QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.079004v1 313/1/24    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peng, J.-H. Articles by Lukas, R. J. Search for Related Content PubMed PubMed Citation Articles by Peng, J.-H. Articles by Lukas, R. J.

NEUROPHARMACOLOGY

High-Affinity Epibatidine Binding of Functional, Human 7-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed de Novo in Human SH-EP1 Cells

Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (J.-H.P., J.D.F., K.M.S., A.A.G., S.M., R.J.L.); Pharmacia Corporation, Kalamazoo, Michigan (R.S.H., V.E.G.); and Department of Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado (S.S.L.)

Received October 7, 2004; accepted December 6, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Human nicotinic acetylcholine receptor (nAChR) 7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows 7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies ¹²⁵I-labeled -bungarotoxin (I-Bgt) binding sites corresponding to human 7 (h7)-nAChRs on the surface of most cells. I-Bgt binds to h7-nAChRs in membrane fractions with a typical apparent K_D value of 5 nM and B_max value of 1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed h7-nAChRs is evident as rapid, transient inward current responses to (–)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to 100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing 3 or 4 subunits (K_D values of about 100 or 10 pM, respectively). h7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of ³H-labeled epibatidine (K_D value of 0.6 nM). Studies of several forms of native or heterologously expressed rat or human 7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and -bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine cytisine > carbamylcholine d-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human 7-nAChRs that exhibit ligand binding and functional properties like native 7-nAChRs and that epibatdine is useful as a probe for human 7-nAChRs.

In the central nervous system, a major nAChR subtype that engages in high-affinity binding of -bungarotoxin (Bgt) is composed of homopentamers of 7 subunits (Chen and Patrick, 1997). Human 7-containing nAChRs (h7-nAChRs) have been stably expressed in mammalian cell lines and function as Bgt-sensitive ion channels exhibiting rapid kinetics of activation and inactivation (Puchacz et al., 1994; Gopalakrishnan et al., 1995). 7-nAChRs are Ca²⁺-permeable (Couturier et al., 1990; Seguela et al., 1993; Peng et al., 1994; Puchacz et al., 1994; Gopalakrishnan et al., 1995), making it possible that they participate in additional forms of nicotinic signaling other than or in addition to classical excitatory neurotransmission.

Epibatidine (EBDN) is an alkaloid obtained from skin extracts of the poisonous, Ecuadorian frog Epipedobates tricolor (Daly, 1995). EBDN effectively competes with nicotine and cytisine in rat brain preparations and acts as a potent nicotinic agonist (Badio and Daly, 1994; Houghtling et al., 1994, 1995; Alkondon and Albuquerque, 1995; Briggs et al., 1995). EBDN has been promoted as a ligand with selectivity for nAChRs containing 4 or 3 subunits (the accepted nomenclature classifies these as 4*- or 3*-nAChRs, respectively; Lukas et al., 1999). However, because interactions of EBDN with 7-nAChRs are less well understood, we investigated such interactions using native nAChR-null SH-EP1 human epithelial cells as hosts for constitutive, stable, heterologous expression of human 7-nAChRs (h7-nAChRs).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Culture. Cells of the SH-EP1 human epithelial cell line or the SH-SY5Y human neuroblastoma line (kindly provided by Dr. June Biedler, Sloan Kettering Institute for Cancer Research, New York, NY; Biedler et al., 1973) were grown in Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (all from Invitrogen, Carlsbad, CA) plus 5% fetal bovine serum (Hyclone Laboratories, Logan, UT) in a humidified atmosphere containing 5% CO₂ in air at 37°C (Lukas, 1986; Lukas et al., 1993).

Construction of Human 7/pCEP4 Plasmid and Generation of Stable Cell Lines. cDNA encoding a human nAChR 7 subunit (h7 subunit; Breese et al., 1997) was excised from the pcDNAI vector at the BamHI restriction site and subcloned into the BamHI site of the plasmid pCEP4 (Invitrogen). Final constructs were verified by restriction mapping and complete sequencing of the insert. SH-EP1 cells (or SH-SY5Y cells) were transfected using electroporation with a Bio-Rad Gene Pulsar at 960 µF and 0.20 kV/cm (t = 28–36 ms) with the pCEP4-h7 construct. Forty-eight hours after transfection, culture medium was supplemented with 0.25 mg/ml hygromycin. Growth was monitored until ring cloning was used to isolate single, transfected cell colonies, which were then expanded. ¹²⁵I-Labeled -bungarotoxin (I-Bgt) binding assays (Bencherif and Lukas, 1993) were used to screen stable transfectants for expression of h7-nAChRs. Three lines (clones 12, 16, and 28) studied in greater detail stably express h7-nAChRs of indistinguishable properties. The clonal line derived from transfected SH-EP1 cells used for most of the studies described herein was clone 12 and was named the SH-EP1-pCEP4-h7 line. One of the stably transfected SH-SY5Y cell clones that expressed h7-nAChRs at high levels was also used for the studies described herein, and this cell line was named the SH-SY5Y-pCEP4-h7 line. The previously described SH-EP1-Toff-h7 cell line (Peng et al., 1999) that stably expresses h7-nAChRs de novo under control of a tetracycline-sensitive promoter and the previously described SH-SY5Y-pCEP4-r7 cell line (Puchacz et al., 1994) that heterologously and stably expresses rat 7 subunits and high levels of 7-nAChRs above a background of human, native 3*-nAChR and native h7-nAChR expression also were used in the current studies. Wild-type SH-SY5Y cells were maintained as described previously (Lukas et al., 1993), and rat brains were obtained from Pel-Freeze (Rogers, AR).

RNA Isolation and Northern Blot Analysis. Total cytoplasmic RNA was extracted from transfected cells using the Fast Track (Invitrogen) technique. RNA was separated on 1% formaldehyde/agarose gels and transferred to nylon blotting membrane by pressure blotting. Blots were probed with ³²P-radiolabeled, full-length, human nAChR 7 subunit cDNA (Bencherif et al., 1995).

Immunocytochemistry. To visualize nAChR 7 subunits, SH-EP1-pCEP4-h7 cells were plated onto 22 x 22-mm glass coverslips and grown for 2 days. Cells were then rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min, and rinsed again before blocking for 20 min with PBS containing 4% normal horse serum, 1% bovine serum albumin, and 0.4% Triton X-100. Mouse monoclonal antibody 306 (Sigma-Aldrich, St. Louis, MO), targeting an intracellular epitope (due to a lack of suitable anti-7 antibodies targeting extracellular epitopes, immunofluorescent assessment of cell surface 7 subunit expression was not a viable option), was added overnight at 4°C in blocking solution, or primary antibody was omitted from control samples. Cells were rinsed again and incubated for 30 min with 1% bovine serum albumin-PBS containing biotinylated secondary antibody (horse anti-mouse IgG; Vector Laboratories, Burlingame, CA) and then for 30 min with avidin-Alexa 488 (Molecular Probes, Eugene, OR), with PBS rinses between steps. Staining was visualized using epifluorescence (Olympus IX70; Olympus, Melville, NY).

¹²⁵I-Labeled -Bungarotoxin Binding Autoradiography. Cultured cells grown for 2 days on Lab Tek II CC2 8-chambered, multi-well slides (Nalge-Nunc International, Rochester, NY) were rinsed at room temperature in PBS for 5 min and then incubated in fresh binding buffer (118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, and 20 mM HEPES, pH 7.5 with NaOH) containing 5 nM I-Bgt (±10 mM nicotine to define nonspecific/total radioligand binding to cell-surface sites) for 60 min at 22°C. After radioligand incubation, samples were rinsed twice in 1x binding buffer at 22°C, once in 1x buffer at 4°C for 5 min, once in 0.1x buffer at 4°C for 10 s, and then in 5 mM HEPES at 4°C for 10 s. Subsequently, slides were fixed in 4% paraformaldehyde (50:50 solution: 8% paraformaldehyde and 0.2 M phosphate buffer, pH 7.2) for 20 min at 22°C before being rinsed briefly in H₂O and air-dried at 22°C. Samples were then subjected to electronic isotope counting using an Instant Imager (PerkinElmer Life and Analytical Sciences, Boston, MA) to quantify specific (total minus nonspecific) radioligand binding (i.e., integrating counts across defined areas of sections or cultures containing equivalent amounts and densities of cells). After radioligand binding quantification, samples were dipped in Kodak NTB-3 nuclear track emulsion and allowed to dry overnight at 22°C in a dust-free environment. Slides were then stored at 4°C for exposure periods ranging from 2 to 5 days (showing a linear grain development response while revealing sites of low level radioligand binding). After exposure, slides were developed for 3 min at 22°C with Kodak D-19 developer and fixed for 3 min at 22°C with Kodak fixer. Slides were counter-stained with 0.5% cresyl violet acetate and serially dehydrated with 50, 75, 85, and 95% ethanol. After ethanol treatments, slides were dipped twice in xylene for 5 min each at 22°C and dry-mounted with Permount. Images were captured using an Olympus IX70 inverted microscope and MagnaFire camera and software and then stored and maximized for Hi Gauss clarity and sharpness using ImagePro Plus (Media Cybernetics, Inc., Silver Spring, MD).

