCl, 7% SDS, 1 mM EDTA and 50% formamide containing 5 × 105 cpm/ml heat-denatured probe. Probes encompassing the entire coding regions of α2, α3, α4 and β4 were labeled with [32P] using the Prime-It RMT Random Primer Kit (Stratagene, La Jolla, CA). Blots were hybridized overnight at 42°C, rinsed briefly in 1× SSPE (150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4), 0.2% SDS and then washed three times in 0.1× SSPE, 0.1% SDS at 65°C. Blots were exposed to X-ray film (Kodak Biomax) with an intensifying screen.

Membrane preparation and immunoblot analysis.

Cells were harvested from 10-cm plates and washed with PBS (140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). Washed cells were resuspended in 50 mM Tris pH 7.4, 1 mM EDTA containing a cocktail of protease inhibitors (Complete, Boehringer Mannheim, Indianapolis, IN) and homogenized with a Dounce homogenizer. The homogenate was centrifuged at 1000 × g for 5 min to remove cellular debris, and the supernatant fraction was centrifuged at 100,000 ×g for 120 min to pellet the membranes. The membranes were resuspended in RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 1% SDS) containing the protease inhibitor cocktail. Protein concentration was determined using a Bio-Rad Protein Assay. Aliquots of membranes were stored at −70°C.

For immunoblot analysis, membranes were solubilized in Tris-Glycine SDS Sample Buffer (Novex, San Diego, CA) containing 5% 2-mercaptoethanol and heated at 65°C for 10 min. Solubilized proteins were separated by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Hy-Bond ECL, Amersham, Arlington Heights, IL). Blots were rinsed once in PBS, 0.1% Tween-20 (wash buffer) and then blocked for 3 h in 5% Carnation non-fat dry milk dissolved in wash buffer (blocking buffer).

Human α2 and α3 subunit proteins were probed with a sheep anti-rat α3 polyclonal antibody (rα3) at 20 μg/ml, the α4 subunit was probed with a sheep anti-rat α4 polyclonal antibody (rα4) at 20 μg/ml, and the β4 subunit was probed with a sheep anti-rat β4 polyclonal antibody (rβ4) at 5 μg/ml. In western blots, the rα3 antibody recognizes both the human α2 and α3 subunits but not the α4 or β4 subunits. The rα4 antibody recognizes human α4 subunits specifically, and the rβ4 antibody recognizes human β4 subunits but not α2, α3 or α4 subunits. The primary antibodies were diluted in blocking buffer and incubated with the nitrocellulose membranes for 3 h at room temperature. The membranes were washed three times in wash buffer. The secondary antibody was peroxidase-conjugated donkey anti-sheep IgG (Sigma, St. Louis, MO) diluted in blocking buffer and incubated with membranes for 45 min at room temperature, followed by five changes of wash buffer. The antibody signal was visualized using the ECL developing system (Amersham) according to the manufacturer’s directions, and the molecular weights of detected proteins were determined by comparison to prestained protein standards.

Fluorescence-based measurements of [Ca++]i.

For the measurement of [Ca++]i in cell populations, cells were plated on poly-d-lysine-coated 96-well microtiter plates at a density of 2 × 105 cells/well. Twenty-four hours after plating, the cells were washed with HBK (155 mM NaCl, 4.6 mM KCl, 1.2 mM MgSO4, 1.8 mM CaCl2, 6 mM glucose and 20 mM HEPES, pH 7.4). Washed cells were incubated with 20 μM fluo-3-acetoxymethylester (Molecular Probes Inc., Eugene, OR) for 1.5 to 2 h at 20°C. Dye not taken up by cells was removed by aspiration followed by washing with 250 μl HBK. Fluorescence measurements were performed at 0.33-s intervals using a 96-well microtiter plate-reading fluorometer (Cambridge Technology, Inc., Watertown, MA). Ten basal fluorescence readings were recorded before activation with nAChR agonists. Responses after the addition of agonists were recorded for approximately 60 s. Antagonists were tested after a preincubation period of 5 to 10 min. Maximal fluorescence (Fmax) was determined after lysing the cells with 0.25% Triton X-100, and minimal fluorescence (Fmin) was determined after subsequent quenching with 10 mM MnCl2. Calculation of [Ca++]i was performed as described by Kaoet al. (1989). Cellular responses were quantitated either by calculating the ratio of peak [Ca++]i after agonist addition to basal [Ca++]i before agonist addition or by calculating the difference between peak [Ca++]i and basal [Ca++]i. EC50 and IC50 values were calculated using the ratio of peak [Ca++]i to basal [Ca++]i. Curve fitting was performed by Prism software (GraphPad, Inc., San Diego, CA) using the equation for a single-site sigmoidal dose-response curve with a variable slope,Y=Ymax/[1+(EC50/X)n]or Y=Ymax−Ymax/[1+(IC50/X)n] where Y is the response amplitude, X is the concentration of agonist or antagonist and n is the Hill coefficient. For determination of rank orders of potency, EC50 or Kb values (converted to log values) were compared by one-way ANOVA with Student-Newman-Keuls (SNK) multiple comparisons test (P < .05; SigmaStat, Jandel Scientific, Inc., San Rafael, CA). Other statistical comparisons also utilized a one-way ANOVA with SNK multiple comparisons test, as indicated in the text. Hill slopes were analyzed by Prism using a one-samplet test compared with a theoretical mean of 1.0 for agonists and of −1.0 for antagonists. The IC50 values for DHβE were converted into Kb values using the Leff-Dougall (Leff and Dougall, 1993) variant of the Cheng-Prusoff equationKb=IC50/[2+([A]/A50)n]1/n−1, where [A] is the agonist concentration, A50 is the EC50 value for the agonist and n is the Hill coefficient for the agonist. EC50, IC50 andKb values are presented in the text as geometric means (−S.D., +S.D.).

