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• W2002244218 abstract "The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing α4 and β2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat α4β2 nAChR. Functional recombinant α4β2 nAChRs, showing high affinity binding of nicotinic radioligands (Kd = 41 ± 22 pm for [3H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of α4β2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 °C. At 30 °C, however, cells expressing α4β2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to “wild-type” α4 and β2 subunits, chimeric nicotinic/serotonergic subunits (“α4χ” and “β2χ”) are expressed very efficiently on the cell surface (at 30 °C or 37 °C), either as hetero-oligomeric complexes (e.g. α4χ+β2 or α4χ+β2χ) or when expressed alone. Compared with α4β2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein. The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing α4 and β2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat α4β2 nAChR. Functional recombinant α4β2 nAChRs, showing high affinity binding of nicotinic radioligands (Kd = 41 ± 22 pm for [3H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of α4β2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 °C. At 30 °C, however, cells expressing α4β2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to “wild-type” α4 and β2 subunits, chimeric nicotinic/serotonergic subunits (“α4χ” and “β2χ”) are expressed very efficiently on the cell surface (at 30 °C or 37 °C), either as hetero-oligomeric complexes (e.g. α4χ+β2 or α4χ+β2χ) or when expressed alone. Compared with α4β2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein. nicotinic acetylcholine receptor dithiobis-sulfosuccinimidylpropionate horseradish peroxidase 5-hydroxytyptamine monoclonal antibody bovine serum albumin Dulbecco's modified Eagle's medium phosphate-buffered saline polyacrylamide gel electrophoresis Hanks' buffered saline solution lysis buffer In addition to the relatively well characterized nicotinic acetylcholine receptor (nAChR)1 expressed at the vertebrate neuromuscular junction, a family of pharmacologically distinct “neuronal” nAChRs is expressed within the central and peripheral nervous system (1Green W.N. Millar N.S. Trends Neurosci. 1995; 18: 280-287Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 2McGehee D.S. Role L.W. Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (896) Google Scholar). Whereas the muscle-type nAChR is a pentameric complex of known subunit composition (α2βγδ in fetal muscle and α2βεδ in adult), the precise subunit composition of the various neuronal nAChR subtypes is less certain. To date, 11 neuronal nAChR subunits (α2–α9 and β2–β4) have been identified and cloned. There is evidence to suggest that the predominant neuronal nAChR subtype expressed in the vertebrate brain contains the α4 and β2 subunits (3Whiting P. Esch F. Shimasaki S. Lindstrom J. FEBS Lett. 1987; 219: 459-463Crossref PubMed Scopus (104) Google Scholar, 4Flores C.M. Rogers S.W. Pabreza L.A. Wolfe B.B. Kellar K.J. Mol. Pharmacol. 1992; 41: 31-37PubMed Google Scholar). When co-expressed in Xenopus oocytes, α4 and β2 co-assemble to form functional nAChRs (5Boulter J. Connolly J. Deneris E. Goldman D. Heinemann S. Patrick J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7763-7767Crossref PubMed Scopus (353) Google Scholar) with a subunit stoichiometry of (α4)2(β2)3 (6Anand R. Conroy W.G. Schoepfer R. Whiting P. Lindstrom J. J. Biol. Chem. 1991; 266: 11192-11198Abstract Full Text PDF PubMed Google Scholar, 7Cooper E. Couturier S. Ballivet M. Nature. 1991; 350: 235-238Crossref PubMed Scopus (400) Google Scholar). Several studies have demonstrated that relatively high levels of functional nAChRs are expressed on the cell surface of mammalian fibroblasts transfected with muscle (α2βγδ or α2βεδ) nAChR subunit cDNAs (8Sine S.M. Claudio T. J. Biol. Chem. 1991; 266: 13679-13689Abstract Full Text PDF PubMed Google Scholar, 9Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar). In contrast, it appears that some neuronal nAChR subunit combinations are expressed considerably less efficiently when expressed heterologously in mammalian cell lines. In particular, the neuronal nAChR α7 and α8 subunits, which readily form functional homo-oligomeric nAChRs when expressed in Xenopus oocytes, appear to fold and assemble very inefficiently in many mammalian cell types (10Cooper S.T. Millar N.S. J. Neurochem. 