Immunoprecipitation and Western Blot Analysis. Membrane preparations (see below) from SH-EP1-pCEP4-h7 cells were suspended in Ringer's buffer (150 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1.3 mM MgCl₂, and 33 mM Tris, pH 7.4) supplemented with 1% Triton X-100, incubated for 10 min at room temperature, and centrifuged at 45,000g for 10 min at 4°C. The supernatant containing detergent-solubilized h7-nAChRs was incubated for 1 h at room temperature and overnight at 4°C with Omnisorb (Calbiochem, La Jolla, CA) previously complexed with goat anti-7 subunit antisera (C-20, sc-1447; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The sample was subjected to centrifugation at 5000g for 10 min to recover immunoaffinity-purified h7-nAChRs in the pellet, which was extracted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer. The immunoisolate was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred via electroblotting to a nylon membrane. The membrane was incubated with rabbit anti-7 subunit antisera (H-302, sc-5544; Santa Cruz Biotechnology, Inc.), rinsed free of unbound antibody, and probed with ¹²⁵I-labeled protein A (Amersham Biosciences Inc., Piscataway, NJ). The labeled immunoisolate was visualized using an Instant Imager, and its mobility was determined relative to mobilities of molecular mass standards (high mass standards; Bio-Rad, Hercules, CA).

Membrane Preparation and I-Bgt Binding Assays. Confluent, stably transfected or untransfected cells were rinsed with ice-cold Ringer's buffer supplemented with 0.1 mg/ml sodium azide, mechanically dislodged and harvested from dishes, and homogenized using a Polytron (setting 65, 45 s). Rat brain tissue was similarly homogenized. Homogenates were centrifuged at 45,000g for 10 min at 4°C, and pellets containing membrane fractions including all but the smallest microsomes were resuspended in ice-cold Ringer's buffer. I-Bgt (Amersham Biosciences Inc.; diluted with 500 nM unlabeled Bgt to achieve a typical specific activity of 45–65 cpm/fmol) binding to membranes was carried out as described previously, using a centrifugation-based protocol and gamma detection (Tracor 1191 gamma-counter; Lukas, 1984). Incubations were carried out in a final volume of 200 µl containing 50 µl of I-Bgt at a given, indicated concentration (typically 40 nM) and 100 µl of cell membrane suspension for 1 h at room temperature in the presence or absence of unlabeled drugs. Nonspecific binding was defined using samples supplemented with 1 µM native Bgt and was subtracted from total binding in other samples to compute specific binding. For I-Bgt binding saturation studies, I-Bgt final concentrations were varied from 10 pM to 40 nM.

I-Bgt Binding to Cell Surface Sites. Based on a previously described assay (Bencherif and Lukas, 1993), cells in culture, sometimes treated over different periods with different concentrations of (–)-nicotine, were rinsed twice with Ringer's buffer before being incubated with 10 nM I-Bgt for 1 h at room temperature in the absence or presence of 1 µM unlabeled Bgt. Binding was terminated by removal of medium followed by three 1-ml rinses in Ringer's buffer. Cells were harvested in 500 µl to 1 ml of 0.1 N NaOH and 0.1% SDS, with shaking, and gamma counting was conducted to quantitate bound I-Bgt.

[³H]EBDN Binding Assays. Membranes prepared as described above were used for ³H-labeled (±)-epibatidine ([³H]EBDN; Amersham Biosciences Inc.) binding assays (modified after Houghtling et al., 1994, 1995) by incubation for 1 to 3 h (or for the indicated time for association rate studies) at room temperature in a final volume of 200 µl containing 50 µl of [³H]EBDN at a given, indicated concentration (typically 4 nM but varying for saturation analyses, for some competition assays, and for kinetics studies) and 100 µl of cell membrane suspension. Samples were supplemented with 50 µl of the specified drug at the indicated concentration for competition assays, with 50 µl of buffer to define total binding, or with 50 µl of nicotine (to a final concentration of 300 µM) to define nonspecific binding. Nonspecific binding was subtracted from total binding in other samples to compute specific binding. Dissociation rate studies were done using bulk (large-volume) master stock samples that were incubated with 1 nM [³H]EBDN for 3 h (other studies done using an initial concentration of 4 nM are not illustrated) and divided into 200-µl aliquots before being diluted 21-fold in assay buffer (physical dilution) or supplemented to 1, 2, or 4 µM(±)-EBDN (chemical dilution) for specified periods before assay termination. To accomplish assay termination, free [³H]EBDN was separated from bound [³H]EBDN using polyethyleneimine-coated GF/B glass fiber filtration and a Whatman manifold (Lukas, 1990), and filters were incubated while shaking overnight in liquid scintillation fluid before sample [³H]EBDN was quantified using liquid scintillation counting.

Whole-Cell Current Recording. Nicotine-evoked currents were recorded in the whole-cell configuration using an Axopatch 200B amplifier (Axon Instruments Inc., Union City, CA). Currents were digitized and recorded using pCLAMP software (Axon Instruments Inc.). Cells were continuously superfused with an external bath solution containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 10 mM HEPES, and 10 mM glucose (pH 7.4, 290–300 mmol/kg). Nicotine (3, 30, 100, and 300 µM) was delivered for 1 s once every 30 s via a multibarrel fast perfusion system (Warner Instrument, Hamden, CT). Patch pipettes were pulled from borosilicate capillary glass tubing using a Flaming/Brown micropipette puller (P97; Sutter Instrument Company, Novato, CA) and filled with an internal pipette solution consisting of 130 mM CH₃SO₃K, 10 mM KCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 5 mM EGTA, 10 mM HEPES, 3 mM Mg²⁺-ATP, and 0.3 mM Na-GTP (pH 7.2, 280 mmol/kg). The resistances of the patch pipettes when filled with internal solution ranged between 3 and 6 M. Experiments were conducted at room temperature.

Data Analysis. Parameters (dissociation constant, K_D, and maximum binding level, B_max) for radioligand binding were determined from nonlinear curve-fitting analysis of binding saturation isotherms (GraphPad Software, Inc., San Diego, CA). In competition experiments, 50% inhibition constants (IC₅₀ values), levels of maximum and minimum binding as a percentage of control specific binding, and Hill coefficients (n_H) were determined from plots of specific binding as a function of the logarithm of the concentration of competing ligand (GraphPad Software, Inc.) using the standard logistic equation for one or two binding sites. In some cases, the Hill coefficient was fixed at –1 for radioligand binding competition assays. In cases where specific radioligand binding was incompletely blocked, the extent of inhibition as a percentage of specific binding in control samples was determined based on fits to the logistic equation. However, in most cases, levels of maximum or minimum binding were within 1 standard error of 100 or 0%, respectively, of control specific binding. Mean ± S.E.M. values are provided unless otherwise noted.

Materials. All other techniques and commercial sources for reagents were as indicated previously (Lukas, 1984, 1986, Bencherif and Lukas, 1993; Peng et al., 1999).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Screening of Transfectants. In our previous studies, we demonstrated that SH-EP1 human epithelial cells do not express any endogenous human nAChR subunits as messenger RNA or as radioligand binding sites (Lukas et al., 1993). Colonies derived from SH-EP1 cells transfected with the pCEP4 plasmid containing the h7 subunit cDNA fragment were selected based on their resistance to hygromycin, subcloned by limiting dilution, and propagated. Screening studies using I-Bgt binding revealed that 20 of the 24 clones expressed I-Bgt binding sites de novo. Absolute levels of expression of I-Bgt binding sites varied across and within these clones for reasons that were not systematically investigated. However, three clones showing the most stable, high-to-moderate levels of expression were selected for further study (clones 12, 16, and 28). Unless otherwise noted, studies reported here were done using clone 12, the cell line named SH-EP1-pCEP4-h7.

