J Biol Chem, Vol. 274, Issue 38, 27145-27152, September 17, 1999

Up-regulation of Cell-surface 42 Neuronal Nicotinic Receptors by Lower Temperature and Expression of Chimeric Subunits^*

Sandra T. Cooper, Patricia C. Harkness, Elizabeth R. Baker, and Neil S. Millar

From the Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing 4 and 2 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 42 nAChR. Functional recombinant 42 nAChRs, showing high affinity binding of nicotinic radioligands (K_d = 41 ± 22 pM for [³H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of 42 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 °C. At 30 °C, however, cells expressing 42 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" 4 and 2 subunits, chimeric nicotinic/serotonergic subunits ("4" and "2") are expressed very efficiently on the cell surface (at 30 °C or 37 °C), either as hetero-oligomeric complexes (e.g. 4+2 or 4+2) or when expressed alone. Compared with 42 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1, 2). Whereas the muscle-type nAChR is a pentameric complex of known subunit composition (₂ in fetal muscle and ₂ in adult), the precise subunit composition of the various neuronal nAChR subtypes is less certain. To date, 11 neuronal nAChR subunits (2-9 and 2-4) have been identified and cloned. There is evidence to suggest that the predominant neuronal nAChR subtype expressed in the vertebrate brain contains the 4 and 2 subunits (3, 4). When co-expressed in Xenopus oocytes, 4 and 2 co-assemble to form functional nAChRs (5) with a subunit stoichiometry of (4)₂(2)₃ (6, 7).

Several studies have demonstrated that relatively high levels of functional nAChRs are expressed on the cell surface of mammalian fibroblasts transfected with muscle (₂ or ₂) nAChR subunit cDNAs (8, 9). 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 7 and 8 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 (10-15). In contrast, chimeric subunits containing the extracellular domain of the 7 or 8 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 (11, 12, 14, 16, 17).

Functional expression of recombinant 42 nAChRs in mammalian cell lines has been demonstrated previously (18-20), 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 42 nAChRs (21-23), and correlates with an up-regulation (by ~2-fold) of the number of cell-surface nAChRs (21). However, despite up-regulation of cell-surface nAChRs, chronic treatment with nicotine has been reported to result in persistent functional inactivation of both recombinant 42 and native nAChRs (21, 24, 25). 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 42 nAChR has also been reported as a consequence of treatments which elevate intracellular cAMP (26). 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 (27, 28).

In this study we have examined factors that dramatically influence the efficiency of cell-surface expression of the rat neuronal 42 nAChR expressed heterologously in mammalian cell lines. We constructed two subunit chimeras, which contain the N-terminal domain of the 4 or 2 nAChR subunits and the C terminus of the 5HT₃ receptor subunit, similar to the 7/5HT₃ chimera described previously (16). 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 4 and 2 nAChR subunits.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Rat neuronal nAChR 4 and 2 subunit cDNAs (29, 30) in the plasmid vector pcDNAI/Neo (Invitrogen) were provided by Dr. Jim Patrick (Baylor College of Medicine, Houston, TX). The mouse 5HT₃ cDNA (31) was provided by Dr. David Julius (University of California San Francisco). Monoclonal antibody (mAb) mAb 270, which recognizes an extracellular epitope on the nAChR 2 subunit (32), 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 4 subunit (33), 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 (34), 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 (35), were obtained from Dr. William Green (University of Chicago). Mouse fibroblast L929 cells were obtained from the European Collection of Cell Cultures (no. 85011425).