For the measurement of [Ca++]i signals in single cells, cells were plated on poly-d-lysine-coated glass coverslips at a density of 4 to 6 × 105cells/35-mm dish. Imaging experiments were performed at room temperature using a Zeiss Axovert microscope and a 100-W mercury lamp, an intensified CCD camera (Dage-MTI, Michigan City, IN) and IMAGE-1 hardware and software (Universal Imaging Corp., West Chester, PA). Cells were incubated with 1 μM fura-2-acetoxymethylester (Molecular Probes, Inc.) for 1 h and washed with mammalian Ringer’s solution to remove excess dye. Cells were transferred to a recording chamber (110 μl, Warner Instruments, Hamden, CT) and continuously superfused with Ringer’s solution at a rate of 8 to 10 ml/min. Agonists were applied by switching between reservoirs. Cells were alternatively excited at 350 and 380 nm (0.2 Hz), and background-subtracted ratio images were averaged for an approximately 12-by-12 pixel area over each cell. Data were further analyzed using IGOR software (WaveMetrics, Lake Oswego, OR). The 350/380 fluorescence ratios were converted into [Ca++]i using the relationship described byGrynkiewicz et al. (1995). To calibrate the fura-2 signals in intact cells, we determined Rmin andRmax values by bathing the cells in 10 mM EGTA, 1 μM ionomycin in Ringer’s solution (no added Ca++) or in buffer containing 10 mM Ca++, 1 μM ionomycin, respectively.

Binding of [3H]epibatidine.

Membranes were prepared by scraping cells from culture dishes in the growth medium and centrifuging for 10 min at 1200 × g. The cells were resuspended in an assay buffer containing 50 mM Tris pH 7.4, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2, homogenized with a Polytron and centrifuged for 15 min at 2000 ×g. The resulting pellet was frozen at −20°C until use. For saturation analysis, cell membranes (6–100 μg protein) were incubated with 5 pM to 1.0 nM [3H](±)epibatidine (total volume 2–5 ml) on ice for 2 h, followed by rapid filtration through Whatman GF/C filters presoaked in 0.5% polyethyleneimine for at least 30 min. Nonspecific binding was determined in the presence of 25 nM Epi. Protein concentrations were adjusted so that specifically bound ligand was always less than 10% of the total ligand in the assay and in most cases was less than 1%. Accordingly, in Scatchard plots the free concentration of ligand was considered to be equivalent to the total concentration.

Electrophysiological methods.

Cells were plated on poly-d-lysine-coated glass coverslips at a density of 1.5 × 105 cells/35-mm dish. One to two days after plating, recordings were performed with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) using the whole-cell voltage-clamp configuration (Hamill et al., 1981). Membrane potential was held at −80 mV. The external recording solution (mammalian Ringer’s solution) consisted of 160 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 11 mM glucose, 1 μM atropine and 5 mM HEPES, pH 7.3. Ringer’s solution was superfused at a rate of ≈3.0 ml/min (110-μl recording chamber). The recording pipette solution was composed of 135 mM CsCl, 10 mM EGTA, 1 mM MgCl2, 4 mM Mg-ATP and 10 mM HEPES, pH 7.3. Agonists were applied either by pressure ejection (200–700 ms pulses, General Valve Corp., Fairfield, NJ) from a glass micropipette (approximately 3–5 μm in diameter) positioned 20 to 60 μm above the cell or by a rapid application system (Jonas, 1995) using a triple-barrel glass pipette attached to an electromechanical switching device (piezo-electric drive, Winston Electronics, Millbrae, CA). Experiments were performed at room temperature. The current-voltage (I-V) relation of agonist-induced currents was determined by the application of 70 to 100-ms voltage ramps from −100 to +40 mV in the absence and in the presence of agonist. Ramps from +40 to −100 mV were also tested in some cells, resulting in the same I-V relation. The net agonist-induced current was obtained by subtracting the current observed in the absence of agonist from that measured in the presence of agonist.

Materials.

Polyclonal antisera were purchased from Bethyl Laboratories (Montgomery, TX) and were generated by immunizing sheep with the fusion proteins expressing the extracellular domains of rat neuronal nAChRs α3, α4 and β4 described by Neff et al. (1995). Epi and DHβE were purchased from Research Biochemicals International (Natick, MA). NEN Life Science Products (Boston, MA) was the supplier of [3H](±)epibatidine. All other compounds were obtained from Sigma Chemical Company (St. Louis, MO).

Results

Identification of stable cell lines.

HEK293 cells transfected with cDNAs encoding human nAChR subunits in the pairwise combinations α2β4, α3β4 and α4β4 were grown in selection medium for 2 weeks. On the basis of the reported Ca++ permeability of nAChRs (Mulle et al., 1992; Trouslard et al., 1993; Vernino et al., 1994) cell colonies resistant to antibiotic(s) were tested for functional nAChRs by measuring elevations in [Ca++]i upon stimulation with 100 to 300 μM Nic. Colonies from each pairwise receptor combination with the largest Nic-induced [Ca++]i signal were subcloned by limiting dilution. Cell lines A2B4, A3B4.2 and A4B4 that displayed the most robust and consistent Nic-stimulated elevations of [Ca++]i (fig.1) were selected for more detailed characterization of α2β4, α3β4 and α4β4 nAChRs, respectively. The magnitude of Nic-induced [Ca++]i signals was stable in each cell line for at least 6 months in continuous culture (data not shown).