Transgene Expression as Message and Protein. Northern blot analyses using a panel of full-length rat or human nAChR subunit cDNAs as probes confirmed lack of expression of any nAChR subunit as messenger RNA in untransfected SH-EP1 cells. However, Northern analysis using full-length h7 subunit cDNA as a probe showed that a 2100-base pair mRNA is expressed in transfected SH-EP1-pCEP4-h7 cells, but not in untransfected SH-EP1 cells (data not shown). Western analysis of immunoaffinity-purified h7-nAChRs identified an anti-7 subunit immunoreactive protein migrating with an apparent molecular mass of 53 kDa (based on mobility of the set of mass standards used). No such band was not evident upon immunoprecipitation of extracts from untransfected cells, but a polypeptide from SH-EP1-pCEP4-h7 cell extracts of the same mobility also was evident after -cobratoxin affinity purification (data not shown).

Immunofluorescence studies using a monoclonal antibody targeting a cytoplasmic epitope of the 7 subunit indicated specific staining [no staining was evident in untransfected SH-EP1 cells (data not shown) or in the absence of primary antibody (Fig. 1a)] of virtually every SH-EP1-pCEP4-h7 cell (Fig. 1b). Staining was more intense in perinuclear areas, but generally filled the cytoplasm, even in processes emanating from soma.

View larger version (87K): [in this window] [in a new window]   Fig. 1. Expression of 7 subunits and I-Bgt binding. SH-EP1-pCEP4-h7 cells were probed using a primary monoclonal antibody against an intracellular epitope of 7 subunits (b) or in the absence of primary antibody (a) to execute immunofluorescence staining as described under Materials and Methods. Epifluorescence images were taken at 200x magnification (scale bar, 50 µm). As shown in this representative set of images from dozens of fields examined in at least three separate studies, primary antibody-specific staining is seen in all cells. No staining was observed (images not shown) with the primary antibody when applied to untransfected cells. Other samples of SH-EP1-pCEP4-h7 cells were treated with 5 nM I-Bgt in the presence of 10 mM nicotine to define nonspecific binding (c and e) or with I-Bgt alone (d and f) to define total binding for samples subjected to microautoradiographic analysis as described under Materials and Methods. Red arrows indicate cells exhibiting specific I-Bgt binding, and black arrows indicate unlabeled cells (d and f). Higher levels of diffuse autoradiographic grain development (d and f) as well as dense grain development over labeled cell bodies are both hallmarks of samples showing specific I-Bgt binding. Images were taken at 200x (c and d) or 600x (e and f) magnification (scale bars for each image, 50 µm) and are representative of images from dozens of fields examined in more than three separate studies.

h7-nAChR Expression as -Bungarotoxin Binding Sites. Cell surface I-Bgt-receptor binding microautoradiography (Fig. 1, c and e) conducted in the presence of nicotine to define nonspecific binding showed minimal silver halide grain development over cell bodies and very low levels of background grain development. By contrast, diffuse, heightened grain development over the entire slide and very dense grain development over cell bodies characterized the labeling pattern in SH-EP1-h7 cell samples exposed to I-Bgt only (Fig. 1, d and f), but not in untransfected SH-EP1 cells (data not shown). Silver grain development was evident at many SH-EP1-h7 cell-cell boundaries (i.e., on cells that were in proximity) as well as on solitary cells. More than 80% of SH-EP1-h7 cells showed labeling (30% of cells were densely labeled), but the remaining cells showed no or very low surface I-Bgt binding despite positive selection for transfectants and immunostaining evidence for expression of at least internal pools of 7 subunit epitope in all cells.

I-Bgt saturation assays showed that the established, transfected clonal lines possess moderate-to-high levels (B_max value of 0.3–3.5 pmol/mg membrane protein) of high-affinity binding sites presumed to represent h7-nAChRs (Fig. 2). The apparent K_D value of 7 nM for the data set shown is a preequilibrium value likely underestimating the true K_D value due to slow association of I-Bgt with its sites and slow dissociation of I-Bgt from radioligand-receptor complexes. The specific, apparent K_D value for any experiment varied somewhat (2–7 nM), apparently reflecting differences in biological activity of radiolabeled material across commercial I-Bgt preparations. Although not studied systematically, variation in B_max values reflected differences in cell passage (sometimes higher passage leads to loss of nAChR expression) and/or cell plating density at the time of harvest for assay (highest expression occurs for cells near confluence), as we have noted in studies of other naturally or heterologously expressed nAChRs in human cell lines. No specific I-Bgt binding was evident in untransfected SH-EP1 cells. Studies of the cellular distribution of I-Bgt binding sites indicated that 62% of the total number of I-Bgt binding sites isolated in membrane fractions (which are obtained under conditions where binding sites on very small microsomes might escape detection) are typically expressed on the cell surface of SH-EP1-pCEP4-h7 cells (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Saturation analysis for I-Bgt binding to a SH-EP1-pCEP4-h7 cell membrane preparation. Reaction mixtures containing 190 µg of membrane protein from SH-EP1-pCEP4-h7 cells and I-Bgt at the indicated final concentrations (abscissa; total radioligand; nanomolar) were processed for 1 h to determine specific I-Bgt binding (; ordinate; femtomoles per milligram of membrane protein). Nonspecific binding (subtracted from total binding to define specific binding) determined using samples containing 1 µM Bgt was defined by the equation ((2.36[I-Bgt] (nM)) + 15.32) fmol/mg and was 20% of total binding at 10 nM I-Bgt. Results from the illustrated studies of three representative preparations yielded a mean (±S.E.M.), apparent KD value of 7.0 ± 3.1 nM, and Bmax value of 0.35 ± 0.04 pmol/mg membrane protein for this clone. Bmax values determined from saturation binding assays using other preparations from this and other clones were as high as 3.5 pmol/mg membrane protein, but all had apparent KD values of about 2 to 7 nM. Reasons for variation across preparations in levels of specific I-Bgt binding have not yet been elucidated, but cell plating density and passage affect radioligand binding levels.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Cell surface expression of heterologously expressed h7-nAChR. SH-EP1-pCEP4-h7 cells were subjected to I-Bgt binding assays in situ (surface binding) or using membrane preparations (membrane binding). Specific I-Bgt binding site levels (ordinate; femtomoles per milligram of membrane protein from sister samples processed in parallel) represent means (±S.E.M.) from five to six replicate determinations over two separate experiments. Nonspecific binding was 10 to 20% of specific binding for determinations using cells in situ and was 20% for determinations using membrane preparations. Samples used for assays typically contained 111 µg of total cell protein or 200 µg of membrane protein.

Screening for Functional h7-nAChR Expression. Whole-cell current recordings using transfected SH-EP1 cells (clone 28) revealed functional h7-nAChRs mediating inward currents stimulated by (–)-nicotine (Fig. 4; see Zhao et al., 2001, 2003 for results of other studies of functional h7-nAChRs in SHEP1-pCEP4-h7 cells) that were not evident in parallel studies of untransfected SH-EP1 cells (data not shown). The inward current responses in SH-EP1-h7 cells were transient, reflecting the rapid activation and desensitization of h7-nAChRs.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Function of heterologously expressed h7-nAChR. Whole-cell current responses to 1-s pulses of 30 µM (top) or 300 µM (bottom) (–)-nicotine showed rapid, transient inward currents indicating functional expression of h7-nAChR in transfected SH-EP1 cells (clone 28). Representative records are shown (horizontal scale, 500 ms; vertical scale, 10 pA). Nine of 13 cells tested showed such responses and an average inward peak current of 40 pA in response to 30 µM nicotine at a holding potential of –60 mV.

Nicotine Exposure-Induced Up-Regulation of h7-nAChRs Measured as Toxin Binding Sites. Exposure of SH-EP1-pCEP4-h7 cells for 2 days to (–)-nicotine resulted in a significant increase (up to 100%) in I-Bgt binding sites (Fig. 5). The effect of nicotine was both dose-dependent (half-maximal effect at 100 µM; Fig. 5) and time-dependent (half-maximal increase at about 12 h of drug exposure; data not shown).

View larger version (61K): [in this window] [in a new window]   Fig. 5. Up-regulation of h7-nAChR on chronic exposure to nicotine. SH-EP1-pCEP4-h7 cells were treated for 2 days in the presence of the indicated concentrations of (–)-nicotine (abscissa; micromolar) before being processed to membrane fractions for determination of specific I-Bgt binding (ordinate; percentage of control). Samples typically contained 200 µg of membrane protein, and results are from two preparations and two to four samples per condition. The half-maximal increase in binding site density occurred at 100 µM nicotine, although a two phase increase (36% increase with an EC50 of 300 nM and 47% increase with an EC50 of 300 µM) also fit the data.