1997; 68: 2140-2151Crossref PubMed Scopus (130) Google Scholar, 11Cooper S.T. Millar N.S. J. Neurochem. 1998; 70: 2585-2593Crossref PubMed Scopus (36) Google Scholar, 12Blumenthal E.M. Conroy W.G. Romano S.J. Kassner P.D. Berg D.K. J. Neurosci. 1997; 15: 6094-6104Crossref Google Scholar, 13Kassner P.D. Berg D.K. J. Neurobiol. 1997; 33: 968-982Crossref PubMed Scopus (45) Google Scholar, 14Rangwala F. Drisdel R.C. Rakhilin S. Ko E. Atluri P. Harkins A.B. Fox A.P. Salman S.B. Green W.N. J. Neurosci. 1997; 17: 8201-8212Crossref PubMed Google Scholar, 15Chen D. Dang H. Patrick J.W. J. Neurochem. 1998; 70: 349-357Crossref PubMed Scopus (54) Google Scholar). In contrast, chimeric subunits containing the extracellular domain of the α7 or α8 subunits, together with the transmembrane and intracellular regions of the 5HT3 receptor subunit, produce very high levels of cell-surface expression in all cell types examined (11Cooper S.T. Millar N.S. J. Neurochem. 1998; 70: 2585-2593Crossref PubMed Scopus (36) Google Scholar, 12Blumenthal E.M. Conroy W.G. Romano S.J. Kassner P.D. Berg D.K. J. Neurosci. 1997; 15: 6094-6104Crossref Google Scholar,14Rangwala F. Drisdel R.C. Rakhilin S. Ko E. Atluri P. Harkins A.B. Fox A.P. Salman S.B. Green W.N. J. Neurosci. 1997; 17: 8201-8212Crossref PubMed Google Scholar, 16Eiselé J.-L. Bertrand S. Galzi J.-L. Devillers-Thiéry A. Changeux J.-P. Bertrand D. Nature. 1993; 366: 479-483Crossref PubMed Scopus (360) Google Scholar, 17Quiram P.A. Sine S.M. J. Biol. Chem. 1998; 273: 11001-11006Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Functional expression of recombinant α4β2 nAChRs in mammalian cell lines has been demonstrated previously (18Whiting P. Schoepfer R. Lindstrom J. Priestley T. Mol. Pharmacol. 1991; 40: 463-472PubMed Google Scholar, 19Buisson B. Gopalakrishnan M. Arneric S.P. Sullivan J.P. Bertrand D. J. Neurosci. 1996; 16: 7880-7891Crossref PubMed Google Scholar, 20Ragozzino D. Fucile S. Giovannelli A. Grassi F. Mileo A.M. Ballivet M. Alema S. Eusebi F. Eur. J. Neurosci. 1997; 9: 480-488Crossref PubMed Scopus (34) Google Scholar), but detailed characterization has been hindered somewhat by relatively low levels of cell-surface expression. Chronic exposure to nicotine has been shown to result in an increase in radioligand binding sites in cell lines expressing recombinant α4β2 nAChRs (21Peng X. Gerzanich V. Anand R. Whiting P.J. Lindstrom J. Mol. Pharmacol. 1994; 46: 523-530PubMed Google Scholar, 22Bencherif M. Fowler K. Lukas R. Lippiello P.M. J. Pharmacol. Exp. Ther. 1995; 275: 987-994PubMed Google Scholar, 23Zhang X. Gong Z.-H. Hellstrom-Lindahl E. Nordberg A. Neuroreport. 1995; 6: 313-317Crossref PubMed Scopus (35) Google Scholar), and correlates with an up-regulation (by ∼2-fold) of the number of cell-surface nAChRs (21Peng X. Gerzanich V. Anand R. Whiting P.J. Lindstrom J. Mol. Pharmacol. 1994; 46: 523-530PubMed Google Scholar). However, despite up-regulation of cell-surface nAChRs, chronic treatment with nicotine has been reported to result in persistent functional inactivation of both recombinant α4β2 and native nAChRs (21Peng X. Gerzanich V. Anand R. Whiting P.J. Lindstrom J. Mol. Pharmacol. 1994; 46: 523-530PubMed Google Scholar, 24Lukas R.J. J. Neurochem. 1991; 56: 1134-1145Crossref PubMed Scopus (74) Google Scholar, 25Marks M.J. Grady S.R. Collins A.C. J. Pharmacol. Exp. Ther. 1993; 266: 1268-1276PubMed Google Scholar). It has been suggested that this “persistent inactivation” may be a consequence of the receptor adopting a long-lasting desensitized state. A 2-fold up-regulation in the level of cell-surface α4β2 nAChR has also been reported as a consequence of treatments which elevate intracellular cAMP (26Rothhut B. Romano S.J. Vijayaraghavan S. Berg D.K. J. Neurobiol. 