Construction and Subcloning of Chimeric nAChR/5HT₃R Subunit cDNAs-- Chimeric nicotinic/serotonergic subunit cDNAs were constructed in the expression vector pRK5, described previously (36), which contains a cytomegalovirus promoter and SV40 termination and polyadenylation signals. Polymerase chain reaction fragments were amplified from pcDNAI/Neo-4 and pcDNAI/Neo-2 by use of a 5' primer to the pcDNAI/Neo T7 priming site and a 3' primer, specific to either the 4 or 2 cDNAs, which introduced a silent BclI site at a position equivalent to the BclI site in pRK5-7^(V201)/5HT₃, described previously (11). Polymerase chain reaction fragments were digested with EcoRI and BclI and ligated into pRK5-7/5HT₃, which had been digested with EcoRI and BclI to remove the 7 cDNA fragment to create pRK5-4/5HT₃ and pRK5-2/5HT₃ (also referred to here as pRK5-4 and pRK5-2, respectively). The 4 and 2 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.

Cell Culture and Transfection-- Cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 2 mM L-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 (37) or by Effectene^TM 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-4 and pMSG-2 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 (38). Expression of 4 and 2 mRNA in clonal L929-42 cells was induced by the addition of dexamethasone (1 µM final concentration) to the culture medium, typically for 5-10 days.

Radioligand Binding-- 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 (28). 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.

Intracellular Calcium Measurement-- 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 (10). 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 mM final concentration), respectively. Agonist-evoked responses were normalized to the maximum fluorescence level to enable comparison between experiments.

Pulse-Chase Metabolic Labeling-- 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 and L-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 of L-Met, 48 mg of L-Cys, and 10% fetal calf serum.

Immunoprecipitation-- Metabolically labeled cells were rinsed with PBS and solubilized in ice-cold lysis buffer (LB) containing protease inhibitors (LB; 150 mM NaCl, 50 mM Tris/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 (10).

Sucrose Gradient Centrifugation-- 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 ²t = 9.00 × 10¹¹ rad²/s (approximately 14 h). Fifteen fractions of 320 µl were taken from the top of the gradient.

Confocal Immunofluorescence Microscopy-- 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.

Enzyme-linked Assay of Cell-surface Expression Levels-- 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 Cross-linking-- 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 mM DTSSP 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 mM dithiothreitol and the distribution of 4 and 2 protein determined by SDS-PAGE, followed by immunoblotting with anti-5HT₃ serum (34), as described below.

Immunoblotting-- 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--rat IgG (Amersham Pharmacia Biotech) or goat--rabbit IgG (Pierce), and processed using the ECL detection system (Amersham Pharmacia Biotech).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Functional Rat 42 Neuronal nAChRs in Transfected Cell Lines-- Functional recombinant 42 nAChRs, showing high affinity binding of nicotinic radioligands (K_d = 41 ± 22 pM for [³H]epibatidine), were detected in human embryonic kidney TSA201 cells after co-transfection with rat neuronal nAChR 4 and 2 subunit cDNAs (Fig. 1A). In contrast, neither functional nAChRs nor specific binding of nicotinic radioligands could be detected when either 4 or 2 subunits were expressed individually. Despite clear evidence for the co-assembly of 4 and 2 subunits into functional nAChRs, as shown by agonist-induced elevations in intracellular calcium (Fig. 1B), we could detect only moderate levels of total radioligand binding (B_max = 195 ± 67 fmol/mg, mean of five separate transfections). We have detected significantly higher B_max 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 B_max value seen when 4 and 2 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 4 and 2 (B_max = 148 ± 30 fmol/mg) and in several other mammalian cell lines transiently co-transfected with 4 and 2 (data not shown). It is also similar to the B_max value (~100 fmol/mg), which we determined previously in a stable L929 cell line co-transfected with nAChR 3 and 4 subunit cDNAs (38).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Expression of functional recombinant 42 nAChRs showing high affinity binding of [3H]epibatidine. A, saturation binding of [3H]epibatidine to cell membrane preparations from TSA201 cells transiently transfected with rat neuronal nAChR 4 and 2 subunit cDNAs. Nonspecific binding, determined in the presence of 2 mM nicotine, has been subtracted. Data points represent the mean of triplicate samples and are typical of four separate determinations resulting in an estimated Kd = 41 ± 22 nM and Bmax = 195 ± 67 fmol/mg. B, functional expression of 42 nAChRs shown by quantitative ratiometric measurement of intracellular calcium levels in cells loaded with the calcium-sensitive dye fura2-AM. Elevation of intracellular calcium, shown as an increase in the measured 340 nm/380 nm ratio, in TSA201 cells transiently transfected with 4 and 2 subunit cDNAs in response to the application of 2 µM epibatidine. No response was detected in untransfected cells. C, maximum and minimum fluorescence values were determined after agonist application by the subsequent addition of Triton X-100 (0.1% final concentration) and MnCl2 (10 mM final concentration). Agonist-evoked responses were normalized to the maximum fluorescence level to enable comparison between experiments.