Epibatidine Binding of h7-nAChRs. [³H]EBDN binding saturation assays also showed that SH-EP1-pCEP4-h7 cells expressed sites with picomolar affinity (macroscopic K_D value of 0.6 nM; B_max value up to 3.5 pmol/mg) for this radioligand, which binds in a Bgt-sensitive manner (Fig. 6). No specific binding of [³H]EBDN occurs for untransfected SH-EP1 cells, consistent with their previous characterization as native nAChR-null (Lukas et al., 1993). As was the case for determination of specific I-Bgt binding sites, variability in B_max values reflected influences of cell passage number and/or cell plating density at the time of harvest for assay. However, for paired studies, numbers of [³H]EBDN and I-Bgt binding sites in the same preparations agreed within 10% after correction for radioligand dissociation during sample processing (e.g., 3.3 and 3.5 pmol/mg membrane protein, respectively).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Saturation analysis for [3H]EBDN binding to a SH-EP1-pCEP4-h7 cell membrane preparation. Reaction mixtures containing 10 µg of membrane protein from SH-EP1-pCEP4-h7 cells and [3H]EBDN at the indicated final concentrations (abscissa; total radioligand; nanomolar) were processed for 1 h to determine specific [3H]EBDN binding (; ordinate; femtomoles per milligram of membrane protein). Nonspecific binding (subtracted from total binding to define specific binding) determined using samples containing 100 µM nicotine was defined by the equation ((350[[3H]EBDN] (nM)) + 6) fmol/mg and was 14% of total binding at 1 nM [3H]EBDN. Results from the illustrated studies are from two separate preparations and yielded a mean (±S.E.M.) KD value of 0.64 ± 0.04 nM and Bmax value of 3.51 ± 0.06 pmol/mg membrane protein (r2 = 0.99). Bmax values determined from saturation binding assays using other preparations from this and other clones varied between 0.23 and 3.5 pmol/mg membrane protein, but all had KD values of 0.4 to 1.0 nM. Reasons for variation across preparations in levels of specific [3H]EBDN binding have not yet been elucidated, but cell passage number and plating density can influence levels of expression. Also shown are results using the same membrane preparation in reaction mixtures also containing 1 µM Bgt (; + Bgt; ordinate; femtomoles per milligram of membrane protein). Specific [3H]EBDN binding in these samples was reduced to 0.46 ± 0.03 pmol/mg membrane protein with a KD value of 0.43 ± 0.10 nM (r2 = 0.95).

Association rate kinetics studies showed that [³H]EBDN binding to h7-nAChRs in SH-EP1-pCEP4-h7 cells can be described as a monoexponential process (Fig. 7, top). Observed rates of [³H]EBDN binding at concentrations of 0.1, 0.3, and 1 nM varied between 6.5 and 9.5 min^–1 nM^–1 (half-times of 4.4–6.4 s). A plot (not shown) of observed association rates versus [³H]EBDN concentration yielded a slope corresponding to k₁ of 3.1 min^–1 nM^–1 and a y-intercept corresponding to k_–1 of 6.3 min^–1, yielding a microscopic _KD value of 2.0 nM, in reasonably good agreement with the macroscopic K_D value of 0.6 nM determined using saturation binding assays.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Kinetics of [3H]EBDN binding to h7-nAChR. Top, [3H]EBDN association rate studies. Reaction mixtures containing SH-EP1-pCEP4-h7 cell membrane preparations (typically containing 350 µg of protein) and 0.1 (), 0.3 (), or 1 nM () [3H]EBDN were incubated for the indicated times (abscissa; minutes) before being processed for determination of specific [3H]EBDN binding (ordinate; femtomoles per milligram). Lines drawn through the data points from one representative study of three using different preparations are best fit curves yielding half-times for association of between 4.4 and 6.4 s (k1obs values between 6.5 and 9.5 min–1 nM–). Plots of k1obs as a function of [[3H]EBDN] have a slope (±S.E.M.) of 3.09 ± 1.28 min–1 nM–1 and an y-intercept of 6.28 min–1. Bottom, [3H]EBDN dissociation rate studies. Reaction mixtures containing SH-EP1-pCEP4-h7 cell membrane preparations (typically containing 170 µg of protein) and 1 nM [3H]EBDN in a volume of 200 µl were incubated for 3 h before being either physically diluted () by adding 4 ml of Ringer's buffer, chemically diluted by adding 1 µM (), 2 µM (not shown), or 4 µM () of unlabeled EBDN at "time 0" before being filtered over GF/B discs to resolve free from bound [3H]EBDN at the indicated times after physical or chemical dilution (abscissa; minutes) to allow determination of specific [3H]EBDN binding (ordinate; percentage of control). Lines drawn through the data points from three studies are best fit curves yielding apparent rate constants for dissociation of 1.26 ± 0.07 min–1 for physical dilution, and 1.47 ± 0.22, 2.64 ± 0.30, and 4.38 ± 0.16 min–1 for chemical dilution in 1, 2 (curve not shown), or 4 µM EBDN, respectively.

Independent empirical studies of dissociation kinetics revealed a dependence of the apparent dissociation rate on the method used in the study. A comparatively slow rate of dissociation occurred for physical dilution, in which reaction samples were diluted 21-fold before separation of bound from free radioligand (k_–1^app = 1.3 min^–1; Fig. 7, bottom). Observed rates of dissociation of bound [³H]EBDN were faster under conditions of chemical dilution, in which reaction mixtures were supplemented with unlabeled EBDN to promote radioligand dissociation (k_–1^app = 1.5–4.4 min^–1; Fig. 7, bottom), and in the presence of higher concentrations of unlabeled drug. These findings are hallmarks of negative cooperativity in radioligand binding to interacting sites, possibly reflecting the presence of five possible binding sites on the homopentamer but weakening of binding if more than two sites are occupied at once. Calculations done using empirically derived rates of [³H]EBDN dissociation gave microscopic K_D values between 0.41 nM (for physical dilution-based measures of k_–1) and 1.4 nM (for chemical dilution-based measures of k_–1), also in good agreement with determinations of macroscopic K_D values.

To assess whether [³H]EBDN binds specifically to h7-nAChRs from other preparations, [³H]EBDN binding saturation assays also were done using membrane fractions from SH-EP1-Toff-h7 cells (Peng et al., 1999) transfected with h7 subunit cDNA under control of a tetracycline-regulated promoter and heterologously expressing h7-nAChRs (Fig. 8). The results showed that heterologously expressed h7-nAChRs in this cell line also have high affinity for [³H]EBDN (K_D = 0.46 nM). Other [³H]EBDN binding saturation studies were done using membranes from SH-SY5Y-pCEP4-h7 cells generated for this study and heterologously expressing recombinant h7 subunits in a natural background of h7 subunits and human nAChRs containing 3 subunits (3*-nAChRs; Fig. 8). Specific [³H]EBDN binding in these cells defined using sensitivity to nicotine blockade suggested expression of high-affinity [³H]EBDN binding, and analysis of specific binding defined using sensitivity to 1 µM Bgt indicated expression of sites with a K_D value of 1.2 nM, in close agreement with K_D values for [³H]EBDN binding to h7-nAChRs expressed de novo in transfected SH-EP1 cells (Fig. 8). Sites showing insensitivity to blockade by Bgt are likely to be naturally expressed human 3*-nAChRs found in SH-SY5Y cells.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Saturation analysis for [3H]EBDN binding to a SH-SY5Y-pCEP4-h7 cell or SH-EP1-Toff-h7 cell membrane preparations. Reaction mixtures containing 10 µg of membrane protein from SH-SY5Y-pCEP4-h7 () or SH-EP1-Toff-h7 () cells and [3H]EBDN at the indicated final concentrations (abscissa; total radioligand; nanomolar) were processed for 1 h to determine specific [3H]EBDN binding (ordinate; femtomoles per milligram of membrane protein). Results from the illustrated duplicate studies yielded a mean (±S.E.M.) KD value of 1.04 ± 0.16 nM and Bmax value of 6.67 ± 0.26 pmol/mg membrane protein for the preparation from SH-SY5Y-pCEP4-h7 cells (varied between 1.6 and 10.1 pmol/mg across preparations) and a KD value of 0.46 ± 0.06 nM and Bmax value of 1.91 ± 0.07 pmol/mg membrane protein for the preparation from SH-EP1-Toff-h7 cells (varied between 0.74 and 3.2 pmol/mg across preparations). Also shown are results obtained using the same membrane preparation from SH-SY5Y-pCEP4-h7 cells in reaction mixtures also containing 1 µM Bgt to define Bgt-sensitive [3H]EBDN binding (), which yielded a Bmax value of 3.97 ± 0.08 pmol/mg membrane protein with a KD value of 1.17 ± 0.08 nM.