1996; 29: 115-125Crossref PubMed Scopus (30) Google Scholar). It has also been shown previously that subunit folding and assembly of some nAChRs, notably those of invertebrates and of cold water fish such as Torpedo, when expressed in mammalian cell lines, is more efficient at lower temperatures (27Paulson H.L. Claudio T. J. Cell Biol. 1990; 110: 1705-1717Crossref PubMed Scopus (32) Google Scholar, 28Lansdell S.J. Schmitt B. Betz H. Sattelle D.B. Millar N.S. J. Neurochem. 1997; 68: 1812-1819Crossref PubMed Scopus (72) Google Scholar). In this study we have examined factors that dramatically influence the efficiency of cell-surface expression of the rat neuronal α4β2 nAChR expressed heterologously in mammalian cell lines. We constructed two subunit chimeras, which contain the N-terminal domain of the α4 or β2 nAChR subunits and the C terminus of the 5HT3receptor subunit, similar to the α7/5HT3 chimera described previously (16Eiselé J.-L. Bertrand S. Galzi J.-L. Devillers-Thiéry A. Changeux J.-P. Bertrand D. Nature. 1993; 366: 479-483Crossref PubMed Scopus (360) Google Scholar). We have shown that substitution of chimeric subunits for wild-type subunits can increase levels of radioligand binding and cell-surface expression by up to ∼20-fold. In addition, we also demonstrate that lower temperature (30 °C) increases total radioligand binding (∼12-fold) and results in an up-regulation of cell-surface receptors (∼5-fold) in mammalian cells transfected with wild-type α4 and β2 nAChR subunits. Rat neuronal nAChR α4 and β2 subunit cDNAs (29Goldman D. Deneris E. Luyten W. Kochhar A. Patrick J. Heinemann S. Cell. 1987; 48: 965-973Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 30Deneris E.S. Connolly J. Boulter J. Wada E. Wada K. Swanson L.W. Patrick J. Heinemann S. Neuron. 1988; 1: 45-54Abstract Full Text PDF PubMed Scopus (236) Google Scholar) in the plasmid vector pcDNAI/Neo (Invitrogen) were provided by Dr. Jim Patrick (Baylor College of Medicine, Houston, TX). The mouse 5HT3 cDNA (31Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (882) Google Scholar) was provided by Dr. David Julius (University of California San Francisco). Monoclonal antibody (mAb) mAb 270, which recognizes an extracellular epitope on the nAChR β2 subunit (32Whiting P. Lindstrom J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 595-599Crossref PubMed Scopus (245) Google Scholar), was purified from the hybridoma cell line HB189 (obtained from the American Type Culture Collection, Rockville, MD). mAb 299, which recognizes an extracellular epitope on the nAChR α4 subunit (33Whiting P.J. Lindstrom J.M. J. Neurosci. 1988; 8: 3395-3404Crossref PubMed Google Scholar), was provided by Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia, PA). A polyclonal antiserum, raised against a fusion protein containing the intracellular loop region of the mouse 5HT3 receptor subunit (34Turton S. Gillard N.P. Stephenson F.A. McKernan R.M. Mol. Neuropharmacol. 1993; 3: 167-171Google Scholar), was provided by Dr. Ruth McKernan (Merck Sharp and Dohme Research Laboratories, Harlow, UK). TSA201 cells, a derivative of the human embryonic kidney HEK293 cell line, which expresses the simian virus 40 large T-antigen (35Heinzel S.S. Krysan P.J. Calos M.P. DuBridge R.B. J. Virol. 1988; 62: 3738-3746Crossref PubMed Google Scholar), were obtained from Dr. William Green (University of Chicago). Mouse fibroblast L929 cells were obtained from the European Collection of Cell Cultures (no. 85011425). Chimeric nicotinic/serotonergic subunit cDNAs were constructed in the expression vector pRK5, described previously (36Schall T.J. Lewis M. Koller K.J. Lee A. Rice G.C. Wong G.H.W. Gatanaga T. Granger G.A. Lentz R. Raab H. Kohr W.J. Goeddel D.V. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (844) Google Scholar), which contains a cytomegalovirus promoter and SV40 termination and polyadenylation signals. Polymerase chain reaction fragments were amplified from pcDNAI/Neo-α4 and pcDNAI/Neo-β2 by use of a 5′ primer to the pcDNAI/Neo T7 priming site and a 3′ primer, specific to either the α4 or β2 cDNAs, which introduced a silent Bcl I site at a position equivalent to the Bcl I site in pRK5-α7(V201)/5HT3, described previously (11Cooper S.T. Millar N.S. J. Neurochem. 1998; 70: 2585-2593Crossref PubMed Scopus (36) Google Scholar). Polymerase chain reaction fragments were digested with Eco RI and Bcl I and ligated into pRK5-α7/5HT3, which had been digested with Eco RI and Bcl I to remove the α7 cDNA fragment to create pRK5-α4/5HT3 and pRK5-β2/5HT3 (also referred to here as pRK5-α4χ and pRK5-β2χ, respectively). The α4 and β2 subunit cDNAs were subcloned from pcDNAI/Neo into pRK5 (to enable comparison of transient expression levels in TSA201 cells to those of chimeric subunits in identical expression vectors) and into pMSG (Amersham Pharmacia Biotech), to enable establishment of an inducible stable cell line in L929 cells (see below). All plasmid constructs were verified by restriction mapping and nucleotide sequencing. Cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 2 mml-glutamine, plus 10% heat-inactivated fetal calf serum (Sigma), penicillin (100 units/ml), streptomycin (100 μg/ml) and maintained in a humidified incubator containing 5% CO2 at either 37 °C or 30 °C. Human TSA201 cells were transfected either by a modified calcium phosphate co-precipitation method (37Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4816) Google Scholar) or by Effectene™ transfection reagent (Qiagen) according to the manufacturer's instructions. In all cases, cells were transfected overnight and assayed for expression approximately 42–44 h after transfection. Mouse L929 cells were stably transfected with pMSG-α4 and pMSG-β2 by calcium phosphate co-precipitation, and clonal cell lines were selected by serial dilution in the presence of mycophenolic acid and aminopterin, as has been described previously (38Lewis T.M. Harkness P.C. Sivilotti L.G. Colquhoun D. Millar N.S. J. Physiol. 1997; 505: 299-306Crossref PubMed Scopus (97) Google Scholar). Expression of α4 and β2 mRNA in clonal L929-α4β2 cells was induced by the addition of dexamethasone (1 μm final concentration) to the culture medium, typically for 5–10 days. Binding studies with [3H]epibatidine (NEN Life Science Products; specific activity 1.25 TBq/mmol) were performed on cell membrane preparations as has been described previously (28Lansdell S.J. Schmitt B. Betz H. Sattelle D.B. Millar N.S. J. Neurochem. 1997; 68: 1812-1819Crossref PubMed Scopus (72) Google Scholar). Curves for equilibrium binding were fitted by weighted least-squares (CVFIT program, David Colquhoun, University College London). Amounts of total cellular protein were determined by a Bio-Rad DC protein assay using BSA standards. Fluorescent ratiometric intracellular calcium measurements were performed on cell populations (typically 5 × 106 cells) loaded with 4 μm fura2-AM (Molecular Probes) using a Perkin-Elmer LS-50B fluorescence spectrometer fitted with a stirred cuvette holder and fast filter accessory. Details of cell loading and fluorimetry have been described previously (10Cooper S.T. Millar N.S. J. Neurochem. 1997; 68: 2140-2151Crossref PubMed Scopus (130) Google Scholar). The excitation wavelength was alternated rapidly between 340 and 380 nm and emitted fluorescence detected at 510 nm. A 340 nm/380 nm ratio was calculated every 40 ms and data averaged over four ratio data measurements. Maximum and minimum fluorescence levels were determined by addition of Triton X-100 (0.1% final concentration) and MnCl2 (10 mmfinal concentration), respectively. Agonist-evoked responses were normalized to the maximum fluorescence level to enable comparison between experiments. Cells were transiently transfected overnight in 6-cm tissue culture dishes, as described above. To starve cells of methionine, cells were washed twice with, and bathed for 10 min in l-methionine (Met)-free andl-cysteine (Cys)-free DMEM (Sigma) containing 10 mm HEPES and 0.37 mg/liter NaHCO3. Cells were labeled with 125 μCi of Redivue