Lower Temperature Increases 42 nAChR Expression-- A dramatic increase (~12-fold) in the level of total [³H]epibatidine binding was observed when TSA201 cells, co-transfected with 4 and 2, were incubated at 30 °C (Fig. 2A) with no change in affinity for epibatidine (K_d = 38 ± 2 pM). Evidence that this corresponds to an increase in the level of assembled receptor was obtained by sucrose-gradient sedimentation (Fig. 2B). In addition, we have consistently observed ~2-fold larger agonist-induced elevations in intracellular calcium levels in TSA201-42 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 L929-42 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.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   [3H]Epibatidine binding to TSA201 cells transiently transfected with wild-type and chimeric nAChR subunits. Total specific binding to transfected TSA201 cells was determined with a saturating concentration of [3H]epibatidine (3 nM). A, an increase (~12-fold) in specific binding was observed in cells transfected with 42 when maintained at 30 °C. B, sucrose gradient profiles of 42 nAChRs. Cells maintained at 37 °C and 30 °C were solubilized in lysis buffer containing 1% Triton and 2 mg of total protein sedimented on a linear 5-20% sucrose gradient. Fractions were removed from the top of the gradient and assayed for [3H]epibatidine binding. The positions of the native electric organ pentameric monomer (9 S) and disulfide-linked dimer (13 S) are shown. The inset shows the data from cells maintained at 37 °C, re-plotted with an expanded y axis. C, a dramatic increase in total specific binding was seen in cells transfected with pairwise subunit combinations containing either 4 or 2. In contrast, no significant radioligand binding was detected (with 3 nM [3H]epibatidine) in cells transfected with either 4 or 2 alone. D, a further increase in total specific radioligand binding is observed in cells transfected with subunit combinations containing either 4 or 2 when maintained at 30 °C (data presented in terms of -fold increase in binding at 30 °C, compared with that at 37 °C). In A, C, and D, nonspecific binding (determined by the addition of 2 mM nicotine to triplicate samples) has been subtracted and data points represent the mean of four separate transfections, each performed in triplicate.

Influence of Chimeric Subunits-- We and others have shown previously that the profound host cell-dependent folding of 7 and 8 nAChR subunits is not observed with chimeric subunits containing the extracellular domain of the nicotinic 7 or 8 subunits together with the putative transmembrane and intracellular regions of the serotonin receptor 5HT₃ subunit (11, 16, 17). We have constructed two similar subunit chimeras (4/5HT₃ and 2/5HT₃, which will be referred to subsequently as 4 and 2, respectively). Chimeric (4 and 2) and wild-type (4 and 2) 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 4 + 2, or 4 + 2, resulted in an ~20-fold up-regulation in specific [³H]epibatidine binding sites, compared with levels obtained following co-expression of wild-type 4 + 2 subunits (Fig. 2C). Co-expression of 4 + 2 resulted in a 5-fold up-regulation in specific [³H]epibatidine binding, compared with levels obtained with wild-type 42 (Fig. 2C).

