Introduction
Nicotinic acetylcholine (ACh) receptors are members of the pentameric ligand-gated ion channel superfamily, and are expressed at neuromuscular junctions and within the central and peripheral nervous system, where they are activated by nicotine and the endogenous neurotransmitter, ACh. A total of 17 nicotinic ACh receptor subunits have been identified in vertebrates (α1–α10, β1–β4, γ, δ and ε), and these subunits can assemble into a variety of heteropentameric and homopentameric receptors (1,2).
Of the nicotinic ACh receptor subtypes, the homopentameric α7 nicotinic ACh receptor is known to be the most permeable to calcium ions (Ca2+). Calcium influx through the α7 nicotinic ACh receptor is involved in increasing cytoplasmic calcium levels, which in turn triggers a series of calcium-dependent intracellular processes. Following the suggestion that the α7 nicotinic ACh receptor regulates inflammation, it has been the focus of intense investigation since the early 21st century (3). Consequently, there has been substantial interest in the identification and characterization of the α7 nicotinic ACh receptor.
However, the expression of functional recombinant α7 nicotinic ACh receptors in mammalian cell types, including human embryonic kidney (HEK) 293 cells, has been problematic, as the assembly of the α7 nicotinic ACh receptor is a slow and inefficient process. Individual subunits require appropriate transmembrane topology and undergo a series of critical post-translational modifications (4). In addition, to enable folding into the correct conformation, the receptors require appropriate inter-subunit interactions. The early steps of receptor folding and assembly occur within the endoplasmic reticulum, an intracellular compartment containing several proteins required for efficient protein folding and post-translational modification (4). Although there have been reports of the successful functional expression of the recombinant α7 nicotinic ACh receptor in certain mammalian cell lines (5–8), measurable levels of functional receptors have been difficult to achieve in several cell types. This effect appears to be host-cell dependent (9,10), as functional α7 nicotinic ACh receptors can be generated in mammalian cell lines when co-expressed with either Caenorhabditis elegans resistance to inhibitors of cholinesterase 3 (CeRIC-3) or its human homolog (hRIC-3).
To the best of our knowledge, the functional expression of recombinant human α7 nicotinic ACh receptors in HEK 293 cells co-expressing hRIC-3 has not been reported. In the present study, heterologously expressed nicotinic ACh receptors in HEK 293 cells were investigated and the functional expression levels of recombinant α7 nicotinic ACh receptors in the HEK 293 cells were examined, in order to aid in the development of novel pharmaceutical agents.
Materials and methods
Drugs
The drugs used in the present study were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. Fluo-4 AM (1 mM) and Pluronic® F-127 (5%) were prepared in dimethyl sulfoxide and stored at −20°C. All solutions were prepared and diluted appropriately prior to experimentation.
Cell culture and transfection
Transfection was performed, as described previously (10). Expression plasmids (hRIC-3 and hα7) containing complementary DNA sequences for hRIC-3 and α7 nicotinic ACh receptor subunits, respectively, were used. The subunits were subcloned into pcDNA3.1+ (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). HEK 293 cells were cultured at 2×104 cells/ml in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO2 incubator. The medium was renewed every 3 days. The HEK 293 cells were then transfected with the expression plasmids using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The transfected cells were incubated for 24 h prior to obtaining recordings.
Immunohistochemistry
The transfected HEK 293 cells (Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai) were grown on glass chamber slides. The cells were washed twice, (5 min for each wash) in 0.05 M phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature. Following washing five times with 0.05 M PBS, the cells were incubated in a blocking solution [10% normal goat serum (Sigma-Aldrich) and 1% Triton™ X-100 in PBS] for 1 h at room temperature. The cells were then incubated for 4 h at 4°C with rabbit anti-human α7 nicotinic ACh receptor polyclonal antibody (cat. no. sc-5544; Santa-Cruz Biotechnology Inc, Santa Cruz, CA, USA) at a dilution of 1:500 in 0.05 M PBS containing 1% Triton™ X-100 and 2% normal goat serum. The slides were then washed with 0.05 M PBS four times, (5 min for each wash). The washed sections were then incubated for 2 h at 37°C with 5% CO2 with a secondary antibody (goat anti-rabbit Alexa Fluor 488; Molecular Probes, Invitrogen; Thermo Fisher Scientific, Inc.) at a dilution of 1:1,000 in 10% normal goat serum/PBS/Triton™ X-100 solution. The cells were then washed with 0.05 M PBS four times (5 min for each wash) prior to incubation with 4′,6-diamidino-2-phenylindole (1:15,000) for 5 min. The slides were stored at 4°C until further use. The cells were visualized with the fluorescence microscope (Leica DMI4000 B; Leica Microsystems GmbH, Wetzlar, Germany) and an optical microscope (BX51, U-TV0.5XC-3; Olympus Corporation, Tokyo, Japan) and camera (DFC320; Leica Microsystems GmbH).