Pro-mix (Amersham Pharmacia Biotech; a mixture of [35S]Met and [35S]Cys) in 1.5 ml of Met/Cys-free DMEM for 30 min. Samples were washed three times and then chased with 8 ml of complete DMEM containing 30 mg ofl-Met, 48 mg of l-Cys, and 10% fetal calf serum. Metabolically labeled cells were rinsed with PBS and solubilized in ice-cold lysis buffer (LB) containing protease inhibitors (LB; 150 mm NaCl, 50 mmTris/Cl, pH 8.0, 5 mm EDTA, 1% Triton X-100, 0.25 mm phenylmethylsulfonyl fluoride, 100 μm N-ethylmaleimide, and 10 μg/ml each of leupeptin, aprotinin, and pepstatin). Samples were immunoprecipitated with mAb 270 or mAb 299 followed by Protein G-Sepharose (Calbiochem) as has been described (10Cooper S.T. Millar N.S. J. Neurochem. 1997; 68: 2140-2151Crossref PubMed Scopus (130) Google Scholar). Cells were solubilized in LB and 250 μl layered onto a 5-ml, linear 5–20% sucrose gradient prepared in lysis buffer. Gradients were centrifuged in a Beckman XL-80 Ultracentrifuge at 4 °C using a SW-55 Ti swing-out rotor at 40,000 rpm to ω2 t = 9.00 × 1011 rad2/s (approximately 14 h). Fifteen fractions of 320 μl were taken from the top of the gradient. Cells were transiently transfected on poly-l-lysine-coated glass coverslips. Coverslips were washed once in Hanks' balanced saline solution (HBSS; Life Technologies, Inc.), blocked for 5–10 min in HBSS containing 2% bovine serum albumin (BSA), and incubated with primary antibody in HBSS + BSA in a humidified chamber for 1–2 h at room temperature. Samples were washed four times in PBS, fixed for 10 min in PBS containing 3% paraformaldehyde, and washed three times. Coverslips were blocked in HBSS + BSA and then incubated with rhodamine-conjugated goat anti-rat IgG (Pierce) for 1 h, washed four times, and mounted in Fluorsave (Calbiochem). Levels of cell-surface immunofluorescent staining were examined with a Leica TCS SP laser-scanning confocal microscope with a 63 × 1.32 numerical aperture oil-immersion PlanApo objective using identical photomultiplier tube settings for all fluorescent images. Digital images (512 × 512 pixels) were captured using Leica TCS NT software. Cells grown on glass coverslips were transfected, incubated in primary antibody, and fixed as described for immunofluorescent microscopy (see above). Coverslips were then incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Amersham Pharmacia Biotech), washed six times and incubated with 500 μl of 3,3′,5,5′-tetramethylbenzidine (Sigma) for exactly 1 h. The supernatant was transferred to a cuvette and absorbance determined at 655 nm in a Beckman DU650 spectrophotometer. Cell-surface receptors were cross-linked with the thiol-cleavable reagent dithiobis-sulfosuccinimidylpropionate (DTSSP; Pierce). Transfected cells were washed twice with PBS and incubated in 2.5 mmDTSSP in PBS for 10 min at room temperature. After washing three times in PBS, cells were solubilized in LB and subjected to sucrose-gradient centrifugation, as described above. Individual gradient fractions were mixed with 2× sample buffer containing 100 mmdithiothreitol and the distribution of α4χ and β2χ protein determined by SDS-PAGE, followed by immunoblotting with anti-5HT3 serum (34Turton S. Gillard N.P. Stephenson F.A. McKernan R.M. Mol. Neuropharmacol. 1993; 3: 167-171Google Scholar), as described below. Samples from sucrose gradient fractions (50–70 μl) or samples of total cellular material (250 μg) were separated by 7.5% SDS-PAGE. Gels were equilibrated for 20 min in transfer buffer (25 mm Tris, 192 mm glycine, 20% methanol, pH 8.3) and then electroblotted onto Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech). Membranes were blocked by incubation with PBS containing 0.1% Tween 20 and 5% nonfat milk powder, then incubated with primary antibody in blocking solution for 1–2 h at room temperature. The nitrocellulose membrane was washed thoroughly, incubated with 1:5000 dilution of HRP-conjugated goat-α-rat IgG (Amersham Pharmacia Biotech) or goat-α-rabbit IgG (Pierce), and processed using the ECL detection system (Amersham Pharmacia