Figure 1
Figure 1

Nicotine-induced changes of [Ca++]i in cells stably expressing human nicotinic receptor subunits. The A2B4, A4B4.2 and A4B4 cell lines were loaded with the Ca++-sensitive fluorescent dye fluo-3 as described in “Materials and Methods.” Data points were measured at approximately 0.33-s intervals. Each point is the mean [Ca++]i value (nM) of four individual wells from a 96-well plate (error bars were omitted for clarity) and represents the average signal of approximately 200,000 cells. After ten measurements of basal [Ca++]i, the lid of the fluorometer was opened briefly, and nicotine (∼EC90–95; hatched bar) was added manually, which accounts for the gap in each record before time 0. Shown are representative responses from each cell line. Detailed kinetic analyses of the rising and falling phases of the [Ca++]i signal were not performed, but in each case the peak [Ca++]i was attained 8 to 20 s after the addition of agonist. The agonist-induced [Ca++]i responses were dependent on the presence of external Ca++ (data not shown).

The homogeneity of each cell line was assessed by measuring agonist-stimulated increases of [Ca++]i in single cells loaded with fura-2. Upon application of 30 to 300 μM Nic, an increase of [Ca++]i greater than 60 nM was observed in 98% of A2B4 cells, 98% of A3B4.2 cells and 90% of A4B4 cells, and this response to Nic was blocked by coapplication with 10 to 100 μM DHβE (data not shown). These data support the conclusion that the A2B4, A3B4.2 and A4B4 cell lines predominantly contain cells expressing functional nAChRs.

Validation of stable cell lines by analysis of mRNA and protein.

Northern blot analysis showed that the cell lines A2B4, A3B4.2 and A4B4 expressed RNAs that hybridized to the appropriate subunit-specific probes (to α2 and β4, to α3 and β4 and to α4 and β4, respectively) and were of the predicted sizes for full-length transcripts (fig. 2). The RNAs encoding human α2, α3, α4 and β4 subunits were not detectable in HEK Neo/Zeo cells. The levels of RNA for each subunit were stable in each cell line for at least 6 months in continuous culture (data not shown).

Figure 2
Figure 2

Northern blot of total RNA isolated from cell lines A2B4, A3B4.2, A4B4 and HEK Neo/Zeo, showing the presence of RNA for human α2, α3, α4 and β4 subunits. Blots were hybridized under high stringency with DNA probes specific for the coding sequence of each subunit (see “Methods and Materials”). Hybridizing bands were detected corresponding to α2 and β4 transcripts in A2B4 cells, to α3 and β4 transcripts in A3B4.2 cells and to α4 and β4 transcripts in A4B4 cells. No RNAs hybridizing to the α2, α3, α4 or β4 probes were detected in HEK Neo/Zeo cells. Arrows indicate the predicted size of the full-length polyadenylated mRNA for the corresponding subunit. The larger transcripts, which are less prominent, are likely to contain the entire coding sequence and possibly some additional expression vector sequence.

The expression of α and β subunit polypeptides was examined by immunoblot analysis using subunit-specific antibodies. As shown in figure 3, membranes from HEK Neo/Zeo cells displayed no immunoreactivity for the rα3, rα4 or rβ4 antibodies that recognize human α2 and α3, α4 or β4 polypeptides, respectively. In contrast, immunoreactivity consistent with the presence of α2 and β4, of α3 and β4 or of α4 and β4 subunits was detected in membranes prepared from A2B4, A3B4.2 and A4B4 cells, respectively. The subunits migrate at a larger apparent molecular weight than predicted from the primary amino acid sequence because of glycosylation of the polypeptides (data not shown). Taken together, these data confirm the stable expression of human recombinant α2β4, α3β4 and α4β4 subunits in the A2B4, A3B4.2 and A4B4 cell lines, respectively.

Figure 3
Figure 3

Western blot of total membrane proteins from A2B4, A3B4.2 and A4B4 cells, showing the presence of α2, α4 and β4 subunit proteins. Membranes were isolated and the Western blot performed as described in “Materials and Methods.” HEK293 cells stably transfected with plasmids containing only the neomycin and Zeocin resistance genes (HEK Neo/Zeo) were used for controls. The α2, α3, α4 and β4 subunits migrated at apparent molecular weights of 70, 64, 78 and 60 kDa, respectively. The molecular weights predicted for α2, α3, α4 and β4 are 59, 53, 67 and 52 kDa, respectively. Migration of subunit proteins at apparent molecular weights larger than predicted by the primary amino acid sequence is due to N-linked glycosylation (data not shown).

Quantitation of receptor expression by binding of [3H]epibatidine.

As a means to quantitate and characterize the nAChRs expressed in each stable cell line, we measured the binding of [3H]epibatidine to cell membranes. We detected no specific binding to membranes prepared from HEK Neo/Zeo cells (data not shown). Specific and saturable binding of [3H]epibatidine was detected in membranes prepared from A2B4, A3B4.2 and A4B4 cells (fig. 4). Scatchard analysis revealed that binding was to a single, high-affinity site in each cell line (fig. 4, insets). The results summarized in table 1 show that the binding affinity was significantly greater in A2B4 cells than in A3B4.2 or A4B4 cells.Bmax values ranged from 1100 ± 338 fmol/mg protein in the A2B4 cells to 3683 ± 1450 in the A4B4 cells, but no statistical differences were detected (table 1). The presence of high-affinity specific binding sites for [3H]epibatidine in A2B4, A3B4.2 and A4B4 cells is consistent with the expression of assembled heteromeric nAChRs (Wang et al., 1996).