As we observed with wild-type 4 or 2 subunits, no high affinity [³H]epibatidine binding could be detected when either of the chimeric (4 or 2) subunits were expressed alone (Fig. 2C). We were, however, able to detect very low affinity binding of [³H]epibatidine to 4 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 (4+2 or 4+2) showed high affinity binding of [³H]epibatidine. Hetero-oligomeric complexes containing either 4 or 2 showed no significant difference in their affinity for [³H]epibatidine (K_d = 51 ± 31 pM for 4+2) compared with that determined for the 42 nAChR (K_d = 41 ± 22 nM). These data demonstrate that formation of a high affinity nicotinic binding site requires the co-assembly of both the 4 and 2 subunit extracellular domains, and that the nicotinic radioligand binding site is preserved in the chimeric subunits.

The Influence of Temperature on nAChRs Containing Chimeric (4 or 2) Subunits-- As described earlier, a 12-fold increase in [³H]epibatidine binding was observed when cells transfected with wild-type 42 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 4+2 (Fig. 2D). A less pronounced effect was detected with cells expressing 4+2 or 4+2 (Fig. 2D). This indicates that nAChR combinations containing the 4, 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 4 subunit (4+2 and 4+2), where levels of radioligand binding are lower at 37 °C.

Influence of Chimeric Subunits and Temperature on Cell-surface Expression-- Despite evidence for the expression of functional nAChRs in mammalian cells transfected with 4 and 2, 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. 3A. We have routinely observed similarly low levels of cell-surface nAChRs in other mammalian cell types transfected with 4 and 2, 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 2 subunit, followed by a rhodamine-conjugated second antibody. Very bright cell-surface staining of the 2 subunit was observed in cells expressing 4+2 (Fig. 3C), which was considerably brighter than when 2 was co-expressed with the wild-type 4 subunit (Fig. 3A). A clear elevation in levels of cell-surface 2 expression was also observed when cells transfected with 42 were incubated at 30 °C (Fig. 3B), rather than 37 °C. No cell-surface staining could be detected when either wild-type 4 or 2 subunits were expressed alone (with mAbs 299 and 270, respectively). However, when either 4 or 2 were expressed alone, very bright cell-surface immunofluorescent staining was detected (data not shown).

View larger version (128K): [in this window] [in a new window]   Fig. 3.   Cell-surface nAChR expression determined by confocal immunofluorescent microscopy. Levels of cell-surface 2 on transfected TSA201 cells were determined by labeling intact cells with mAb 270 (which recognizes an epitope on the extracellular domain of 2). After fixation and staining with rhodamine-conjugated second antibody, levels of cell-surface immunofluorescent staining were examined with a Leica TCS SP laser-scanning confocal microscope using identical photomultiplier tube settings for all fluorescent images. Cells were transfected with either 4+2 (A and B) or 4+2 (C) and incubated at either 37 °C (A and C) or 30 °C (B). Transmission images of the same fields are shown in lower panels. No cell-surface staining was detectable in cells transfected with 2 alone (data not shown). Scale bar, 10 µm.

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 incubated with subunit-specific monoclonal antibodies (mAb 270 or 299, both directed against extracellular epitopes), followed by a HRP-conjugated second antibody. Compared with 42 nAChR (expressed at 37 °C), we observed a 4-5-fold up-regulation in cell-surface expression of 42 (with either mAb 270 or mAb 299) when cells were maintained at 30 °C (Fig. 4). We were unable to detect significant levels of cell-surface 4 or 2 when either subunit was transfected individually but, as seen by immunofluorescent microscopy, 4 and 2 chimeric subunits (transfected individually) were expressed at very high levels on the cell surface (15-20-fold higher than seen with 42 expressed at 37 °C; see Fig. 4). Therefore, in order to determine cell-surface levels of hetero-oligomeric combinations containing both chimeric and non-chimeric subunits, intact cells were assayed with an antibody specific for the non-chimeric subunit. When 2 was co-transfected with 4 (4+2 at 37 °C), it was expressed on the cell surface at levels 15-30-fold higher than when co-transfected with 4 (4+2 at 37 °C; see Fig. 4). In contrast, we can detect little difference in the level of 4 expressed on the cell surface, whether co-transfected with 2 or 2 (Fig. 4). It appears, therefore, that 4 has a more pronounced effect, both on cell-surface expression and on radioligand binding levels, than does 2.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Cell-surface nAChR expression determined by enzyme-linked antibody assay. Transfected TSA201 cells were labeled with either mAb 270 (A) or mAb 299 (B), fixed, and then incubated with an HRP-conjugated second antibody and substrate (3,3',5,5'-tetramethylbenzidine). After 1 h, the intensity of the colorimetric product was determined by absorbance at 655 nm. Data points represent the mean of three separate experiments, each performed in triplicate with three individually transfected coverslips. Background staining in each case was determined by three mock-transfected coverslips (for both 37 °C or 30 °C), and has been subtracted. To enable comparison of results from cells cultured at 37 °C or 30 °C, data have been normalized to levels of total cellular protein determined by protein assay and are presented as -fold increase in absorbance intensity, relative to that seen with 42 at 37 °C.