Biotech). Functional recombinant α4β2 nAChRs, showing high affinity binding of nicotinic radioligands (Kd = 41 ± 22 pm for [3H]epibatidine), were detected in human embryonic kidney TSA201 cells after co-transfection with rat neuronal nAChR α4 and β2 subunit cDNAs (Fig.1 A). In contrast, neither functional nAChRs nor specific binding of nicotinic radioligands could be detected when either α4 or β2 subunits were expressed individually. Despite clear evidence for the co-assembly of α4 and β2 subunits into functional nAChRs, as shown by agonist-induced elevations in intracellular calcium (Fig. 1 B), we could detect only moderate levels of total radioligand binding (Bmax = 195 ± 67 fmol/mg, mean of five separate transfections). We have detected significantly higher Bmax values in TSA201 cell lines transfected with other ligand-gated ion channels, such as the mouse serotonin receptor 5HT3 subunit, where we can routinely detect 10–20-fold higher total radioligand ([3H]GR65630) binding, using the same expression vector (pRK5) under identical transfection conditions. The relatively low Bmaxvalue seen when α4 and β2 are co-expressed in TSA201 cells is similar to the levels of radioligand binding we have seen in a mouse fibroblast (L929) cell line stably co-transfected with α4 and β2 (Bmax = 148 ± 30 fmol/mg) and in several other mammalian cell lines transiently co-transfected with α4 and β2 (data not shown). It is also similar to the Bmax value (∼100 fmol/mg), which we determined previously in a stable L929 cell line co-transfected with nAChR α3 and β4 subunit cDNAs (38Lewis T.M. Harkness P.C. Sivilotti L.G. Colquhoun D. Millar N.S. J. Physiol. 1997; 505: 299-306Crossref PubMed Scopus (97) Google Scholar). A dramatic increase (∼12-fold) in the level of total [3H]epibatidine binding was observed when TSA201 cells, co-transfected with α4 and β2, were incubated at 30 °C (Fig.2 A) with no change in affinity for epibatidine (Kd = 38 ± 2 pm). Evidence that this corresponds to an increase in the level of assembled receptor was obtained by sucrose-gradient sedimentation (Fig.2 B). In addition, we have consistently observed ∼2-fold larger agonist-induced elevations in intracellular calcium levels in TSA201-α4β2 cells maintained at 30 °C compared with cells maintained at 37 °C (1.7 ± 0.4-fold increase, mean of seven independent determinations). We have also observed a similar increase in the magnitude of intracellular calcium responses in stably transfected L929-α4β2 cells maintained at 30 °C, rather than 37 °C (data not shown). Although this would appear to indicate a significant increase in the number of functional nAChRs in cells maintained at 30 °C, a more rigorous assay to determine the relative number of functional channels will require detailed whole-cell and single-channel electrophysiological characterization. We and others have shown previously that the profound host cell-dependent folding of α7 and α8 nAChR subunits is not observed with chimeric subunits containing the extracellular domain of the nicotinic α7 or α8 subunits together with the putative transmembrane and intracellular regions of the serotonin receptor 5HT3 subunit (11Cooper S.T. Millar N.S. J. Neurochem. 1998; 70: 2585-2593Crossref PubMed Scopus (36) Google Scholar, 16Eiselé J.-L. Bertrand S. Galzi J.-L. Devillers-Thiéry A. Changeux J.-P. Bertrand D. Nature. 1993; 366: 479-483Crossref PubMed Scopus (360) Google Scholar,17Quiram P.A. Sine S.M. J. Biol. Chem. 1998; 273: 11001-11006Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We have constructed two similar subunit chimeras (α4/5HT3 and β2/5HT3, which will be referred to subsequently as α4χ and β2χ, respectively). Chimeric (α4χ and β2χ) and wild-type (α4 and β2) subunit cDNAs, subcloned into the same mammalian expression vector (pRK5), were transiently transfected into TSA201 cells under identical conditions. Transfection of pairwise combinations of chimeric and wild-type subunits resulted in the formation of high levels of specific [3H]epibatidine binding sites. Co-expression of α4χ + β2, or α4χ + β2χ, resulted