Western blot analysis
Cell lysates were prepared by incubating the HEK 293 cells with lysis buffer, containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris (pH 7.4), 0.02% NaN3, 1% Triton™ X-100 and protease inhibitor cocktail, on ice for 90 min. Protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal quantities (5 µg) of total protein were subjected to 12% SDS-polyacrylamide electrophoresis and were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline prior to western blot analysis. The membranes wer incubated with the rabbit anti-human α7 nicotinic ACh receptor polyclonal primary antibody (1:2,000; cat. no. sc-5544; Santa-Cruz Biotechnology, Inc.) at 4°C overnight, followed by incubation at 37°C for 2 h with the horseradish peroxidase-conjugated secondary antibodies (1:5,000; ab6721; Abcam, Cambridge, MA, USA). The signals were detected using enhanced chemiluminescence reagent (EMD Millipore). The expression of each target protein was relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and was calculated based on the grey level.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was prepared from the in vitro HEK 293 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RT-PCR analysis was performed using a PrimeScript RT reagent kit and SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. The reaction mixture (25 µl) was comprised of 2X Subgreen mix (12.5 µl), forward and reverse primers (each 10 µM; 1 µl), cDNA (1 µg; 0.5 µl) and diethylpyrocarbonate-treated ddH2O (10 µl). The primer sets for reverse transcription were as follows: RIC-3, forward 5′-TTCAGACTGTATCAAGCGTAGGC-3′ and reverse 5′-TGGATCACACGAGGTAACAGAA-3′; GAPDH, Forward 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse 5′-GCCATCACGCCACAGTTTC-′3. Cycling was conducted using and ABI 7900 cycling machine (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the conditions were as follows: 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec.
Calcium imaging
Changes in cytosolic free calcium concentration were measured using fluorescence imaging with the Ca2+-sensitive dye, Fluo-4. The transfected HEK 293 cells were treated with 2 µM Fluo-4 AM for 30 min at 37°C, in a medium containing 120 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM glucose and 10 mM HEPES (pH 7.3, adjusted with Tris) prior to imaging. Following treatment with the dye, the cells were observed under an inverted microscope (Leica DMI4000 B) and images were captured using a charge-coupled device camera (Leica DF350, Leica Microsystems GmbH), as shown in Fig. 1. The fluorescence intensities of individual cells in regions of interest were recorded and analyzed using Leica Advanced Florescence Application software (AF6000; Leica Microsystems GmbH). Nicotine (10, 30, 100, 300 and 1,000 nM) and adenosine diphosphate (ADP; 10 µM) were applied to the cells by gravity using a microperfusion apparatus. Between each drug application for 5 sec, a 15-min washout period with fresh medium was included to allow clearance of the drug.
Statistical analysis
Data are presented as the mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Two-tailed unpaired Student's t-tests were used for all comparisons, unless otherwise indicated.
Results
Surface α<sub>7</sub> receptors are detected on the surface of HEK 293 cells expressing the human α<sub>7</sub> receptor and/or co-expressing hRIC-3
To detect the protein expression of α7, HEK 293 cells were incubated and transfected, and the HEK 293 cells were treated with antibody directed against the α7 protein. This was followed by incubation with a fluorescent-labeled secondary antibody. No discernible binding of the anti-α7 antibody to the HEK 293 control cells was observed (Fig. 1). Immunostaining of the HEK 293 cells co-transfected with hRIC-3 revealed binding of anti-α7 antibodies to the surface of the co-transfected cells, suggesting that these cells expressed α7 nicotinic ACh receptors on their membrane surface.
α<sub>7</sub> protein is detected in HEK 293 cells expressing human α<sub>7</sub> receptors and/or co-expressing hRIC-3
To detect the protein expression of α7, the present study examined HEK 293 cells, transfected HEK 293 cells and co-transfected HEK 293 cells using western blot analysis with antibody against α7 protein. As shown in Fig. 2, α7 protein was detected in the transfected and co-transfected HEK 293 cells.
hric3 mRNA is expressed in co-transfected HEK 293 cells, and is absent in HEK 293 cells and transfected HEK 293 cells
RT-PCR analysis was used to examine the expression of hric3 in HEK 293 cells, transfected HEK 293 cells and co-transfected HEK 293 cells. As shown in Fig. 3, hric3 transcripts were detected in the co-transfected HEK 293 cells only; hric3 transcripts were not detected in the HEK 293 cells or HEK 293 cells transfected with α7 nicotinic ACh receptor alone.
Nicotine does not induce calcium transients in HEK 293 cells expressing human α<sub>7</sub> receptors
Subsequently, to analyze the changes in the concentration of cytosolic free calcium induced by the opening of the α7 nicotinic ACh receptors, HEK 293 cells expressing human α7 receptors were treated with various concentrations of nicotine for 30 sec. Following 15 min of washout with fresh medium, the cells were treated with 10 µM ADP. Nicotine did not induce calcium influx in the HEK 293 cells expressing human α7 receptors at any concentration (Fig. 4).
Nicotine induces calcium transients in HEK 293 cells co-expressing hRIC-3 and human α<sub>7</sub> receptors
The present study then analyzed the changes in the concentration of cytosolic free calcium induced by the opening of α7 nicotinic ACh receptors in HEK 293 cells transiently co-expressing hRIC-3 and the human α7 nicotinic ACh receptor. In these cells, high levels of functional α7 nicotinic ACh receptors were expressed; the activity of these receptors has been confirmed in a previous study using whole-cell patch-clamp recording (7).
To assess the effect of nicotine on functional α7 nicotinic ACh receptors, the co-transfected HEK 293 cells were treated with different concentrations of nicotine for 30 sec. Nicotine induced calcium influx in a concentration-dependent manner (Fig. 5). The data obtained were fitted to a logistic equation, and the nicotine concentration required to elicit 50% of the maximal response was calculated to be 29.42 µM, with a 95% confidence interval of 13.32–65.42 µM).
Discussion
Although the α7 subunit is able to generate functional nicotinic ACh receptors when expressed in Xenopus oocytes, considerable difficulty has been encountered in the efficient expression of functional α7 nicotinic ACh receptors in cultured mammalian cell lines (9,11–13). Previous studies (10,14) have demonstrated that α7 can efficiently generate functional nicotinic ACh receptors in mammalian cell lines when co-expressed with either CeRIC-3 or its human homolog, hric-3. RIC-3 is required for efficient receptor folding, assembly and functional expression of homomeric α7 nicotinic ACh receptors (15).
In the present study, the expression of functional recombinant nicotinic ACh receptors in HEK 293 cells was induced by co-expression with hRIC-3. In addition, immunohistochemistry and western blot analysis were performed to confirm the protein expression of the human α7 nicotinic ACh receptor, and RT-PCR analysis was used to detect hric3 transcripts. Native HEK 293 cells did not express the human α7 nicotinic ACh receptor pr hric-3 transcripts, whereas the human α7 nicotinic ACh receptor was detected following transfection. Even in the absence of hric-3, α7 protein was detected in the HEK 293 cells transiently expressing α7.
Using the calcium dye, Fluo-4, to record intracellular calcium signals generated by the opening of α7 nicotinic ACh receptors, images of calcium transients were captured in the transfected HEK 293 cells expressing a high level of α7 nicotinic ACh receptors. It was demonstrated that these HEK 293 cells did not express detectable levels of hric-3 transcripts, which was associated with a lack of functional human α7 nicotinic ACh receptors. Subsequently, images of calcium transients were captured in co-transfected HEK 293 cells with high expression levels of α7 nicotinic ACh receptors and hric-3 transcripts. The observed calcium transients were predominantly derived from the opening of membrane α7 nicotinic ACh receptors, as evidenced by the following observations: (i) the signals were induced by treatment with nicotine and (ii) human α7 nicotinic ACh receptors were detectable in the co-transfected HEK 293 cells using immunohistochemical and western blot analyses.
In conclusion, the findings of the present study suggested that hRIC-3, when co-expressed with human α7 nicotinic ACh receptors in HEK 293 cells, supported the functional expression of α7 nicotinic ACh receptors. These observations may aid in the development of treatment strategies for inflammation.