Assembly of Chimeric Subunits into Pentameric Complexes-- nAChRs generated by the co-expression of wild-type and chimeric subunits were examined by sucrose gradient centrifugation. The distribution of co-assembled subunits after sedimentation on a 5-20% sucrose gradient was examined by [³H]epibatidine binding to individual gradient fractions. All subunit combinations examined (4+2, 4+2, 4+2, and 4+2) showed a clear peak of radioligand binding at a position similar to that of the pentameric muscle and Torpedo electric organ nAChR (Fig. 5). Combinations containing wild-type 4 migrated at a higher apparent buoyant density than those containing the 4, consistent with the considerably larger predicted and apparent molecular weight of the 4 subunit.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Sucrose gradient profiles receptors containing wild-type and chimeric subunits. Transfected TSA201 cells were solubilized in lysis buffer containing 1% Triton and sedimented on a linear 5-20% sucrose gradient. Fractions were removed from the top of the gradient and assayed for [3H]epibatidine binding. The data shown are from single experiments, which have been repeated twice with identical results. The positions of the native electric organ pentameric monomer (9 S) and disulfide-linked dimer (13 S) are shown.

Because of the inability of chimeric subunits to bind nicotinic radioligands with high affinity when expressed alone, we could not examine their distribution on a sucrose gradient by [³H]epibatidine binding. We were interested to examine whether the efficient cell-surface expression of 4 and 2 (when expressed individually) indicated that they were assembled into homo-oligomeric pentameric complexes. The distribution of 4 and 2 after sucrose-gradient centrifugation was determined by immunoblotting of individual gradient fractions. Surprisingly, the distribution of 4 and 2 resembled that of unassembled subunits (Fig. 6). However, after covalent cross-linking of cell-surface receptors (with DTSSP), 4 and 2 migrated at a position typical of fully assembled pentamers (Fig. 6). It appears, therefore, that 4 and 2 do assemble into pentameric complexes when expressed individually, but that the subunit-subunit interactions within such homo-oligomeric complexes are not resistant to sucrose-gradient centrifugation after solubilization in 1% Triton.

View larger version (87K): [in this window] [in a new window]   Fig. 6.   Sucrose gradient profiles of 4 and 2 subunits expressed individually. Transfected TSA201 cells, either untreated or after cross-linking of cell-surface receptors with DTSSP, were solubilized in lysis buffer containing 1% Triton X-100 and sedimented on a linear 5-20% sucrose gradient. Samples from individual gradient fractions were reduced in dithiothreitol and separated by SDS-PAGE. The distribution of chimeric 4 and 2 subunit protein was determined by immunoblotting with an antiserum that recognizes the intracellular loop region of the 5HT3 receptor subunit (34). Sucrose-gradient fractions were taken from the top of the gradient and correspond to increasing buoyant density. The positions of the native electric organ pentameric monomer (9 S) and disulfide-linked dimer (13 S) are shown.