in an ∼20-fold up-regulation in specific [3H]epibatidine binding sites, compared with levels obtained following co-expression of wild-type α4 + β2 subunits (Fig. 2 C). Co-expression of α4 + β2χ resulted in a 5-fold up-regulation in specific [3H]epibatidine binding, compared with levels obtained with wild-type α4β2 (Fig.2 C). As we observed with wild-type α4 or β2 subunits, no high affinity [3H]epibatidine binding could be detected when either of the chimeric (α4χ or β2χ) subunits were expressed alone (Fig.2 C). We were, however, able to detect very low affinity binding of [3H]epibatidine to α4χ expressed alone (0.5 μm [3H]epibatidine produced only low levels of specific binding and failed to saturate). In contrast, saturation binding studies with hetero-oligomeric nAChRs containing chimeric subunits (α4χ+β2 or α4+β2χ) showed high affinity binding of [3H]epibatidine. Hetero-oligomeric complexes containing either α4χ or β2χ showed no significant difference in their affinity for [3H]epibatidine (Kd = 51 ± 31 pm for α4χ+β2) compared with that determined for the α4β2 nAChR (Kd = 41 ± 22 nm). These data demonstrate that formation of a high affinity nicotinic binding site requires the co-assembly of both the α4 and β2 subunit extracellular domains, and that the nicotinic radioligand binding site is preserved in the chimeric subunits. As described earlier, a 12-fold increase in [3H]epibatidine binding was observed when cells transfected with wild-type α4β2 were maintained at a lower temperature. We examined the influence of temperature upon levels of [3H]epibatidine binding to cells expressing receptors containing chimeric subunits. A 3-fold increase in total [3H]epibatidine binding was observed with cells transfected with α4+β2χ (Fig. 2 D). A less pronounced effect was detected with cells expressing α4χ+β2 or α4χ+β2χ (Fig. 2 D). This indicates that nAChR combinations containing the α4χ, which express very efficiently at 37 °C, are not influenced as greatly by lower temperature. In contrast, lower temperature has a more pronounced effect on nAChR combinations containing the wild-type α4 subunit (α4+β2 and α4+β2χ), where levels of radioligand binding are lower at 37 °C. Despite evidence for the expression of functional nAChRs in mammalian cells transfected with α4 and β2, we have consistently detected only very low levels of cell-surface receptors by confocal immunofluorescent microscopy. The low level of cell-surface staining (with mAb 270) in transiently transfected TSA201 cells at 37 °C is illustrated in Fig.3 A. We have routinely observed similarly low levels of cell-surface nAChRs in other mammalian cell types transfected with α4 and β2, including stably transfected mouse fibroblast L929 cells (data not shown), confirming that low levels of cell-surface expression is not a phenomenon exclusive to transfected TSA201 cells. We examined the influence of lower temperature and of chimeric subunits upon the level of cell-surface nAChR expression in TSA201 cells. Cells transfected with various subunit combinations were incubated with mAb 270, which recognizes an extracellular epitope on the β2 subunit, followed by a rhodamine-conjugated second antibody. Very bright cell-surface staining of the β2 subunit was observed in cells expressing α4χ+β2 (Fig.3 C), which was considerably brighter than when β2 was co-expressed with the wild-type α4 subunit (Fig. 3 A). A clear elevation in levels of cell-surface β2 expression was also observed when cells transfected with α4β2 were incubated at 30 °C (Fig. 3 B), rather than 37 °C. No cell-surface staining could be detected when either wild-type α4 or β2 subunits were expressed alone (with mAbs 299 and 270, respectively). However, when either α4χ or β2χ were expressed alone, very bright cell-surface immunofluorescent staining was detected (data not shown). In order to obtain a more quantitative estimate of the relative levels of cell-surface nAChRs, we employed an enzyme-linked antibody assay. Transfected cells were incubat" @default.
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