Epibatidine Binding to Other 7-nAChRs. Other [³H]EBDN binding saturation analyses using SH-SY5Y-pCEP4-r7 cells heterologously expressing rat 7 subunits in a natural background of h7 subunits and human 3*-nAChRs revealed Bgt-sensitive [³H]EBDN binding sites with K_D values of 4 to 16 nM and Bgt-insensitive [³H]EBDN sites with K_D values of about 120 pM (data summarized but not shown). Additional [³H]EBDN binding saturation studies revealed Bgt-sensitive [³H]EBDN binding in rat brain membranes with an apparent K_D value of 8 nM and Bgt-insensitive [³H]EBDN sites with a K_D value of 20 pM (data summarized but not shown). Similarly, [³H]EBDN binding saturation studies done using untransfected SH-SY5Y cells indicated expression of Bgt-sensitive [³H]EBDN binding sites with an apparent K_D value of 1 nM and Bgt-insensitive [³H]EBDN sites with a K_D value of 40 pM (data summarized but not shown). In some cases, two site fits to [³H]EBDN saturation curves (not shown) obtained using nicotine to define specific binding to rat brain, wild-type SH-SY5Y cell, or transfected SH-SY5Y cell preparations allowed clear separation of two classes of binding sites with K_D values like those for Bgt-sensitive and Bgt-insensitive sites. Thus, analyses of specific [³H]EBDN saturation binding studies using wild-type or transfected SH-SY5Y cells were consistent with expression of Bgt-sensitive, lower affinity [³H]EBDN binding sites corresponding to human or rat 7-nAChRs at different levels of expression, either naturally expressed in wild-type cells, or representing a mixture of naturally and heterologously expressed forms in transfected cells. In addition, these analyses were consistent with expression of Bgt-insensitive, higher affinity [³H]EBDN binding sites corresponding to naturally expressed 3*-nAChRs found in wild-type or transfected SH-SY5Y cells. Similarly, saturation analyses using rat brain indicated the presence of Bgt-sensitive, lower affinity [³H]EBDN binding sites corresponding to 7-nAChRs and a predominant population of Bgt-insensitive, higher affinity [³H]EBDN binding sites presumably correlated with 4*-nAChRs. However, heterologous de novo expression of h7 subunits in transfected SH-EP1 cells gave the clearest indications of Bgt-sensitive [³H]EBDN binding properties of h7-nAChRs.

Mutual Inhibition of Epibatidine and -Bungarotoxin Binding to 7-nAChRs. Results indicating that EBDN and Bgt bind at overlapping sites on h7-nAChRs in transfected SH-EP1-pCEP4-h7 cells and that h7-nAChRs have higher affinity for EBDN than for Bgt come from a series of radioligand binding competition studies. Specific I-Bgt binding at 10 nM was inhibited by EBDN with an IC₅₀ value of 13 nM (Fig. 9A) and by Bgt with an IC₅₀ value of 35 nM (Fig. 9C; Table 1). However, whereas all of specific I-Bgt binding was blocked in the presence of Bgt, a subset (20%) of specific I-Bgt binding sites were not blocked by EBDN at the highest concentration tested (Fig. 9, A and C; Table 1). Nevertheless, "small drug-insensitive but Bgt-sensitive" I-Bgt binding sites were observed when using other drugs (Fig. 12) and other 7-nAChR expression systems (Peng et al., 1999; see other results in Fig. 9; Table 1). Similarly, specific [³H]EBDN binding at 1 nM to sites in SH-EP1-pCEP4-h7 cells was completely blocked in the presence of Bgt with an IC₅₀ value of 45 nM (Fig. 10C), but specific [³H]EBDN binding was even more sensitive to autologous (and complete) blockade by EBDN (IC₅₀ value of 3.2 nM; Fig. 10A; Table 1).

View larger version (35K): [in this window] [in a new window]   Fig. 9. EBDN and Bgt compete for I-Bgt binding to 7-nAChR. Reaction mixtures containing membrane protein from rat brain (), SH-EP1-pCEP4-h7 cells (), or SH-SY5Y-pCEP4-h7 cells () (A and C), from SH-EP1-Toff-h7 cells () (B), or from wild-type SH-SY5Y cells () or SH-SY5Y-pCEP4-r7 cells ) (B and D), with 10 nM I-Bgt, and either unlabeled EBDN (A and B) or Bgt (C and D) at the indicated final concentrations (abscissas; nanomolar) were processed for 1 h to determine I-Bgt binding (ordinate; percentage of control specific binding; n = 2–3). Continuous lines represent fits to the Hill equation (nH = –1). IC50 or log IC50 values and extents of ligand inhibition of I-Bgt binding are given in the text and/or in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Parameters [log IC50 (molar) and extent of inhibition (ExIn; percentage of control specific binding) ± S.E.M.] for EBDN or Bgt competition toward specific [3H]EBDN or I-Bgt binding to 7-nAChR Radioligand binding competition assays were conducted as described under Materials and Methods and in the legends to Figs. 9 and 10. Results were fit to the logistic equation with Hill coefficients set to -1 to determine log IC50 values and maximum or minimum levels of binding relative to control specific binding. Maximum binding was within one standard error of 100% for all samples, and minimum binding was within one standard error of 0% for samples unless the extent of inhibition (ExIn) is shown (ExIn for EBDN versus I-Bgt binding for SH-EP1-Toff-h7 cells is >73% because a full dose-response was not conducted).

View larger version (25K): [in this window] [in a new window]   Fig. 12. Drug competition profiles for blockade of specific I-Bgt binding to sites on SH-EP1-pCEP4-h7 cells. Reaction mixtures containing SH-EP1-pCEP4-h7 cell membrane preparations (typically containing 28 µg of protein), 10 nM I-Bgt, and the indicated drugs were used to assess the concentration dependence (abscissa; molar, log scale) for competition toward specific I-Bgt binding (ordinate; percentage of control). Results are the averages three separate experiments. Top, competition profiles for DMPP (), cytisine (cyt; ), or d-tubocurarine (d-TC; ). Bottom, competition profiles for MLA (), nicotine (nico; ), or carbamylcholine (carb; ). Log IC50 values and Hill coefficients (±S.E.M.) are provided in Table 2, and IC50 values are indicated in the text. Upper and lower limits for sigmoid curves were fixed at 100 and 19%, reflecting submaximal inhibition of I-Bgt binding by small ligands (extent of inhibition determined from fitting of the data without a fixed lower limit is 83 ± 3% for DMPP, 84 ± 3% for cyt, 82 ± 3% for d-TC, 77 ± 3% for MLA, 83 ± 2% for nicotine, and 83 ± 5% for carb; all results significantly different from 100% inhibition at the 95% confidence level or better).

View larger version (36K): [in this window] [in a new window]   Fig. 10. EBDN and Bgt compete for [3H]EBDN binding to 7-nAChR. Reaction mixtures containing membrane protein from rat brain (), SH-EP1-pCEP4-h7 cells (), or SH-SY5Y-pCEP4-h7 cells () (A and C), from SH-EP1-Toff-h7 cells () (D), or from wild-type SH-SY5Y cells () or SH-SY5Y-pCEP4-r7 cells () (B and D), with 1 nM [3H]EBDN, and either unlabeled EBDN (A and B) or Bgt (C and D) at the indicated final concentrations (abscissas; nanomolar) were processed for 1 h to determine [3H]EBDN binding (ordinate; percentage of control specific binding; n = 2–3). Continuous lines represent fits to the Hill equation (nH = –1). IC50 or log IC50 values and extents of ligand inhibition of [3H]EBDN binding are given in the text and/or in Table 1.

View this table: [in this window] [in a new window]   TABLE 2 Parameters [log IC50 values (±S.E.M. in boldface) above Hill coefficients (±S.E.M. in italics)] for drug competition toward specific [3H]EBDN or I-Bgt binding to h7-nAChR in SH-EP1-pCEP4-h7 cells Radioligand binding competition assays were conducted as described under Materials and Methods and in the legends to Figs. 11 and 12. Results were fit to the logistic equation to determine Hill coefficients and log IC50 values.

Other radioligand binding competition studies showed that specific I-Bgt binding at 10 nM to sites on SH-EP1-Toff-h7 cells (heterologously expressing h7-nAChRs) was inhibited by EBDN (IC₅₀ = 14 nM; Fig. 9B; Table 1). Moreover, all specific [³H]EBDN binding sites at 1 nM on SH-EP1-Toff-h7 cells were blocked by Bgt with an IC₅₀ value of 47 nM (Fig. 10D; Table 1). Similar studies demonstrated that all I-Bgt binding sites in SH-SY5Y cells transfected with either human or rat 7 subunits or in wild-type SH-SY5Y cells were blocked by Bgt (IC₅₀ values of 21, 20, and 26 nM, respectively; Fig. 9, C and D; Table 1). All I-Bgt binding sites on these cells were also blocked by EBDN (IC₅₀ values of 22, 36, and 17 nM, respectively, for SH-SY5Y-pCEP4-h7, SH-SY5Y-pCEP4-r7, or wild-type SH-SY5Y cells; Fig. 9, A and B; Table 1). Whereas all specific [³H]EBDN binding sites on these cells were blocked by EBDN (IC₅₀ values of 7.0, 8.4, and 2.8 nM, respectively, for SH-SY5Y-pCEP4-h7, SH-SY5Y-pCEP4-r7, or wild-type SH-SY5Y cells; Fig. 10, A and B), only a subset of those [³H]EBDN binding sites were blocked by Bgt in transfected SH-SY5Y cells (56% inhibition and 9.3 nM IC₅₀ value for SH-SY5Y-pCEP4-h7 cells; Fig. 10C; 35% inhibition and 8.2 nM IC₅₀ value for SH-SY5Y-pCEP4-r7 cells; Fig. 10D) or in wild-type SH-SY5Y cells (only 6% inhibition; Fig. 10D; Table 1). The remaining sites that were insensitive to Bgt block would correspond to naturally expressed 3*-nAChRs characterized in these cells (Lukas et al., 1993; Ke et al., 1998). Similarly, studies done using rat brain indicated that nearly all I-Bgt binding sites are blocked by EBDN (87% inhibition, 6.7 nM IC₅₀ value; Fig. 9A), whereas all were blocked by Bgt (55 nM IC₅₀ value; Fig. 9C; Table 1). All [³H]EBDN binding sites in rat brain preparations were blocked by EBDN (2.2 nM IC₅₀ value; Fig. 10A), but only a small percentage (8%) were blocked by Bgt (38 nM IC₅₀ value; Fig. 10C; Table 1).

Additional studies showed that proportions of Bgt-sensitive [³H]EBDN binding sites in membranes from rat brain, SH-SY5Y-pCEP4-r7, or wild-type SH-SY5Y cells were higher for assays done in the presence of 10 nM [³H]EBDN than in the presence of 1 nM [³H]EBDN (Table 1). These findings indicated that Bgt-insensitive, higher affinity binding sites for [³H]EBDN (K_D values of 10 pM for 4*-nAChRs in rat brain or K_D values of 100 pM for 3*-nAChRs naturally found in SH-SY5Y cells) were already saturated when assays were done using 1 nM [³H]EBDN, but only a fraction of Bgt-sensitive, lower affinity sites (K_D value of 1 nM) for [³H]EBDN corresponding to rat or human 7-nAChRs in these preparations were labeled at 1 nM [³H]EBDN. By contrast, Bgt-insensitive 3*- or 4*-nAChR binding sites remained saturated in the presence of 10 nM [³H]EBDN, but a higher fraction of Bgt-sensitive 7-nAChRs became labeled when assay [³H]EBDN concentrations were increased from 1 to 10 nM, and the proportion of [³H]EBDN binding sites corresponding to Bgt-insensitive, lower affinity 7-nAChRs also increased. Other studies demonstrated that -cobratoxin also inhibited specific [³H]EBDN (1 nM) binding to these preparations to the same extent as did Bgt (inhibition by 88 ± 9% for SH-EP1-pCEP4-h7 cells, 57 ± 6% for SH-SY5Y-pCEP4-h7 cells, 37 ± 5% for SH-SY5Y-pCEP4-r7 cells, and 5 ± 3% for rat brain; data summarized but not shown).

Similar Pharmacological Profiles for Epibatidine and -Bungarotoxin Binding Sites on h7-nAChRs. Another series of radioligand binding competition studies revealed that specific [³H]EBDN binding sites on heterologously expressed h7-nAChRs in SH-EP1-pCEP4-h7 cells have a nicotinic pharmacological profile (Fig. 11; Table 2). Rank order inhibitory potency and IC₅₀ values for ligands acting to block [³H]EBDN binding were 0.047 µM methyllycaconitine (MLA) > 0.27 µM 1,1-dimethy-4-phenyl-piperazinium (DMPP) > 0.43 µM nicotine > 0.89 µM cytisine > 5.9 µM carbamylcholine 7.1 µM d-tubocurarine. This profile closely matched rank order inhibition potency and IC₅₀ values for nicotinic ligand competition toward specific I-Bgt binding to SH-EP1-pCEP4-h7 cell membranes of: 0.035 µM MLA > 0.087 µM DMPP > 0.91 µM cytisine 1.0 µM nicotine > 7.2 µM d-tubocurarine 8.3 µM carbamylcholine (Fig. 12; Table 2; although "small drug-insensitive but Bgt-sensitive" I-Bgt binding sites were evident, perhaps representing incompletely assembled receptors). These profiles also are like those for blockade of specific I-Bgt binding to naturally expressed h7-nAChRs found in SH-SY5Y human neuroblastoma cells (Lukas et al., 1993) or heterologously expressed h7-nAChRs in SH-EP1-Toff-h7 cells (Peng et al., 1999). Moreover, these findings are consistent with at least some overlap between Bgt, EBDN, and other small drug binding sites on h7-nAChRs. Lending further support to the notion that both naturally expressed 3*-nAChRs and either naturally or a combination of naturally and heterologously expressed 7-nAChRs are found on wild-type or transfected SH-SY5Y cells, MLA competition toward specific [³H]EBDN binding was best fit by a two-site model for either wild-type SH-SY5Y cells or SH-SY5Y-pCEP4-h7 cells (data not shown). High-affinity (IC₅₀ value of 30 nM) blockade occurred for about 85% of specific [³H]EBDN binding sites on SH-SY5Y-pCEP4-h7 cells and for about 34% of the sites on wild-type SH-SY5Y cells, whereas the remaining sites were inhibited by MLA with lower affinity (IC₅₀ value of 3 µM). The sites with high affinity for MLA that are expressed at higher levels in transfected SH-SY5Y cells would seem to be h7-nAChRs, whereas the lower affinity MLA sites would seem to be 3*-nAChRs.

View larger version (24K): [in this window] [in a new window]   Fig. 11. Drug competition profiles for blockade of specific [3H]EBDN binding to sites on SH-EP1-pCEP4-h7 cells. Reaction mixtures containing SH-EP1-pCEP4-h7 cell membrane preparations (typically containing 28 µg of protein), 1 nM [3H]EBDN, and the indicated drugs were used to assess the concentration dependence (abscissa; molar, log scale) for competition toward specific [3H]EBDN binding (ordinate; percentage of control). Results are the averages three separate experiments. Top, competition profiles for DMPP (), cytisine (cyt; ), or d-tubocurarine (d-TC; ). Bottom, competition profiles for MLA (), nicotine (nico; ), or carbamylcholine (carb; ). Log IC50 values and Hill coefficients (±S.E.M.) are provided in Table 2, and IC50 values are indicated in the text. Upper and lower limits, respectively, for sigmoid curves were 102 ± 3 and 1 ± 3% for MLA, 97 ± 5 and 3 ± 4% 1 for DMPP, 103 ± 4 and 1 ± 4% for nicotine, 100 ± 1 and 1 ± 1% for cytisine, 101 ± 2 and 3 ± 4% for d-tubocurarine, and 97 ± 2 and –1 ± 3% for carbamylcholine (not different from 100 or 0% at the 95% confidence interval).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The major findings of this study are that human nAChR 7 subunit CDNAs introduced into native nAChR-null SH-EP1 human epithelial cells lead to production of human 7 subunit message and protein and that subunits assemble to form functional h7-nAChRs that have high affinity for the radioligands [³H]EBDN and I-Bgt. As we observed in previous studies using a different expression vector (Peng et al., 1999), heterologously expressed h7-nAChR are found on the cell surface and undergo nicotine exposure-induced increases in expression measured as membrane-bound radioligand binding sites. Based on several criteria, mutually exclusive binding of unlabeled or radiolabeled EBDN or Bgt occurs to a single class of sites on heterologously expressed h7-nAChRs.

Although we cannot formally exclude the possibility that entities composed of 7 subunits plus other endogenous components could give rise to [³H]EBDN-binding sites, Bgt and EBDN binding profiles for h7-nAChRs expressed in SH-EP1-pCEP4-h7 cells match those for other preparations heterologously or naturally expressing h7-nAChRs or rat 7-nAChRs but using I-Bgt as a radioprobe (Peng et al., 1994, 1997, 1999; Gopalakrishnan et al. 1995). High sensitivities to MLA and DMPP seem to be distinguishing characteristics of human nAChR composed of 7 as opposed to other subunits. The current finding that EBDN inhibits binding of I-Bgt or [³H]EBDN to h7-nAChR with higher affinity than Bgt suggests that although EBDN binds with higher affinity to 3*- or 4*-nAChR than to 7-nAChR, it can be used as a radioligand for 7-nAChRs. Furthermore, autoradiographic studies and radioligand binding studies using [³H]EBDN and brain or other preparations containing a mixture of nAChR subtypes need to consider interactions with 7-nAChRs.

Functional studies using heterologous or natural expression systems indicated that EBDN is a reasonably potent agonist at 7-nAChRs (see Zhao et al., 2001, 2003, for results of additional studies of functional h7-nAChRs in SHEP1-pCEP4-h7 cells). The functional EC₅₀ found for (–)- or (+)-EBDN at human 7-nAChRs heterologously expressed in Xenopus oocytes was 1.1 to 1.2 µM (Gerzanich et al., 1995). Briggs et al. (1995) reported that functional potency for (±)-EBDN was 64-fold higher than that for (–)-nicotine (EC₅₀ values = 1.30 ± 0.11 and 83 ± 10 µM, respectively) for h7-nAChRs heterologously expressed in oocytes. Alkondon and Albuquerque (1995) reported that EBDN elicited type IA currents presumably mediated by 7-nAChRs in rat hippocampal neurons grown in culture [EC₅₀ values of 2.9 and 4.3 µM for (–)- and (+)-enantiomers, respectively]. It is widely appreciated that nicotinic agonists show higher affinities for many nAChR subtypes when assessed using radioligand binding saturation or competition assays than when assessed based on acute functional potencies. This presumably reflects higher affinity agonist interactions with (or stabilization of) nAChRs in a functionally desensitized compared with lower affinity agonist interactions with nAChRs in a functionally ready state. However, it was not appreciated that the affinity difference would be 1000-fold for 7-nAChR interactions with EBDN, although a 3 order of magnitude difference in binding and functional affinities for EBDN also is a feature of human 42-nAChRs (Eaton et al., 2003). The likelihood that these findings reflect differences in EBDN binding affinity of recombinant 7-nAChRs heterologously expressed in oocyte as opposed to SH-EP1 cells is diminished because of the findings using rat hippocampal neurons.

IC₅₀ values for unlabeled EBDN or Bgt homologous competition toward 1 nM [³H]EBDN or 10 nM I-Bgt binding, respectively, were 3 and 30 nM, and application of the Cheng-Prusoff correction (radioligand concentrations were 2–3 times higher than their K_D values as determined in radioligand binding saturation analyses) thus brings radioligand binding homologous competition K_i values much closer to K_D values, but not completely. Commercially provided radioligand specific activities and/or concentrations are not always as represented, possibly confounding accurate determinations of radioligand concentration, but there is little reason to question the purity and stock solution concentrations of unlabeled homologous competitors. We also have noted that ligand depletion is a factor in some of our experiments using more concentrated 7-nAChRs, in that homologous competition of unlabeled EBDN for 3 nM [³H]EBDN binding gives IC₅₀ values of 3 nM also. Nevertheless, homologous competition studies serve to calibrate heterologous competition studies. Thus, they show that both Bgt and EBDN are slightly less effective in heterologous competition than in homologous competition studies, possibly suggesting imperfect overlap of binding domains for both ligands. Other than a possibility that there are separate sites interrogated by radioagonist and radiotoxin binding, an alternative explanation would be that Bgt stabilizes a conformer(s) of 7-nAChR that has lower affinity for EBDN than does the conformer(s) stabilized by EBDN and that the converse is also true. The small differences in other ligand apparent affinities for EBDN or Bgt binding sites could also be explained by this circumstance. Interestingly, Gerzanich et al. (1995) found that K_i values for heterologous competition for I-Bgt binding to immunoimmobilized chick 7-nAChR were 590 and 350 nM for (+)- or (–)-EBDN, respectively, and were 9.8 and 3.1 nM, respectively, for competition toward I-Bgt binding to immunoimobilized human 7-nAChR naturally expressed by SH-SY5Y cells [although functional EC₅₀ values for (+)- or (–)-EBDN were only 2-fold higher for chick 7-nAChR than for h7-nAChR]. This suggests that radioligand binding and functional features of 7-nAChR from different species may differ. However, the K_i values for EBDN competition toward SH-SY5Y h7-nAChR I-Bgt binding are close to those observed in the present study.

Recent studies on analgesic, locomotor, hypothermic, cardiovascular, and metabolic activity of EBDN indicate the potential importance of it and related compounds in studies targeting nAChRs for therapeutic purposes (Damaj et al., 1994; Levin and Rosecrans, 1994). nAChRs exist as diverse subtypes with unique distributions and pharmacological profiles. However, profiling of nAChRs of defined compositions is necessary toward a complete understanding of these important entities and toward rational nicotinic drug design. The current studies reinforce the use of the SH-EP1 cell line as a host for heterologous expression of specific nAChR subtypes of defined subunit composition and as models for clarification of the physiological roles and therapeutic potential of nAChR ligands, but they also indicate that 7-nAChRs should be considered as targets of EBDN and related compounds.

	Acknowledgements

 
We thank Dr. June Biedler and Barbara Spengler for SH-EP1 and SH-SY5Y cells.

	Footnotes

 
The work done in Phoenix was supported by endowment and capitalization funds from the Men's and Women's Boards of the Barrow Neurological Foundation and Epi-Hab Phoenix, Inc., by the Arizona Disease Control Research Commission (Grants 9730 and 9615), the National Institutes of Health (Grant NS40417), and the Council for Tobacco Research–U.S.A., Inc. (Grant 4366). Work done in Denver was supported by the Veterans Affairs Medical Research Service and National Institutes of Health Grants DA9457 and DA12241. The contents of this report are solely the responsibility of the authors and do not necessarily represent the views of the aforementioned awarding agencies. This article is dedicated to the memory of Jian-Hong Peng and Michelle Zhang. Portions of this work were presented previously [Peng J-H, Leonard SS, and Lukas RJ (1998) Heterologous expression of epibatidine- and -bungarotoxin-binding human 7-nicotinic acetylcholine receptor in a native receptor-null human epithelial cell line. Soc Neurosci Abstr 24:831].

doi:10.1124/jpet.104.079004.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; 3*-nAChR or 4*-nAChR, accepted nomenclature for nAChR containing 3 or 4 subunits, respectively, as well as other nAChR subunits; Bgt, -bungarotoxin; h7, human 7 subunit(s); h7-nAChR, human 7-nicotinic acetylcholine receptor(s); I-Bgt, ¹²⁵I-labeled -bungarotoxin; PBS, phosphate-buffered saline; MLA, methyllycaconitine; DMPP, 1,1-dimethyl-4-phenyl-piperazinium; EBDN, epibatidine.

¹ Current address: Division of Biology and Medical Sciences, Washington University, St. Louis, MO.

² Current address: Pfizer, Groton, CT.

³ Current address: Phoenix Epidemiology and Clinical Research Branch, National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases, Phoenix, AZ.

⁴ Current address: Institute for Neuroscience, University of Texas at Austin, Austin, TX.

⁵ Current address: College of Medicine, University of Arizona, Tucson, AZ.

⁶ Current address: Pfizer, Kalamazoo, MI.

Address correspondence to: Dr. R. J. Lukas, Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013. E-mail: rlukas{at}chw.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Alkondon M and Albuquerque EX (1995) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. III. Agonist actions of the novel alkaloid epibatidine and analysis of type II current. J Pharmacol Exp Ther 274: 771–782.[Abstract/Free Full Text]

Badio B and Daly JW (1994) Epibatidine, a potent analgetic and nicotinic antagonist. Mol Pharmacol 45: 563–569.[Abstract]

Bencherif M, Fowler K, Lukas RJ, and Lippiello PM (1995) Mechanisms of upregulation of neuronal nicotinic acetylcholine receptors in clonal cell lines and primary cultures of fetal rat brain. J Pharmacol Exp Ther 275: 987–994.[Abstract/Free Full Text]

Bencherif M and Lukas RJ (1993) Cytochalasin modulation of nicotinic cholinergic receptor expression and muscarinic receptor function in human TE671/RD cells: a possible functional role of the cytoskeleton. J Neurochem 61: 852–864.[Medline]

Biedler JL, Helson L, and Spengler BA (1973) Morphology and growth, tumorigenicity and cytogenetics of human neuroblastoma cells in continuous cell culture. Cancer Res 33: 2643–2652.[Abstract/Free Full Text]

Breese CR, Adams C, Logel J, Drebing C, Rollins Y, Barnhart M, Sullivan B, DeMasters BK, Freedman R, and Leonard S (1997) Comparison of the regional expression of nicotinic acetylcholine receptor 7 mRNA and [¹²⁵I]--bungarotoxin binding in human postmortem brain. J Comp Neurol 387: 385–398.[CrossRef][Medline]

Briggs CA, McKenna DG, and Piattoni-Kaplan M (1995) Human alpha 7 nicotinic acetylcholine receptor responses to novel ligands. Neuropharmacology 34: 583–590.[CrossRef][Medline]

Chen D and Patrick JW (1997) The -bungarotoxin-binding nicotinic acetylcholine receptor from rat brain contains only the 7 subunit. J Biol Chem 272: 24024–24029.[Abstract/Free Full Text]

Clementi F, Fornasari D, and Gotti C (2000) Neuronal Nicotinic Receptors Handbook of Experimental Pharmacology, vol 144, Springer, Heidelberg.

Couturier S, Bertrand D, Matter JM, Hernandez MC, Bertrand S, Millar N, Valera S, Barkas T, and Ballivet M (1990) A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX. Neuron 5: 847–856.[CrossRef][Medline]

Daly JW (1995) The chemistry of poisons in amphibian skin. Proc Natl Acad Sci USA 92: 9–13.[Abstract/Free Full Text]

Eaton JB, Peng J-H, Schroeder KM, George AA, Fryer JD, Krishnan C, Buhlman L, Kuo Y-P, Steinlein O, and Lukas RJ (2003) Characterization of human 42-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells. Mol Pharmacol 64: 1283–1294.[Abstract/Free Full Text]

Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, and Boulter J (2001) 10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc Natl Acad Sci USA 98: 3501–3506.[Abstract/Free Full Text]

Gerzanich V, Peng X, Wells G, Anand R, Fletcher S, and Lindstrom J (1995) Comparative pharmacology of epibatidine: a potent agonist for neuronal nicotinic acetylcholine receptors. Mol Pharmacol 48: 774–782.[Abstract]

Gopalakrishnan M, Buisson B, Touma E, Giordano T, Campbell JE, Hu IC, Donnelly-Roberts D, Arneric SP, Bertrand D, and Sullivan JP (1995) Stable expression and pharmacological properties of the human 7 nicotinic acetylcholine receptor. Eur J Pharmacol 290: 237–246.[CrossRef][Medline]

Houghtling RA, Davila-Garcia MI, Hurt SD, and Kellar KJ (1994) [³H]Epibatidine binding to nicotinic cholinergic receptors in brain. Med Chem Res 4: 538–546.

Houghtling RA, Davila-Garcia MI, and Kellar KJ (1995) Characterization of (+)-[³H]epibatidine binding to nicotinic cholinergic receptors in rat and human brain. Mol Pharmacol 48: 280–287.[Abstract]

Ke L, Eisenhour CM, Bencherif M, and Lukas RJ (1998) Effects of chronic nicotine treatment on expression of diverse nicotinic acetylcholine receptor subtypes. I. Dose- and time-dependent effects of nicotine treatment. J Pharmacol Exp Ther 286: 825–840.[Abstract/Free Full Text]

Leonard S and Bertrand D (2001) Neuronal nicotinic receptors: from structure to function. Nicotine Tobacco Res 3: 203–223.

Levin ED and Rosecrans JA (1994) The promise of nicotinic-based therapeutic treatments. Drug Dev Res 31: 1–2.

Lindstrom J (1996) Neuronal nicotinic acetylcholine receptors, in Ion Channels (Narahashi T ed) vol 4, pp 377–450, Plenum Press, New York.[Medline]

Lukas RJ (1984) Properties of curaremimetic neurotoxin binding sites in the rat central nervous system. Biochemistry 23: 1152–1160.[CrossRef][Medline]

Lukas RJ (1986) Characterization of curaremimetic neurotoxin binding sites on membrane fractions derived from the human medulloblastoma clonal line, TE671. J Neurochem 46: 1936–1941.[Medline]

Lukas RJ (1990) Heterogeneity of high affinity nicotinic ³H-acetylcholine binding sites. J Pharmacol Exp Ther 253: 51–57.[Abstract/Free Full Text]

Lukas RJ (1998) Neuronal nicotinic acetylcholine receptors, in The Nicotinic Acetylcholine Receptor: Current Views and Future Trends (Barrantes FJ ed) pp 145–173, Springer, Heidelberg and RG Landes Co., Georgetown, TX.

Lukas RJ, Changeux J-P, Le Novère N, Albuquerque EX, Balfour DJK, Berg DK, Bertrand D, Chiappinelli VA, Clarke PBS, Collins AC, et al. (1999) International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev 51: 397–401.[Abstract/Free Full Text]

Lukas RJ, Norman SA, and Lucero L (1993) Characterization of nicotinic acetylcholine receptors expressed by cells of the SH-SY5Y human neuroblastoma clonal line. Mol Cell Neurosci 4: 1–12.

McGehee DS and Role LW (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu Rev Physiol 57: 521–546.[CrossRef][Medline]

Peng J-H, Lucero L, Fryer J, Herl J, Leonard SS, and Lukas RJ (1999) Inducible, heterologous expression of human 7-nicotinic acetylcholine receptors in a native nicotinic receptor-null human clonal line. Brain Res 825: 172–179.[CrossRef][Medline]

Peng X, Gerzanich V, Anand R, Wang F, and Lindstrom J (1997) Chronic nicotine treatment up-regulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y. Mol Pharmacol 51: 776–784.[Abstract/Free Full Text]

Peng X, Katz M, Gerzanich V, Anand R, and Lindstrom J (1994) Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes. Mol Pharmacol 45: 546–554.[Abstract]

Puchacz E, Buisson B, Bertrand D, and Lukas RJ (1994) Functional expression of nicotinic acetylcholine receptors containing rat 7 subunits in human SH-SY5Y neuroblastoma cells. FEBS Lett 354: 155–159.[CrossRef][Medline]

Sargent PB (1993) The diversity of neuronal nicotinic acetylcholine receptors. Annu Rev Neurosci 16: 403–443.[Medline]

Seguela P, Wadiche J, Dineley-Miller K, Dani JA, and Patrick JW (1993) Molecular cloning, functional properties and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J Neurosci 13: 596–604.[Abstract]

Zhao L, Kuo Y-P, George AA, Peng J-H, Purandare MS, Schroeder KM, Lukas RJ, and Wu J (2003) Functional properties of homomeric, human 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line. J Pharmacol Exp Ther 305: 1132–1141.[Abstract/Free Full Text]

Zhao L, Lukas RJ, and Wu J (2001) Electrophysiological characteristics of heterologously expressed human 7-nicotinic receptors. Soc Neurosci Abstr 27: 375.

This article has been cited by other articles:

				S. Razani-Boroujerdi, R. T. Boyd, M. I. Davila-Garcia, J. S. Nandi, N. C. Mishra, S. P. Singh, J. C. Pena-Philippides, R. Langley, and M. L. Sopori T Cells Express {alpha}7-Nicotinic Acetylcholine Receptor Subunits That Require a Functional TCR and Leukocyte-Specific Protein Tyrosine Kinase for Nicotine-Induced Ca2+ Response J. Immunol., September 1, 2007; 179(5): 2889 - 2898. [Abstract] [Full Text] [PDF]

				J. Wu, Q. Liu, K. Yu, J. Hu, Y.-P. Kuo, M. Segerberg, P. A. St John, and R. J. Lukas Roles of nicotinic acetylcholine receptor {beta} subunits in function of human {alpha}4-containing nicotinic receptors J. Physiol., October 1, 2006; 576(1): 103 - 118. [Abstract] [Full Text] [PDF]

				M. J. Marks, P. Whiteaker, and A. C. Collins Deletion of the {alpha}7, beta2, or beta4 Nicotinic Receptor Subunit Genes Identifies Highly Expressed Subtypes with Relatively Low Affinity for [3H]Epibatidine Mol. Pharmacol., September 1, 2006; 70(3): 947 - 959. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.079004v1 313/1/24    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peng, J.-H. Articles by Lukas, R. J. Search for Related Content PubMed PubMed Citation Articles by Peng, J.-H. Articles by Lukas, R. J.