Steady-state Levels and Turnover of Wild-type and Chimeric Subunits-- We examined the influence of chimeric subunits and of temperature upon subunit half-lives by pulse-chase metabolic labeling and immunoprecipitation with subunit-specific mAbs (Fig. 7 and Table I). Despite the dramatic increase seen in radioligand binding and in levels of cell-surface expression, immunoprecipitation with mAb 299 shows that 4 and 4 subunits have similar half-lives at 37 °C (~2.4 and ~2.6 h, respectively) when co-expressed with 2. Immunoprecipitation with mAb 270 showed that 2 had a similar half-life when expressed alone (~2.1 h), or when co-expressed with 4 (~2.7 h). However, the half-life of 2 was increased about 2-fold when co-expressed with 4. The half-life of all subunits appeared to be increased (by ~1.5-2-fold) when cells were maintained at 30 °C (see Table I), perhaps due to a decreased rate of degradation at the lower temperature.

View larger version (62K): [in this window] [in a new window]   Fig. 7.   Pulse-chase metabolic labeling. TSA201 cells, transfected with various subunit combinations, were metabolically labeled for 30 min using a mixture of [35S]methionine and [35S]cysteine and chased for varying times (as indicated). Cell lysates (solubilized under conditions that maintained subunit-subunit interactions) were immunoprecipitated with either mAb 299, which recognizes an epitope on the extracellular domain of 4 and 4 (A), or mAb 270, which recognizes an epitope on the extracellular domain of 2 (B). Immunoprecipitated samples were analyzed by SDS-PAGE and autoradiography.

View this table: [in this window] [in a new window]   Table I Half-lives of wild-type (4 and 2) and chimeric (4 and 2) subunits TSA201 cells, transfected with wild-type and chimeric subunit cDNAs, were pulse-chase-labeled and immunoprecipitated with subunit-specific monoclonal antibodies (see Fig. 7). The intensity of immunoprecipitated subunit bands at various chase times was determined by densitometry. Intensities were plotted against chase time and half-lives determined by fitting an exponential function. Data are means of two independent determinations, which did not differ by more than 20%.

We examined steady-state levels of subunit proteins by Western blotting of total cell lysates. Immunoblotting with mAb 299 (which recognizes 4 and 4) showed broadly similar levels of 4 or 4 protein for each combination of transfected subunits (Fig. 8A), consistent with our pulse-chase data. Despite the increased half-lives of all subunits at 30 °C, we detected broadly similar steady-state levels of 4 and 4 protein whether cells were maintained at either 37 °C or 30 °C (Fig. 8B). We assume that this reflects a reduced rate of protein synthesis at 30 °C. Although mAb 270 (which was raised against native chick nAChR) has been used previously to detect SDS-denatured 2 subunit purified from rat brain by immunoblotting (32), we have been unable to detect recombinant 2 protein by Western blotting with mAb 270. We have, however, seen broadly similar steady-state levels of 2 (when expressed with either 4 or 4) at both 37 °C and 30 °C by immunoblotting with a polyclonal antibody (sc-1449; Santa Cruz Biotechnology Inc.), which was raised against a C-terminal epitope of 2. Immunofluorescent staining of fixed and permeabilized cells with mAb 270 reveals much brighter fluorescence when 2 is co-expressed with 4 than with 4, suggesting that the total steady-state level of 2 is considerably higher when co-expressed with 4. However, as Western blotting with the anti-2 polyclonal serum indicates that the steady-state level of 2 is similar, whether co-expressed with 4 or 4, it seems probable that the brighter internal immunofluorescent staining with mAb 270 is a consequence of 2 adopting a conformation, when co-expressed with 4, which is recognized more readily by mAb 270 after cell fixation and permeabilization.