Figure 4
Figure 4

Saturation binding of [3H]epibatidine to membranes from A2B4, A3B4.2 and A4B4 cells. Binding of [3H]epibatidine was measured as described in “Materials and Methods.” The figure shows saturation isotherms for specific binding of [3H]epibatidine to membranes from cell lines A2B4 (α2β4, top panel), A3B4.2 (α3β4, middle panel) and A4B4 (α4β4, bottom panel). Scatchard analyses (shown at right) revealed that binding was to a single site in each cell line, with theKd and Bmax values summarized in table 1. The data presented are from one experiment that is representative of three performed in a similar manner.

View this table:
Table 1

Specific binding of [3H]epibatidine to membranes from HEK293 cells stably expressing human recombinant nAChR subunits

Validation of functional nAChRs by electrophysiology.

In voltage-clamped cells (Vh = −80 mV), application of 30 to 300 μM Nic, ACh, DMPP or Cyt elicited inward currents ranging from 13 pA to 14 nA in A2B4 cells (n = 20), from 1.1 to 20 nA in A3B4.2 cells (n = 20) and from 160 pA to 10.9 nA in A4B4 cells (n = 20). These currents desensitize rapidly in the presence of the agonist (see fig.5). Application to A3B4.2 cells of high concentrations of DMPP (30 μM and above) produced a “rebound” inward current upon removal of the agonist from the bath (fig. 5). This rebound current was not produced, or was minimal, in response to ACh, Nic, or Cyt (data not shown for Nic or Cyt) applied at the same concentration in A3B4.2 cells. Also, the rate of decay of current elicited by DMPP (100 μM) was faster compared with that of currents elicited by Nic (300 μM), by Cyt (100 μM; data not shown) and by ACh (300 μM) (P < .05) in A3B4.2 cells. In the A2B4 and A4B4 cells, no rebound current was detected with any agonist at concentrations up to 100 μM. The rebound current in A3B4.2 cells and the accelerated rate of decay of inward current are probably associated with voltage-dependent open-channel block by DMPP, as has been shown for nicotinic agonists on native nAChRs in bovine adrenal chromaffin cells (Machonochie and Knight, 1992) and on other recombinant nAChRs (Bertrand et al., 1992).

Figure 5
Figure 5

Whole-cell currents obtained in A2B4, A3B4.2 and A4B4 cells. Whole-cell patch-clamp recordings were performed as described in “Materials and Methods.” The recordings shown are from six different cells. Agonist was applied during the period indicated by the horizontal bar above each trace. Desensitization of the responses in the continued presence of the agonist is detectable in all three cell lines. The “rebound” current produced upon removal of DMPP in the A3B4.2 cells is probably due to relief of voltage-dependent open-channel block (see text). The mean amplitudes of agonist responses are summarized in the text.

The I-V relation of agonist-induced currents in A2B4, A3B4.2 and A4B4 cells shows a very strong inward rectification (fig. 6). The estimated reversal potential of these agonist-induced currents is −6.5 ± 3.2 mV for A2B4 cells (n = 6), +1.7 ± 7.8 mV for A3B4.2 cells (n = 5) and −4.7 ± 2.7 mV for A4B4 cells (n = 5).

Figure 6
Figure 6

Current-voltage relation of agonist-induced currents in A2B4, A3B4.2 and A4B4 cells. A strong inward rectification was observed in all cells. The Vrev for these currents varied between −12 and +12 mV (n = 16, see text). Voltage ramps and leak subtractions were carried out as indicated in “Materials and Methods.”

Pharmacological characterization of nAChR-mediated increases in [Ca++]i.

Epi, Nic, Cyt, DMPP and Sub stimulated concentration-dependent increases in [Ca++]i in the A2B4, A3B4.2 and A4B4 cells. Kinetics of the changes in [Ca++]i typically showed a rapid rising phase that reached a peak 8 to 20 s after the addition of agonist, followed by a slower relaxation toward basal [Ca++]i levels (see fig. 1). The maximal amplitude of the increases in [Ca++]i varied among cell lines. For example, Epi stimulated a maximal increase in [Ca++]i of 1152 ± 244 nM (mean ± S.E.; n = 4) in A2B4 cells, of 1912 ± 690 nM (n = 4) in A3B4.2 cells and of 880 ± 229 nM (n = 4) in A4B4 cells. ACh also stimulated increases in [Ca++]i, but we did not characterize its effects because of concern over degradation and the necessity of including atropine to block endogenous muscarinic receptors in HEK293 cells.

No agonist-stimulated elevation of [Ca++]iwas detected in untransfected HEK293 cells or in the absence of external Ca++ (data not shown), which indicates that the agonist-evoked increase in [Ca++]i required both recombinant nAChRs and Ca++ entry from the extracellular space. Furthermore, 100 μM CdCl2, a concentration expected to block voltage-gated calcium channels (VGCCs;De Waard et al., 1996) but not nAChRs (Rathouz and Berg, 1994), did not significantly inhibit the nicotine-evoked [Ca++]i signal in A2B4 (81% ± 15% of control, n = 4), A3B4.2 (159% ± 21% of control,n = 4) or A4B4 (117% ± 16% of control,n = 5) cells, which suggests that VGCCs do not participate in the agonist-induced [Ca++]isignal.

In the A2B4 cells, DMPP, Nic and Sub produced bell-shaped concentration-response relationships, whereas the response to Cyt appeared to reach a plateau (fig. 7). It was difficult to assess the shape of the concentration-response relationship for Epi because only one concentration was tested above the maximal response (fig. 7). The concentration of agonist producing a half-maximal increase of [Ca++]i was calculated from curve fits omitting points on the downward side of bell-shaped curves. The EC50 values for Epi, Nic, Cyt, DMPP and Sub are shown in table 2. The rank order of EC50 values in A2B4 cells was Epi > Cyt > Sub = Nic = DMPP (P < .05). Finally, Hill slopes were not significantly different from 1.0.

Figure 7
Figure 7

Concentration-dependent effects of nAChR agonists on [Ca++]i in A2B4, A3B4.2 and A4B4 cells. Cells were stimulated with either (−)nicotine (Nic, ▪), cytisine (Cyt, ○), DMPP (▴), suberyldicholine (Sub, ◊) or (+)epibatidine (Epi, •) at the concentrations indicated. The agonist-induced increase of [Ca++]i was quantitated using fluo-3 as described in “Materials and Methods” and is plotted as a percent of the maximal peak [Ca++]i/basal [Ca++]i response to nicotine in each cell line. Each point represents the mean value (± S.E.) from 3 or 4 experiments, each performed in quadruplicate. The EC50values, Hill coefficients and relative efficacies are summarized in table 2.

View this table:
Table 2

Pharmacological properties of agonist effects on HEK293 cells stably expressing human recombinant nAChR subunits

In the A3B4.2 cells, there was evidence of a bell-shaped concentration-response relationship for Nic and DMPP (fig. 7). For Cyt, a peak response below the highest concentration tested (1 mM) was attained in only one out of three experiments, so the EC50value may be an underestimate. The EC50 values for Epi, Nic, Cyt, DMPP and Sub in A3B4.2 cells are shown in table 2. The rank order of EC50 values in A3B4.2 cells was Epi > DMPP = Cyt = Nic = Sub (P < .05). With the exception of Cyt, none of the agonists displayed a Hill slope different from 1.0.

In the A4B4 cell line, only DMPP produced a marked bell-shaped concentration-response relationship, although higher concentrations of the other agonists were not tested (fig. 7). The EC50values for Epi, Nic, Cyt, DMPP and Sub in the A4B4 cells (table 2) reveal the rank order Epi > Cyt = Sub > Nic > DMPP (P < .05), and none of the agonists had a Hill slope different from 1.0. These data show that each cell line displays a unique rank order of potency for the five agonists.

Most of the agonists also displayed selectivity across cell lines. The rank orders of EC50 values were A2B4 > A4B4 > A3B4.2 for Epi, A4B4 = A2B4 > A3B4.2 for Nic, A2B4 = A4B4 > A3B4.2 for Cyt and A4B4 > A2B4 > A3B4.2 for Sub (P < .05). DMPP displayed no selectivity.

The efficacies of the agonists relative to Nic also varied, as illustrated in figure 7 and summarized in table 2. Cytisine was significantly less efficacious than Nic in the A2B4 cells. Although DMPP also appeared to be less efficacious than Nic in the A2B4 cells, the effect was not significant at the 0.05 level (P = .07). In the A3B4.2 cells, Epi was significantly more efficacious than Nic, whereas Cyt and DMPP did not differ from Nic in efficacy. The efficacy of Sub in the A3B4.2 cells tended to be lower than that of Nic, but this effect did not reach statistical significance (P = .08). In the A4B4 cells, Cyt, DMPP and Sub were less efficacious than Nic.

Figure 8 demonstrates that Nic-evoked elevations of [Ca++]i in A2B4, A3B4.2 and A4B4 cells were blocked by the nicotinic antagonists d-tubocurarine, mecamylamine and DHβE. At the EC80–93 of Nic, the concentrations of d-tubocurarine, mecamylamine and DHβE producing half-maximal inhibition are summarized in table3. There were no significant differences in IC50 value between cell lines for mecamylamine or d-tubocurarine. Only d-tubocurarine in A4B4 cells displayed a Hill slope different from −1.0. The Kb values for the competitive antagonist DHβE are also shown in table 3. On the basis of the Kb values, the rank order of potency for DHβE was A2B4 = A4B4 > A3B4.2 cells.

Figure 8
Figure 8

Effect of nAChR antagonists on agonist-stimulated [Ca++]i elevations in A2B4, A3B4.2 and A4B4 cells. The stimulation-induced increases of [Ca++]i were quantitated using fluo-3 as described in “Materials and Methods.” Cells were incubated with either mecamylamine (Mec, ▪), DHβE (○) or d-tubocurarine (d-Tubo, □) for 5 to 10 min before the addition of agonist. The agonist was 20 μM nicotine for the A2B4 cells (EC90) and was 200 μM nicotine for the A3B4.2 and A4B4 cells (EC81 and EC93, respectively). The data are plotted as percent of the control peak [Ca++]i/basal [Ca++]i response to nicotine. Each point represents the mean value (± S.E.) from 3 or 4 experiments, each performed in quadruplicate.

View this table:
Table 3

Pharmacological properties of antagonist effects on HEK293 cells stably expressing human recombinant nAChR subunits

Discussion

This is the first report describing functional and pharmacological characteristics of human recombinant β4-containing neuronal nAChRs stably expressed in a mammalian cell line. We utilized the Ca++-permeability of nAChRs to develop an assay for functional receptors based on the measurement of [Ca++]i. With this method, HEK293 cells expressing functional recombinant nAChRs were identified by monitoring nicotinic agonist-induced increases in [Ca++]i. The agonist-evoked [Ca++]i signals were mediated by activation of nAChRs, as shown by blockade with nicotinic antagonists.

The agonist-induced [Ca++]i signal in the A2B4, A3B4.2 and A4B4 cell lines derives from the entry of Ca++ through cell surface receptors, because no agonist-induced [Ca++]i responses were detected in the absence of external Ca++ (data not shown). The transient nature of the agonist-stimulated [Ca++]i signal may be explained by a combination of desensitization of the human nAChRs (see fig. 5 andChavez-Noriega et al., 1997) and increased buffering of cytosolic Ca++. Whether the [Ca++]i signal also involves secondary activation of calcium-induced calcium release mechanisms or involves endogenous VGCCs (Berjukow et al., 1996) is not known. However, experiments with Cd++ to block endogenous VGCCs suggest that they do not participate in the agonist-induced elevation of [Ca++]i. Stetzer et al. (1996)have recently demonstrated nicotine-induced [Ca++]i signals in HEK293 cells that stably express both recombinant l-type Ca++ channels and rat α3β4 receptors. It was suggested that the nicotine-induced [Ca++]i signal was amplified by functional coupling of nAChR-mediated depolarization to activation of the recombinant VGCCs. Our results indicate that an ample [Ca++]i signal can be achieved without functional coupling of human β4-containing nAChRs to recombinant VGCCs. The [Ca++]i assay provides a rapid and convenient way to study the function and pharmacology of human nAChRs.

Northern and western blot analyses demonstrated the presence of RNA and protein associated with the human α2 and β4, the human α3 and β4 and the human α4 and β4 subunits in the A2B4, A3B4.2 and A4B4 cells, respectively. The cell lines were stable for at least 6 months in continuous culture and for at least 2 years in frozen storage, as judged by stability in steady-state levels of RNA and agonist-induced [Ca++]i signals.

Specific binding of [3H]epibatidine, a high-affinity ligand for nAChRs (Houghtling et al., 1994), to membranes isolated from A2B4, A3B4.2 and A4B4 cells was saturable and was described by a single site, which is consistent with the presence of a homogeneous population of high-affinity receptors. The affinity for [3H]epibatidine was 4- to 5-fold higher for human α2β4 receptors (Kd = 42 pM) than for α3β4 (Kd = 230 pM) and α4β4 (Kd = 187 pM). These relatively high affinities are consistent with the Ki for (±)epibatidine binding to human α4β2 receptors stably expressed in HEK293 cells (70 pM; Gopalakrishnan et al., 1996). The similarity of affinity values suggests that it might be difficult to resolve putative α2β4, α3β4, α4β4 or α4β2 nAChR subtypes in brain solely on the basis of their relative affinities for [3H]epibatidine binding.

The Kd values for [3H]epibatidine binding were at least 200-fold lower than the EC50 values for Epi-induced [Ca++]i signals, a result consistent with the interpretation that in equilibrium binding assays [3H]epibatidine labels a high-affinity desensitized state of nAChRs (Gerzanich et al., 1995). Interestingly, the similarity in binding affinities at α3β4 and α4β4 receptors (203 and 187 pM, respectively) was not reflected in the relative EC50 values for Epi-induced [Ca++]i signals (151 and 38 nM, respectively). Thus the binding data would not have predicted the 4-fold selectivity in potency of Epi-induced [Ca++]i signals at α3β4 and α4β4 receptors. These results provide evidence that binding assays do not always reflect the functional selectivity of nicotinic agonists on subtypes of nAChRs.

The presence of functional nAChRs on the surface of the A2B4, A3B4.2 and A4B4 cells was confirmed with whole-cell patch-clamp recordings. Previous studies using Xenopus oocytes have shown that human α2, α3, α4 and β4 subunits are functional only when expressed in pairwise combinations of αxβ4 (Elliott et al., 1996). These data are consistent with the interpretation that the functional responses detected in A2B4, A3B4.2 and A4B4 cells derive from pairwise combinations of nAChR subunits.

The maximal agonist-induced current detected in the A2B4, A3B4.2 and A4B4 cell lines (14, 20, and 10.9 nA, respectively) corresponds to the rank order of maximal Epi-induced [Ca++]isignals. The current-voltage relations for agonist-induced currents demonstrated strong inward rectification, a result consistent with the properties reported for native nAChRs recorded from cultured rat sympathetic neurons (Trouslard et al., 1993), bovine adrenal chromaffin cells (Nooney et al., 1992), acutely dissociated rat medial habenula neurons (Mulle et al., 1991) and chick ciliary ganglion neurons (Rathouz and Berg, 1994). Recombinant chick α4β2 nAChRs stably expressed in mouse fibroblasts (Whiting et al., 1991) and human α4β2 nAChRs stably expressed in HEK293 cells (Gopalakrishnan et al., 1996) also display similar rectifying properties.

The [Ca++]i assay was used to examine the pharmacological profile of the human recombinant α2β4, α3β4 and α4β4 receptors stably expressed in HEK293 cells. Some of the nicotinic agonists displayed marked bell-shaped concentration-response relationships. This behavior cannot be explained by Ca++-dependent inactivation of the nAChRs at higher [Ca++]i concentrations, because it was observed with agonists that stimulated both relatively high and relatively low [Ca++]i signals,e.g., Sub and DMPP in the A2B4 cells. It is more likely that the bell-shaped curves result from either rapid desensitization or open-channel block that occurs at higher agonist concentrations. For example, Marshall et al. (1991) found that Sub produced a bell-shaped concentration-response curve on nAChRs at the frog neuromuscular junction, with the downward side of the curve resulting from open-channel block of the receptors. The “rebound” current detected upon washout of DMPP in the A3B4.2 cells (fig. 5) is consistent with relief from open-channel block. More detailed electrophysiological measurements in the stable cell lines may resolve this issue.

The relative potencies of five nicotinic agonists showed that each nAChR subtype possessed a unique agonist profile. The rank order of potency was Epi > Cyt > Sub = Nic = DMPP at α2β4, Epi > DMPP = Cyt = Nic = Sub at α3β4 and Epi > Cyt = Sub > Nic > DMPP at α4β4 receptors. Absolute EC50 value and efficacy were also influenced by receptor subtype. For example, the EC50values for Cyt were at least 50- and 60-fold lower on α4β4 and α2β4 receptors, respectively, than on α3β4 receptors. Also, Sub was as efficacious as Nic on α2β4 receptors but was less efficacious than Nic on α4β4 receptors. These data support the conclusion that human α2, α3 and α4 subunits contribute to the potency, selectivity and efficacy of nicotinic agonists at β4-containing receptors.

Using statistical comparisons of EC50 values, we found that the relative potency of DMPP = Cyt = Nic at human α3β4 receptors expressed in the A3B4.2 cell line is similar to that at rat α3β4 receptors transiently (DMPP = Cyt = Nic; Wonget al., 1995) or stably (Cyt ≅ Nic; Stetzer et al., 1996) expressed in HEK293 cells. Other comparisons reveal that Cyt is as efficacious as Nic at transiently expressed rat α3β4 receptors (Wong et al., 1995) and at human α3β4 receptors stably expressed in the A3B4.2 cell line. However, DMPP was less efficacious than Nic at rat α3β4 receptors (Wong et al., 1995), whereas it was as efficacious as Nic at human α3β4 receptors. Unfortunately, efficacy data were not reported for rat α3β4 receptors stably expressed in HEK293 cells (Stetzer et al., 1996). Another difference is in the Hill slopes for Cyt, DMPP and Nic. These values were greater than unity at rat α3β4 receptors expressed in HEK293 cells (Wong et al., 1995; Stetzeret al., 1996), but at human α3β4 in A3B4.2 cells the Hill slopes for DMPP and Nic were not different from unity, and that for Cyt was less than unity. Different methodologies, or species differences, might explain the differences in efficacy and Hill slope between rat and human α3β4 nAChRs expressed in HEK293 cells.

A panel of human β4-containing nAChRs have recently been examined by transient expression in Xenopus oocytes (Chavez-Noriega et al., 1997). The EC50values for Nic and DMPP at α2β4 receptors expressed in oocytes (21 and 23 μM, respectively; Chavez-Noriega et al., 1997) were close to their values at α2β4 receptors stably expressed in the A2B4 cells (9.9 and 12.2 μM, respectively). The efficacies of DMPP and Cyt were also lower than that of Nic in oocytes expressing α2β4 receptors, and a similar tendency in efficacy was determined for these agonists in the A2B4 cells (although the value for DMPP did not quite achieve statistical significance compared with Nic; P = .07). On the other hand, the EC50 value for Cyt was much lower in the A2B4 cells (0.44 μM) than in the oocytes (39 μM), resulting in a different rank order of potency (Cyt > Nic = DMPP in A2B4 cells, DMPP ≈ Nic > Cyt in oocytes). The reason for this difference remains unclear (but see below for possible explanations).

At human α3β4 receptors stably expressed in the A3B4.2 cells, the rank order of potency of DMPP = Cyt = Nic differed slightly from the rank order of DMPP > Cyt ≈ Nic at human α3β4 receptors expressed in Xenopus oocytes (Chavez-Noriegaet al., 1997). However, the EC50 values for Epi, DMPP, Cyt and Nic in the A3B4.2 cells (151 nM, 12, 26 and 40 μM, respectively) were within 3-fold of the corresponding values in oocytes (73 nM, 19, 72 and 80 μM; Gerzanich et al., 1995,Chavez-Noriega et al., 1997). The efficacy profile also revealed that Cyt and DMPP were as efficacious as Nic in stimulating [Ca++]i increases in the A3B4.2 cells, whereas Cyt was less efficacious than Nic in oocytes (Chavez-Noriegaet al., 1997). Aside from the differences detected with Cyt, there is reasonable agreement in activity among Epi, Nic and DMPP at human α3β4 receptors expressed in the A3B4.2 cells andXenopus oocytes.

For human α4β4 receptors stably expressed in the A4B4 cells, the rank order of potency of Cyt > Nic > DMPP was identical to that for α4β4 receptors expressed in Xenopus oocytes (Chavez-Noriega et al., 1997). The EC50 values for Cyt, Nic and DMPP were similar in A4B4 cells (0.52, 6.7 and 18 μM, respectively) and oocytes (0.9, 5 and 19 μM, respectively). Also, the efficacies of Cyt and DMPP were less than that of Nic in both the A4B4 cells and the oocytes expressing α4β4 receptors. Thus the pharmacological profiles of Cyt, Nic and DMPP were the same for human α4β4 receptors expressed in HEK293 cells or in Xenopusoocytes. The pharmacological differences detected between oocytes and stable cell lines expressing human α2β4 or α3β4 receptors may result from the divergent methodologies employed, from differences in subunit stoichiometries or from intrinsic differences between the two expression systems.

The antagonists d-tubocurarine and mecamylamine displayed no selectivity in potency among α2β4, α3β4 and α4β4 receptors stably expressed in HEK293 cells. That the Hill slope for d-tubocurarine was significantly less than −1.0 on α4β4 receptors may reflect a noncompetitive mechanism of action (Chavez-Noriegaet al., 1996, Mulle et al., 1991). In contrast, the competitive antagonist DHβE displayed subtype selectivity, which is consistent with results reported previously with rat or human α2β4, α3β4 and α4β4 receptors expressed inXenopus oocytes (Harvey et al., 1996; Harvey and Luetje, 1996; Chavez-Noriega et al., 1997). At human α2β4, α3β4 and α4β4 receptors expressed inXenopus oocytes (Chavez-Noriega et al., 1997), the Kb values for DHβE were 3.61, 13.8 and 0.01 μM, respectively. The Kb value determined for α3β4 receptors expressed in oocytes agrees with theKb value determined in the A3B4.2 cells (Kb = 9.0 μM), but theKb values at α2β4 and α4β4 receptors expressed in oocytes lack agreement with the corresponding cell lines. These discrepancies may be related to the lower concentrations of ACh (30 μM, EC16) or Nic (10 μM, EC70) used in the oocyte study compared with the higher concentrations of nicotine used here (EC80–93). High concentrations of nicotinic agonists have been associated with channel-blocking activity (De Fiebreet al., 1995; Machonochie and Knight, 1992; Sine and Steinbach, 1984), which could complicate the inhibitory effects of DHβE. In an observation consistent with this hypothesis, preliminary evidence indicates that the Kb value for DHβE determined in A4B4 cells at 10 μM Nic (EC60) is 72 nM (data not shown), a value closer to that determined in oocytes (Chavez-Noriega et al., 1997). Additionally, it is possible that the Kb for competitive antagonists may change depending on the agonist employed (Brabet et al., 1995).

Recent data indicate a significantly greater number of nAChR subunit combinations in the CNS and the peripheral nervous system than were implicated previously by biochemical studies that assigned dominant roles to α4β2 receptors in the CNS and α3β4 receptors in the peripheral nervous system (McGehee and Role, 1995). Work is now in progress that may make it possible to associate nicotinic responses in neurons with specific nAChR subtypes. For example, although Cyt was reported to be a partial agonist at stimulating [3H]-dopamine release in rat striatal slices or synaptosomes (El-Bizri and Clarke, 1994; Sacaan et al., 1995), Grady et al. (1992) have shown that Cyt acts as a full agonist in evoking [3H]-dopamine release from mouse striatal synaptosomes. The latter result suggests the involvement of β4, because β4 combined with α2, α3 or α4 was shown to be more responsive to Cyt than β2 expressed with the same α subunits (Luetje and Patrick, 1991). Additionally, Marks et al.(1993) reported the rank order of potency for agonist-induced Rb+ efflux from mouse thalamic synaptosomes as Cyt (0.09 μM) > Nic (0.18 μM) > ACh (0.54 μM) ≈ DMPP (0.64 μM). This order does not correlate with the reported rank order of potency for rat or human α4β2 (Chavez-Noriega et al., 1997; Luetje and Patrick, 1991), but it does correlate with human α4β4 expressed in oocytes and the A4B4 cell line. Clearly, stable expression of human recombinant nAChRs containing β4 subunits will prove useful in elucidating the composition of native nAChRs.

In conclusion, the results presented in this report show the A2B4, A3B4.2 and A4B4 cell lines to be powerful tools for examining the properties of human recombinant α2β4, α3β4 and α4β4 nAChRs, respectively. The advantage they offer in the identification of subtype-selective compounds will be valuable both for developing potential therapeutic agents and in probing the physiological function of nAChRs.

Acknowledgments

The authors wish to thank Cecilia Tran for excellent technical assistance and Karen Payne for secretarial help in the preparation of this manuscript.

Footnotes

  • Send reprint requests to: Kenneth A. Stauderman, SIBIA Neurosciences, Inc., 505 Coast Blvd. So., Suite 300, La Jolla, CA 92037-4641.

  • Abbreviations:
    [Ca++]i
    intracellular free Ca++ concentration
    DHβE
    dihydro-β-erythroidine
    HBK
    HEPES-buffered Krebs-saline
    PBS
    phosphate-buffered saline
    SDS
    sodium dodecyl sulfate
    ACh
    acetylcholine
    Epi
    (+)epibatidine
    Nic
    (−)nicotine
    DMPP
    1,1-dimethyl-4-phenylpiperazinium iodide
    Cyt
    cytisine
    Sub
    suberyldicholine
    EC50
    concentration of agonist producing half-maximal effect
    IC50
    concentration of antagonist producing half-maximal inhibition
    • Received June 24, 1997.
    • Accepted October 15, 1997.

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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Characterization of Human Recombinant Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations α2β4, α3β4 and α4β4 Stably Expressed in HEK293 Cells

Kenneth A. Stauderman, L. Scott Mahaffy, Michael Akong, Gönül Veliçelebi, Laura E. Chavez-Noriega, James H. Crona, Edwin C. Johnson, Kathryn J. Elliott, Alison Gillespie, Richard T. Reid, Pamala Adams, Michael M. Harpold and Janis Corey-Naeve
Journal of Pharmacology and Experimental Therapeutics February 1, 1998, 284 (2) 777-789;
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