View larger version (36K): [in this window] [in a new window]   Fig. 8.   Steady-state levels of subunit proteins determined by Western blotting. Total cellular protein (250 µg) from TSA201 cells transfected with various subunit combinations were separated by SDS-PAGE and immunoblotted with mAb 299 (which recognizes an epitope on the extracellular domain of 4 and 4). The positions of molecular size markers are indicated. Specific immunoreactive bands were detected corresponding to the wild-type 4 subunit (~70 kDa) and the 4 subunit (~54 kDa). Broadly similar levels of 4 and 4 subunit protein were detected when expressed individually or with either 2 or 2 (A). Despite the dramatic up-regulation in levels of cell-surface expression and radioligand binding seen at 30 °C, no increase in total steady-state protein levels was observed at lower temperatures (B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Functional nAChRs with high affinity for [³H]epibatidine were detected when rat neuronal nAChR 4 and 2 subunits were co-transfected into either human embryonic kidney TSA201 cells or mouse fibroblast L929 cells. In both systems, despite the expression of functional nAChRs, we have detected only moderate levels of total radioligand binding and low levels of cell-surface receptor at 37 °C. A 12-fold increase in total nicotinic radioligand binding and a 5-fold up-regulation of cell-surface receptors was observed when mammalian cells transfected with rat 4 and 2 were maintained at a lower temperature (30 °C, rather than 37 °C). We have also shown that lower temperature causes in an increase in the magnitude of agonist-induced elevations in intracellular calcium (by ~2-fold). Evidence that these effects are a consequence of a posttranslational phenomenon is provided by the observation that there is a broadly similar total steady-state level of nAChR subunit proteins in cells maintained at either 37 °C or 30 °C. One might expect lower temperature to result in a reduced rate of transcription and protein synthesis, but this appears to be offset by a reduced subunit turnover (increased half-life) at 30 °C, presumably due to reduced protein degradation at the lower temperature.

We and others have reported inefficient folding and assembly of homo-oligomeric (7 and 8) nAChRs in several mammalian cell lines (10-15). In some transfected mammalian cell lines, no specific binding of nicotinic radioligands can be detected, despite the efficient expression of the 7 subunit (10). It appears, therefore, that in some (but not all) mammalian cell types, homo-oligomeric neuronal nAChR subunits fail to fold into a conformation recognized by nicotinic radioligands. In contrast to the wild-type 7 and 8 subunits, chimeric subunits containing the extracellular domain of either 7 or 8, together with the transmembrane and intracellular region of the 5HT₃R subunit, are expressed on the cell surface very efficiently in all cell types examined (11, 12, 14, 16).

We have been able to detect specific radioligand binding after co-transfection of hetero-oligomeric subunit combinations (e.g. 42 and 34) into all cell types examined (including those which are "non-permissive" for 7), as shown here and in previous studies (10, 28, 38). However, we have shown that co-expression of the 2 subunit with 4 (rather than wild-type 4) results in a 20-fold increase in total nicotinic radioligand binding and a similar -fold up-regulation in the amount of cell-surface 2 subunit. Despite the very dramatic increase in binding and surface expression, the 4 and 4 subunits appear to have similar half-lives and similar total steady-state protein levels. The increased binding and surface expression is not, therefore, simply a consequence of increased levels of total protein. We have shown that the increased level of radioligand binding corresponds to an increased level of assembled receptor. As unassembled 4 and 2 subunits are not transported to the cell surface (Fig. 4), it is possible that the up-regulation in cell-surface receptor is largely a consequence of this increase in assembly. Several studies have demonstrated that assembled nAChRs have a longer half-life than unassembled subunits (13, 39, 40), and in this study we have observed a significantly slower turnover of total subunit protein at lower temperature.

Whereas the extracellular domains of the 7/5HT₃ and 8/5HT₃ chimeras are derived from subunits that are capable of generating homo-oligomeric complexes, 4/5HT₃ and 2/5HT₃ (4 and 2) contain an extracellular domain derived from subunits that normally assemble only into hetero-oligomeric complexes. It was of interest, therefore, that both 4 and 2, when transfected alone, are expressed at high levels on the cell surface (although neither chimeric subunit forms a high affinity radioligand binding site). We have shown that both 4 (alone) and 2 (alone) assemble into a complex of a size expected of a pentamer but that the subunit-subunit interaction involved is not resistant to detergent (1% Triton) solubilization and sucrose-gradient centrifugation. This is in contrast to the 7/5HT₃ and 8/5HT₃ chimeras, which oligomerize into complexes in which the subunit-subunit interactions are resistant to solubilization in 1% Triton (10, 11). Similarly, when either 4 or 2 is present in a hetero-oligomeric complex (together with either chimeric or wild-type subunits), they generate subunit-subunit interactions that are resistant to solubilization in 1% Triton.

It is hoped that further studies with chimeras such as those described here will help to identify subunit domains that are important in determining both the specificity and stability of subunit-subunit interactions. Previous studies with the muscle-type nAChR have demonstrated that, whereas the fully assembled (₂) pentameric complex is resistant to solubilization in 1% Triton, some putative assembly intermediates that contain less than the full complement of subunits (e.g. ) form complexes with weaker subunit-subunit interactions, which are disrupted by similar detergent solubilization (9).

The work reported here with chimeric subunits containing the extracellular domain of either 4 or 2, together with previous observations with 7/5HT₃ and 8/5HT₃ chimeras (11, 12, 14, 16, 17), would suggest that inefficient cell-surface expression of neuronal nAChRs is influenced primarily by their intracellular and/or transmembrane regions. It is still not clear why chimeric subunits (e.g. 4/5HT₃, 7/5HT₃ etc.) should generate cell-surface receptors very much more efficiently than wild-type nAChRs subunits. It is possible, however, that this may reflect differences in the ability of these subunits to interact with cytoplasmic and ER-resident proteins. There is increasing evidence that the efficiency of assembly of nAChR subunits may be influenced by interactions with chaperone proteins (41-45). It may, however, be possible to identify more precisely those subunit domains that help to influence these processes by the construction and expression of alternative subunit chimeras. Such an approach is now under way in this and other laboratories (46).²

An intriguing aspect of native neuronal nAChRs, as has been shown in chick ciliary ganglia, is that only a small proportion of the total cell-surface receptors appear to be functional (47). Chronic nicotine treatment has been shown to increase the number of radioligand binding sites in cell lines expressing 42 (21-23) and correlates with an up-regulation (by ~2-fold) of the number of cell-surface nAChRs (21). However, despite up-regulation of cell-surface nAChRs, chronic treatment with nicotine has been reported to result in a persistent functional inactivation of both recombinant 42 and native nAChRs (21, 24, 25). Here we have demonstrated that lower temperature leads to a 5-fold up-regulation of cell-surface 42 nAChRs expressed in transfected mammalian cell lines. By measuring agonist-induced elevations in intracellular calcium, we have obtained evidence to suggest that lower temperature may also lead to an increase in the number of functional nAChRs.

	ACKNOWLEDGEMENTS

We thank Drs William Green, David Julius, Jon Lindstrom, Ruth McKernan, and Jim Patrick for generously providing some of the antibodies, cDNAs and cell lines used in this work. We also thank Darran Clements for assistance with confocal microscopy.

	FOOTNOTES

^* This work was supported by grants from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-207-380-7241; Fax: 44-207-380-7245; E-mail: n.millar@ucl.ac.uk.

² S. T. Cooper, P. C. Harkness, E. R. Baker, and N. S. Millar, unpublished data.

	ABBREVIATIONS

The abbreviations used are: nAChR, nicotinic acetylcholine receptor; DTSSP, dithiobis-sulfosuccinimidylpropionate; HRP, horseradish peroxidase; 5HT, 5-hydroxytyptamine; mAb, monoclonal antibody; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HBSS, Hanks' buffered saline solution; LB, lysis buffer.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURE