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CELLULAR AND MOLECULAR

Functional Properties of Homomeric, Human 7-Nicotinic Acetylcholine Receptors Heterologously Expressed in the SH-EP1 Human Epithelial Cell Line

Divisions of Neurology, Barrow Neurological Institute, Phoenix, Arizona (L.Z., J.W.) and Neurobiology (Y.-P.K., A.A.G., J.-H.P., M.S.P., K.M.S., R.J.L.), Barrow Neurological Institute, Phoenix, Arizona

Received January 3, 2003; accepted March 3, 2003.

	Abstract

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7-Nicotinic acetylcholine receptors (7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human 7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human 7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional 7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human 7-nAChRs. Current-voltage relationships show inward rectification for agonist-induced currents. Human 7-nAChRs exhibit some sensitivity to 7-nAChR antagonists -bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and 7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of 7-nAChR function. Whole-cell current peak amplitudes and half-times for inactivation of 7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca²⁺, suggestive of high Ca²⁺ permeability of the 7-nAChR channel. Thus, heterologously expressed human 7-nAChR in mammalian cells have properties of native 7-nAChR or of 7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of 7-nAChR function.

It is now acknowledged that nAChRs in the brain or autonomic nervous system that bind the curaremimetic neurotoxin -bungarotoxin (Bgt) represent a unique class within the nAChR family and may have functions distinct from the mediation of classical excitatory neurotransmission (Lukas, 1998; Elgoyhen et al., 2001). These Bgt-binding nAChRs were known for years to share many features with other nAChRs, such as 1) a unique but distinctively nicotinic pharmacology, 2) residues that can be affinity labeled with nicotinic ligands, and 3) ability to undergo agonist interaction-induced changes in ligand binding affinity state that are biochemically analogous to physiologically relevant transitions of nAChR from native to active to functionally desensitized states (Clarke, 1992; Lukas and Bencherif, 1992). Critical breakthroughs in our understanding of Bgt-binding nAChRs were the cloning of what is now know as the chick nAChR 7 subunit gene, and technical improvements allowing the detection of transient and novel functional responses of nAChRs made up of 7 subunits (Couturier et al., 1990; Schoepfer et al., 1990). The protein products of chick, rat, or human 7 genes can form homopentameric complexes in Xenopus oocytes that can bind curaremimetic neurotoxins and can mediate very short-lived (lifetimes of tens of milliseconds), nicotine-gated, toxin-sensitive ion channel responses with high Ca²⁺ permeability (Couturier et al., 1990; Li et al., 1993; Seguela et al., 1993; Peng et al., 1994; Palma et al., 1996). Because of their homomeric nature, these 7-nAChRs and related chimeric receptors have proven to be useful in oocyte expression- and mutagenesis-based studies that have provisionally identified structures and residues contributing to ligand binding, subunit interactions, and/or lining of the ligand-gated ion channel (Bertrand et al., 1993; Galzi and Changeux, 1994; Garcia-Guzman et al., 1994; Corringer et al., 1995, 1998; Campos-Caro et al., 1996). By virtue of their unique subcellular localizations, channel kinetics, and Ca²⁺ permeability, 7-nAChRs seem to have novel functional roles in processes such as vicinal control of neurotransmitter release (Lukas, 1998), structuring and maintenance of neurites and synapses (Freeman, 1977; Chan and Quik, 1993; Pugh and Berg, 1994), long-term potentiation (Hunter et al., 1994; Morales et al., 1994), and neuronal viability/death (Renshaw, 1994; Hory-Lee and Frank, 1995; Renshaw and Dyson, 1995; Treinin and Chalfie, 1995).

Previously, our laboratory provided the first report of the successful heterologous expression of functional 7-nAChRs in mammalian cells (Puchacz et al., 1994a,b). Work by us and others has shown that additional cell types can be suitable hosts for expression of 7 subunit transgenes as functional 7-nAChRs (Gopalakrishnan et al., 1995; Puchacz et al., 1995; Lukas et al., 1996; Quik et al., 1996; Peng et al., 1998, 1999). The present study concerns functional characterization of human 7-nAChRs heterologously expressed in the native nAChR-null SH-EP1 human epithelial cell line (see a preliminary report of some of these findings by Zhao et al., 2001).

	Materials and Methods

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Expression of Human 7-nAChRs in SH-EP1 Human Epithelial Cells. Cells of the SH-EP1 human epithelial cell line (kindly provided by Dr. June Biedler, Sloan Kettering Institute for Cancer Research, New York, NY) 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). Cells were transfected using electroporation with cDNA encoding a human 7 subunit (Breese et al., 1997) excised from pcDNAI and subcloned into pCEP4 (Invitrogen; Peng et al., 1998). Positive transfectants were isolated after supplementation of medium 48 h later with 0.25 mg/ml hygromycin, and ring cloning was used to isolate single, transfected cell colonies. The clonal line used for most of the studies described herein stably expressed high quantities of binding sites for radiolabeled Bgt and was named the SH-EP1-h7 line. This cell line has been used for about 5 years and for continuous passages as long as 1 year, and although there is fluctuation for reasons that we do not fully understand in levels of 7-nAChR expression as measured by radioligand binding and current density measurements, there has been no systematic change in these parameters with time, indicating that the cell line is indeed stably transfected.

RNA Preparation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Total cytoplasmic RNA was isolated from cells growing at approximately 80% confluence in a 100-mm culture dish using 2 ml of TRIzol reagent (Bethesda Research Laboratories, Gaithersburg, MD). Before RT-PCR, RNA preparations were treated with DNase I (Ambion, Austin, TX) to remove residue genomic DNA contamination. Typically, 40 µg of RNA was incubated with 4 units of DNase I in a 50-µl reaction at 37°C room temperature for 30 min, and then the DNase I was inactivated by addition of 5 µl of 25 mM EDTA and incubation at 65°C for 10 min. RT was carried out using 2 µg of DNA-free total RNA, oligo d(T)_12–18 primer, and the Superscript II preamplification system (Bethesda Research Laboratories) in a 20-µl reaction. At the end of the RT reaction, reverse transcriptase was deactivated by incubation at 75°C for 10 min, and RNAs were removed by adding 1 unit of RNase H followed by incubation at 37°C for 30 min. A reaction excluding reverse transcriptase was also conducted as RT negative control. PCR was performed using 1 µl of cDNA preparation, 1 µl of 10 µM each of 5' and 3' gene-specific primers, 1 µl of 10 mM dNTP, and 2.5 units of RedTaq (Sigma-Aldrich, St. Louis, MO) in a 50-µl reaction. The primers used in the amplification stage were designed and synthesized based on published gene sequences (GenBank accession no. 7, X70297 [GenBank] , and GAPDH, M32599 [GenBank] ). The primer sequences and their predicted product sizes are as follows: 7 sense, 5'-gttctatgagtgctgcaaagagcc-3'; 7 antisense, 5'-ctccacactggccaggctgcag-3' (product size 497 bp); GAPDH sense, 5'-cgtattgggcgcctggtcaccag-3'; and GAPDH antisense, 5'-gtccttgcccacagccttggcagc-3' (product size 624 bp). Amplification reactions were carried out in a RoboCycler (Stratagene, La Jolla, CA) for 35 amplification cycles at 95°C for 1 min, 55°C for 90 s, and 72°C for 90 s, followed by an additional 4-min extension at 72°C. One-tenth of each RT-PCR product was then resolved on a 1% agarose gel, and sizes or products were determined based on migration relative to mass markers loaded adjacently.

In Situ Hybridization for Cell-Specific Localization of 7 Subunit Message. nAChR 7 subunit messenger RNA primers (forward, 5'-gaggacaaggtgcgcccggc-3'; reverse, 5'-tcctgcacggcgggcaaccc-3') were used to generate PCR products corresponding to a nucleotide sequence coding for a segment of the cytoplasmic domain conserved across human, mouse, or rat 7 subunit sequences but distinct from other gene sequences. PCR products were purified by 1% agarose gel electrophoresis, which also confirmed product size and provided estimates for quantity of DNA. These purified, 7 subunit cytoplasmic domain templates were nondirectionally ligated (Lig'n Scribe; Ambion) to a T7 phage promoter adapter. Subsequent PCR of ligation products using forward or reverse, gene-specific 7 subunit primers in conjunction with Lig'n Scribe PCR adapter primers was done in separate reactions to obtain antisense or sense orientation transcription templates, respectively. Sense or antisense cRNAs for 7, 232 bp in length, were transcribed incorporating biotinylated UTPs using MAXIscript (Ambion) in vitro transcription.

Confluent SH-EP1-h7 cells were trypsinized, resuspended in medium, seeded on Lab Tek II CC2 chambered slides at a target density of 50,000 cells/chamber, and allowed to grow for 2 days to achieve proper morphology and optimal confluence (90%). To begin the procedure, cells were briefly rinsed in 1x phosphate-buffered saline (PBS); fixed in 4% paraformaldehyde; rinsed again in 1x PBS for 5 min; and acetylated, delipidated with chloroform, and serially dehydrated with ethanol (50, 75, 85, and 95%) at 22°C. Samples were then incubated in prehybridization solution containing final concentrations of 250 µg/ml tRNA, 25% formamide, 10% dextran sulfate, 2.5x Denhardt's solution, 0.05 mg/ml salmon sperm DNA, 4x SSCP (10 mM sodium phosphate, 150 mM NaCl, 1 mM ethylenediamine acid), 4 mM EDTA, pH 8.0, for 2 h at 50°C before being incubated in hybridization solution containing antisense or sense probes (final concentration ranging from 0.2 to 0.7 ng/µl) for 20 h at 50°C. After hybridization, samples were rinsed twice in 2x SSC for 5 min each, 1x SSC for 10 min, and 0.5x SSC for 10 min at 22°C and once in 0.1x SSC for 20 min at 60°C before being briefly rinsed in double distilled water at 22°C. Samples were then dehydrated again in a graded ethanol series and vacuum-dried under desiccant, allowing them to be stored if desired. Dry samples were rehydrated by rinsing in 1x PBS for 5 min at 22°C before submersion in a 1:2000 dilution of avidin-Alexa fluorophore complexes (Molecular Probes, Eugene, OR) in 1x PBS supplemented with 0.1% bovine serum albumin for 30 min at 22°C. To end the reaction, samples were rinsed three times in 1x PBS for 15 min each wash at 22°C. Slides were then coverslipped and stored at 4°C in the dark until being subjected to fluorescence microscopy (IX70; Olympus, Melville, NY) and image analysis using ImagePro Plus version 4.1 (Media Cybernetics, Silver Spring, MD).

Patch-Clamp Whole-Cell Current Recordings. Conventional whole-cell current recording coupled with techniques for fast application and removal of agonist (U-tube system) was applied in this study (Wu et al., 1996, 2002; Wu and Partridge, 1998). Briefly, cells plated on poly-lysine-coated 35-mm culture dishes were placed on the stage of an inverted microscope (IX70; Olympus) and continuously superfused with standard external solution. Glass microelectrodes (3–5-M resistance between pipette and extracellular solutions) were used to form tight seals (>1 G) on the cell surface until suction was applied to convert to conventional whole-cell recording. Cells were then voltage-clamped at holding potentials of -60 mV, and ion currents in response to application of ligands were measured (200B amplifier; Axon Instruments, Inc., Foster City, CA). Both pipette and whole-cell current capacitances were minimized, and the series resistance was routinely compensated to 80%. Whole-cell access resistance less than 20 M was accepted. All experiments were performed at room temperature (22 ± 1°C).

Solutions and Drug Application. The standard external solution contained 120 mM NaCl, 3 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 25 mM D-glucose, 10 mM HEPES, pH 7.4, with Tris base. For conventional whole-cell recording, the "K⁺ electrode" solution contained 140 mM KCl, 4 mM MgSO₄, 0.1 mM EGTA, 4 mM ATP, 10 mM HEPES, pH 7.2, with Tris base. The K⁺-free "Tris electrode" solution contained 110 mM Tris phosphate dibasic, 28 mM Tris base, 11 mM EGTA, 2 mM MgCl₂, 0.1 mM CaCl₂, 4 mM Na-ATP, pH 7.3. To initiate whole-cell current responses, under constant superfusion of the recording chamber, nicotinic drugs were rapidly delivered into the bath medium by a U-tube system, in which the applied drug completely surrounds the recorded cell in less than 20 ms [faster than the typical 10–90% rising time of 12.0 ± 1.6 ms for 100 µM nicotine-induced currents, n = 6, holding potential (V_H) = -60 mV]. Times between drug applications (3 min) were adjusted specifically to ensure stability of nAChR responsiveness (absence of functional rundown), and the K⁺-free pipette solutions used in most of the studies described here was made with the same objective. Drugs used in the present study were (-)-nicotine, acetylcholine (ACh), choline, methylycaconitine (MLA), mecamylamine, and Bgt (Sigma-Aldrich). In experiments using ACh as the agonist, 1 µM atropine sulfate was routinely added to standard solution to exclude any possible influences of muscarinic receptors without affecting [mean (±S.E.M.) peak current amplitudes of 100 µM nicotine-induced responses were 340 ± 48 pA in the absence of atropine and 342 ± 54 pA in 1 µM atropine; n = 10 each paired condition; Tris electrodes; -60 mV holding potential] human 7-nAChR-mediated responses.

Data Acquisition and Analysis. All experimental data were recorded using a 200B amplifier (Axon Instruments, Inc.), typically using data filtered at 2 kHz, acquired at 5 kHz, displayed and digitized on-line (Digidata 1200 series A/D board; Axon Instruments, Inc.), and stored to hard drive. Data acquisition and analyses were done by using pClamp8 (Axon Instruments, Inc.), and results were plotted using Origin 5.0 (Microcal, North Hampton, MA). nAChR acute desensitization was analyzed for decay time (), peak current (I_p), and steady-state current (I_s) using fits to the mono-exponential expression I = [(I_p - I_s) e^-kt] + I_s. There was no significant improvement if data were fit to higher order exponential expressions. Data usually were fit over the period between 90 and 10% of the peak inward current. Curve fitting for sigmoid agonist and antagonist data were performed using Origin (Microcal) or Prism (GraphPad Software Inc., San Diego, CA) and the Hill equation.

	Results

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Expression of Human 7-nAChR in Transfected SH-EP1 Epithelial Cells. RT-PCR studies showed expression of nAChR 7 subunit messages in transfected SH-EP1 cells, but not in wild-type SH-EP1 cells (Fig. 1). In situ hybridization studies indicated that no staining of SH-EP1-h7 cells occurred for samples reacted with sense riboprobe (Fig. 2, a and b) or for wild-type SH-EP1 cells reacted with antisense probe (data not shown), but that >80% of cells displayed high hybridization signal when reacted with antisense 7 subunit riboprobes (Fig. 2, a' and b'). Intracellular distribution of 7 transcript was prominently localized in perinuclear regions, although 7 hybridization could be observed throughout entire cell bodies. There were no consistent differences in apparent levels of 7 subunit transcripts as a function of cell confluence or between grouped or solitary cells.

View larger version (55K): [in this window] [in a new window]   Fig. 1. Expression of nAChR 7 subunit transcripts in the SH-EPI-h7 cell line. RT-PCR analysis of products of GAPDH or nAChR 7 subunit messenger RNAs (see labels at bottom of figure) from transfected SH-EPI-h7 cells or from untransfected SH-EP1 host cells (see labels at top of figure) were obtained as described under Materials and Methods and electrophoretically resolved on a 1% agarose gel. Also shown in the first lane are bands from a 100-bp DNA ladder as molecular size markers. The RT (-) control sample in the last lane was derived using SH-EPI-h7 cell total RNA in the absence of reverse transcriptase followed by PCR using nAChR 7 subunit-specific primers.

View larger version (27K): [in this window] [in a new window]   Fig. 2. In situ hybridization showing expression of nAChR 7 subunit transcripts in the SH-EP1-h7 cell line. Following the protocol described under Materials and Methods, SH-EP1-h7 cells were probed either with biotinylated 7 sense-orientation riboprobe (a and b) or with biotinylated antisense 7 riboprobe (a' and b') before being labeled with avidin-Alexa 488 and subjected to epifluorescence microscopic analysis. Images were obtained at 200x (a and a') or 600x (b and b') magnification (calibration bars are 50 µm).

Functional screening using whole-cell current recording confirmed that functional responses to the nAChR agonist choline are evident in transfected SH-EP1-h7 cells, but not in untransfected, wild-type SH-EP1 cells (Fig. 3), indicating that transfected cells express functional human 7-nAChRs.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Functional responses of 7-nAChR in transfected SH-EP1-h7 cells. Wild-type, untransfected SH-EP1 cells (A) or SH-EP1-h7 cells heterologously expressing human 7 subunits (B) were tested for whole-cell current responses to 10 mM choline applied for 1 s (horizontal calibration bar, 1 s) using the protocol described under Materials and Methods. No response was observed in wild-type cells, but an 500-pA peak response was rapidly induced and inactivated in transfected cells (vertical calibration bar, 200 pA; VH = -60 mV; Tris electrode).

Nicotine-Induced Currents in 7-nAChR-Expressing SH-EP1 Cells. Experiments were performed using conventional whole-cell recording in the voltage-clamp mode at a V_H of -60 mV. If patch pipettes contained 140 mM KCl (K⁺ electrode), the extracellular application of 100 µM nicotine to 7-nAChR-expressing transfected SH-EP1 cells induced an inward current (I_nicotine) that exhibited rapid "acute desensitization", defined as a decline in inward current amplitude during the brief period of nicotine application (Fig. 4A). To induce clear functional rundown, repeated exposures to nicotine for periods of 4 s at intervals of 3 min were applied. The peak I_nicotine showed "functional rundown", defined as a loss of peak current amplitude with each, repeated application of agonist, with time (Fig. 4, A and C). Upon removal of internal KCl by use of K⁺-free Tris electrodes substituting Tris and Tris phosphate dibasic for KCl in the recording pipette while maintaining comparable or identical concentrations of free Ca²⁺ or ATP, functional rundown of I_nicotine was eliminated (Fig. 4, B and C). Moreover, the decay half-time () for acute desensitization of responses recorded using K⁺ electrodes was 20.8 ± 3.4 ms (n = 6), but was increased to 49.5 ± 3.5 ms (n = 18) when responses were recorded using Tris electrodes. First, these results indicate that heterologously expressed 7-nAChRs exhibit a rapid phase of inactivation, or rapid acute desensitization, that is a hallmark of vertebrate 7-nAChR behavior. Second, the finding that both acute desensitization and functional rundown of heterologously expressed 7-nAChR-mediated currents can be diminished when recorded using Tris electrodes (mechanisms underlying this phenomena are explored in studies to be reported elsewhere) indicates a practical and empirically useful approach to stabilize 7-nAChR function for more extensive study. Therefore, we used Tris electrodes for the bulk of the studies presented here.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Rundown of nicotine-induced currents mediated by human 7-nAChRs. A, whole-cell current recording of human 7-nAChR responses measured as described under Materials and Methods using K+ electrodes and 4-s exposure (only show initial 1 s) to 100 µM nicotine at 3-min intervals (six successive responses shown by successive traces) shows decay of peak current amplitude. B, with the same procedure, no peak current amplitude rundown is observed for recordings made using Tris electrodes (horizontal calibration bar, 500 ms; vertical calibration bars, 0.5 and 1 nA, respectively, for A and B; VH = -60 mV; representative traces from one study are shown). C, summary of results from functional rundown study showing stability of 7 nAChR-mediated peak currents (ordinate; normalized to the initial response induced at time 0 as 100%) induced by repetitive application of nicotine (abscissa; time of nicotine challenge in minutes) measured using Tris electrodes () or decay of peak current amplitude for measurements made using K+ electrodes (; n = 8–10 cells/condition; data points are means ± S.E.M.; **, p < 0.01).

Concentration-Response Relationship for 7-nAChRs. To characterize functional properties of 7-nAChRs, three nAChR agonists, including choline, reported to be a selective or specific agonist for 7-nAChRs (Papke et al., 1996; Albuquerque et al., 1997), were examined. Peak whole-cell, inward current amplitudes induced by each of the three agonists increased with increasing agonist concentration (Fig. 5, A–C), producing sigmoidal concentration-response curves (Fig. 5D). Peak currents normalized to the response to 100 µM nicotine show that ACh, nicotine, and choline are high-efficacy agonists. Concentrations at which half-maximal peak currents were induced (EC₅₀ values) were 40 µM (n = 8) for nicotine and 850 µM (n = 8) for choline (Hill coefficients of 1.34 and 1.33, respectively; r² values for fits to the single-site Hill equation were 0.99 and 1.00, respectively, for nicotine and choline). The EC₅₀ for ACh was 1.1 mM, and the Hill slope was 0.51 when fit to the single-site Hill equation (r² = 1.00). Although the low Hill coefficient was suggestive of effects at two sites or for negative cooperativity, the two-site logistic equation did not provide a better fit to the data. Agonist efficacies for maximal peak currents normalized to those induced by 100 µM nicotine were 121% for nicotine (1 mM), 167% for choline (30 mM), and 94% for ACh (10 mM). Thus, the peak efficacy of choline was higher, but that of ACh was lower, than nicotine peak efficacy.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Agonist concentration-response relationships for human 7-nAChR-mediated peak whole-cell currents. Functional responses of SH-EP1-h7 cells were measured as described under Materials and Methods using Tris electrodes at a holding potential of -60 mV for 500-ms applications at the indicated concentrations (in millimolar units) of ACh (A), nicotine (B), or choline (C) (horizontal calibration bar, 500 ms; vertical calibration bars, 200 pA for A and B and 500 pA for C; representative traces from one study are shown). D, concentration-response relationship curves showing peak current amplitudes (ordinate; *, percentage of response in the same cell to 10 µM nicotine) as a function of dose (abscissa; in micromolar units; log scale) of ACh (), choline (), or nicotine (). Recordings were made from 5 to 12 cells, and data points are means ± S.E.M. Log molar EC50 values (and Hill coefficients) are -4.40 ± 0.09 (1.34 ± 0.34) for nicotine, -3.07 ± 0.05 (1.33 ± 0.20) for choline, and -2.97 ± 0.20 (0.51 ± 0.09) for ACh, and r2 values for fits to the logistic equation are 0.99, 1.00, and 1.00, respectively.

Current-Voltage Relationship for 7-nAChR-Mediated Currents. Current-voltage relationships determined for agonist-induced inward currents in SH-EP1 cells transfected with 7 subunits show clear inward rectification at the potentials more positive than 20 mV for all three agonists (Fig. 6).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Current-voltage relationships for human 7-nAChR function. Whole-cell currents induced by 500-ms applications of 0.1 mM nicotine (A), 1 mM ACh (B), or 10 mM choline (C) were measured in SH-EP1-h7 cells as described under Materials and Methods using Tris electrodes at the indicated holding potentials (horizontal calibration bar, 500 ms; vertical calibration bars, 200 pA; representative traces from one study are shown). D, current-voltage curves showing peak current amplitudes (*, normalized to the response in the same cell at -100 mV) as a function of holding potentials (abscissa; in millivolts) for recordings made from 6 to 12 cells and for data points as means ± S.E.M.

Antagonism of 7-nAChR-Mediated Currents. A reported characteristic feature of 7-nAChR expressed in rat neurons is its selective sensitivity to functional blockade by MLA (Albuquerque et al., 1997). MLA was a relatively weak antagonist of heterologously expressed human 7-nAChR functional responses to nicotine when applied to transfected cells at the same time as nicotine (Fig. 7, A, B, and D), being ineffective up to a concentration of 10 nM. In contrast, if MLA was applied 2 min before and throughout exposure to nicotine, the 7-nAChR functional response was markedly suppressed (Fig. 7, A, C, and D). Functional block was reversed less than 5 min after washout of MLA (Fig. 7A). The concentration of MLA producing half-maximal inhibition of 7-nAChR function (IC₅₀ value) for the pretreatment condition was 1.2 nM, and the Hill coefficient for blockade was 1.8.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Antagonist concentration-response relationships for MLA acting at human 7-nAChRs. Whole-cell responses to 0.1 mM nicotine applied for 3 s were measured alone (first trace in A–C), in the presence of MLA at the indicated concentrations, in the presence of MLA at the indicated concentrations after 2-min pre-exposure to MLA alone at the same dose used for cotreatment with nicotine (see individual representative traces from single studies in A–C), or to nicotine alone 3 min after removal of 3 nM MLA (A, right trace). Responses were measured using Tris electrodes at a holding potential of -60 mV (horizontal calibration bar, 1 s; vertical calibration bars, 100 pA for A and 200 pA for B and C). D, MLA concentration-response relationship curves showing peak current amplitudes (ordinate; percentage of response in the absence of MLA) as a function of dose (abscissa; nanomolar units; log scale) of MLA without pretreatment () or after 2-min pretreatment (). Recordings were made from five to seven cells, and data points are means ± S.E.M.

7-nAChR are defined in part by their high-affinity binding of, and functional sensitivity to, Bgt. Our other studies show high-affinity binding of radiolabeled Bgt to 7-nAChR heterologously expressed in transfected SH-EP1 cells (Peng et al., 1998, 1999). Pretreatment (2 min) with 3 nM Bgt dramatically reduced functional responses of 7-nAChR to nicotine, and the effect was only slowly reversible, requiring more than 15 min for half-recovery and showing essentially complete recovery after 26 min in Bgt-free medium (Fig. 8A). However, as was the case for MLA and as might be expected given the slower binding of toxin to receptor, without pretreatment (i.e., upon coapplication with nicotine), Bgt showed less ability to block nicotine-induced inward currents at concentrations of 3 nM or lower (Fig. 8B). In contrast, whole-cell current traces indicate that Bgt-mediated functional block of 7-nAChR responses are concentration-dependent in the low nanomolar range when applied during a 2-min pretreatment (Fig. 6C). Dose-response profiles for Bgt blockade of 7-nAChR function (Fig. 8D) yielded IC₅₀ values of 1.3 nM and a Hill coefficient of 1.8 after 2 min of toxin pretreatment and an IC₅₀ value of >10 nM without pretreatment. These results indicate that both MLA and Bgt block heterologously expressed human 7-nAChR function with high affinity, but that the kinetics for conversion to the blocked receptor state is slow compared with the rate of channel opening upon exposure to agonist.

View larger version (22K): [in this window] [in a new window]   Fig. 8. -Bungarotoxin concentration-response profile for blockade of human 7-nAChR function. Whole-cell responses to 0.1 mM nicotine (applied for a period showed by a full horizontal bar above each trace) were measured alone (leftmost trace, A–C); in the presence of 3 nM Bgt for 1 s before and then during nicotine exposure (second trace, A), at different times after Bgt removal (third–fifth traces, A), in the presence of Bgt at the indicated concentrations (second–fifth traces, B), or in the presence of Bgt at the indicated concentrations after 2-min pre-exposure to Bgt alone at the same dose used for cotreatment with nicotine (second–fifth traces, C). Responses were measured using Tris electrodes at a holding potential of -60 mV (horizontal calibration bar, 1 s; vertical calibration bars, 100 pA for A and B and 200 pA for C). With 2-min or 1-s pretreatment, Bgt functional antagonistic potency was markedly increased, and about 25 min was required for full recovery from Bgt block. D, Bgt concentration-response relationship curves showing peak current amplitudes (ordinate; percentage of response in the absence of Bgt) as a function of dose (abscissa; nanomolar units; log scale) of Bgt without pretreatment () or after 2-min pretreatment (). Recordings were made from five to seven cells, and data points are means ± S.E.M.

Mechanism of Functional Block of 7-nAChR by Bgt and MLA. To further examine effects of MLA and Bgt on 7-nAChR, nicotine concentration-response curves were obtained in the absence or presence of MLA or Bgt at concentrations giving some block of responses to 100 µM nicotine to reveal whether antagonism occurred via noncompetitive or competitive mechanisms. Typical whole-cell current response traces in the presence of 1 nM MLA or Bgt (Fig. 9, A and B) differed from those in the presence of nicotine alone. Dose-response profiles indicate a shift to the right (i.e., toward higher agonist concentrations; Fig. 9, C and D) without a change in efficacy of nicotine at higher concentrations. The EC₅₀ value for the nicotine response alone was 36 µM, but was 100 µM in the presence of 1 nM MLA and 250 µM in the presence of 1 nM Bgt.

View larger version (21K): [in this window] [in a new window]   Fig. 9. Mechanism of MLA and Bgt block of human 7-nAChR function. After 2-min pretreatment (open horizontal bars) with 1 nM MLA (A) or 1 nM Bgt (B), whole-cell responses to nicotine (black horizontal bars) at the indicated doses applied for 1 s were measured using Tris electrodes at a holding potential of -60 mV (horizontal calibration bar, 500 ms; vertical calibration bars, 200 pA). Nicotine concentration-response curves alone (, C and D) or in the presence of 1 nM MLA (, C) or Bgt (, D) for peak current amplitudes (ordinate; percentage of response to 1 mM nicotine alone) as a function of nicotine dose (abscissa; micromolar units; log scale) show rightward shifts in the presence of antagonist from an EC50 for nicotine alone of 36 µM to EC50 values of 100 µM in the presence of MLA or 250 µM in the presence of Bgt. Recordings were made from five to seven cells, and data points are means ± S.E.M.

Effects of Extracellular Ca²⁺ on 7-nAChR-Mediated Currents. Several studies have suggested that 7-nAChR have a relatively high permeability to Ca²⁺, suggesting potentially important roles for 7-nAChR in modulation of neurotransmitter release and synaptic plasticity (Seguela et al., 1993; Albuquerque et al., 1997). We examined 7 nAChR-mediated currents induced by nicotine in the presence of different concentrations of external Ca²⁺. Upon removal of extracellular Ca²⁺ (Fig. 10A), the peak whole-cell current response was dramatically reduced from 337 ± 67 to 137 ± 35 pA (p < 0.01, n = 5; Fig. 10B). Reintroduction of external Ca²⁺ was accompanied by an immediate return of peak current responses to control levels (Fig. 10A). Moreover, the time constant for acute desensitization, which was 60.3 ± 8.5 ms for the tested cells under control conditions of 2 mM external Ca²⁺, was reduced to 25.2 ± 2.6 ms (p < 0.01; n = 4) upon removal of external Ca²⁺ (Fig. 10, A and B). These results indicate that there is a substantial modulation of human 7-nAChR by extracellular Ca²⁺ and that removal of Ca²⁺ reduces of peak amplitude and accelerates of receptor desensitization. Using the same experimental approach, we found that less than 5% of the peak current mediated through heterologously expressed human 42-nAChR is lost when extracellular Ca²⁺ is removed (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 10. Effects of extracellular Ca2+ on human 7-nAChR-mediated currents. Whole-cell currents induced by 1-s applications of 0.1 mM nicotine (A) were measured in SH-EP1-h7 cells as described under Materials and Methods using Tris electrodes at -60 mV (horizontal calibration bar, 500 ms; vertical calibration bar, 100 pA; representative traces from one study are shown) in the presence of 2 mM external Ca2+ (left), after perfusion with buffer containing 1 mM EGTA without CaCl2 (middle trace), or after reperfusion with buffer containing 2 mM Ca2+ (right trace). B, summary of studies under the three recording conditions (Ca2+, Ca2+-free, and reperfusion with Ca2+) for peak current amplitude (left ordinate; in picoamperes; closed columns) and decay constant (right ordinate; in milliseconds; open columns). Results are means ± S.E.M. for recordings from five cells (*, p < 0.05; **, p < 0.01).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The major findings of this study are that heterologous, stable expression of functional human 7-nAChRs can be achieved in a human host cell line and that the expressed human 7-nAChRs exhibit many of the properties of naturally or heterologously expressed 7-nAChRs made up of 7 subunits from different species. That is, recombinant human 7-nAChRs expressed in SH-EP1 cells exhibit rapid activation and inactivation upon agonist exposure, responsiveness to choline as well as nicotine and ACh, high Ca²⁺ permeability, and high-affinity competitive antagonism by MLA or Bgt. In addition, the current studies show that functional inhibitory potency of MLA and Bgt is increased when these ligands are applied to cells expressing 7-nAChRs in advance of exposure to agonist.

Stable Expression of Human Neuronal 7-nAChRs in the SH-EP1 Clonal Cell Line. Previous work has shown the utility of SH-EP1 cells as a host for heterologous expression of several nAChR subtypes as functional receptors (Peng et al., 1998, 1999; Eaton et al., 2000). This contrasts with difficulties in getting functional expression of nAChRs in other cell lines and may reflect shared features between SH-EP1 and neuron-like cells (Lukas et al., 2002).

Rapid Desensitization and Functional Rundown of Human Neuronal 7 nAChR. A rapid phase of acute desensitization is one of the distinguishing features of 7-nAChR (Zorumski et al., 1992; Alkondon and Albuquerque, 1993; Gopalakrishnan et al., 1995; Cuevas et al., 2000). Findings presented here indicate that heterologously expressed human 7-nAChRs in SH-EP1 cells have functional responses to nicotinic agonists characterized by rapid kinetics of channel opening (10–90% peak current rise time for a 100 µM nicotine-induced response = 12 ms) and closing (half-time for inward current decay of 20.8 or 49.5 ms for measurements made using K⁺ or Tris electrodes, respectively), with closing occurring even in the continued presence of agonist. These values for channel closing are comparable to those observed the fast transient currents recorded from SH-SY5Y cells stably transfected with the rat 7 cDNA (7 ms; Puchacz et al., 1994a), and for 7-nAChR-mediated inward current responses reported in transfected 7-nAChR in the HEK-293 cell line (6 ms; Gopalakrishnan et al., 1995). Current findings also indicate that heterologously expressed human 7-nAChR-mediated peak current responses to nicotinic agonists applied at 3-min intervals and measured using K⁺ electrodes undergo "rundown", declining in amplitude with each successive application of agonist. Similar features have been observed for 7-nAChR-mediated responses in cultured neurons (Hilmas et al., 2001). Rundown of 7-nAChR-mediated function in transfected SH-EP1 cells seems to differ from that of ACh- or GABA-induced currents in dissociated neurons reported previously (Wakamori et al., 1993; Harata et al., 1997) because it cannot be prevented by addition of ATP (4 mM) to the recording pipette solution (current findings) or by using nystatin-perforated patch recording (data not shown). Thus, rundown of 7-nAChR responses presents a technical difficulty, because it compromises ability to accurately evaluate agonist or antagonist concentration-response relationships, current-voltage-relationships, or other features of the receptor requiring repeated challenges with drugs. However, we have discovered that functional rundown of human 7-nAChR can be eliminated by replacement of recording pipette solutions containing KCl with solutions substituting Tris for KCl. Pipettes with a similar composition of solution are reported to allow stable, whole-cell recording of voltage-gated Ca²⁺ current responses in dissociated thalamic neurons (Huguenard and Prince, 1994) and of GABA-induced currents in dissociated neurons (Kapur et al., 1999). Furthermore, we also have discovered that use of Tris electrodes reduces the rate of 7-nAChR acute desensitization. Mechanisms involved in the influence of whole-cell recording configuration and recording pipette solution composition on acute desensitization and functional rundown of 7- or other nAChR subtypes are explored more thoroughly in another study to be presented elsewhere. However, differences do not seem to be related to internal Ca²⁺ or ATP levels, suggesting that differences are only due to the presence of intracellular K⁺ or Tris. Pragmatically, however, stability of responses recorded using Tris electrodes makes study of 7-nAChR function more feasible.

Agonists Acting on Human 7-nAChRs. Three of the agonists tested, (-)-nicotine, ACh, and choline, induced the same type of inward current mediated via human 7-nAChR, but with distinctive potencies and efficacies. The EC₅₀ value for nicotine-induced current of 40 µM is slightly higher (25 µM) than that for rat 7-nAChR heterologously overexpressed above a native human 7-nAChR background in transfected SH-SY5Y cells (Puchacz et al., 1994a), comparable with that for human 7-nAChRs heterologously expressed in Xenopus oocytes (40 µM; Peng et al., 1994), and slightly lower than that for human 7-nAChR heterologously expressed in HEK-293 cells (49 µM; Gopalakrishnan et al., 1995). Nicotine and ACh display comparable peak (at high agonist doses) efficacies at human 7-nAChR expressed in SH-EP1 cells. However, the EC₅₀ value for ACh-induced current mediated by recombinant, human 7-nAChR in SH-EP1 cells was 1.1 mM. This apparently low potency was surprising, especially compared with EC₅₀ values measured for ACh acting at 7-nAChR in other cell types [values of 129.4 µM for cultured rat hippocampal neurons (Alkondon and Albuquerque 1993; Alkondon et al., 1994); 150 µM for heterologously overexpressed rat 7-nAChR in SH-SY5Y cells (Puchacz et al., 1994a); 79.2 µM for heterologously expressed human 7-nAChRs in Xenopus oocytes (Peng et al., 1994); and 155 µM for transfected human 7-nAChRs in HEK-293 cell line Gopalakrishnan et al., 1995)]. Perhaps an explanation comes from the shallow slope for the ACh dose-response profile obtained in the current studies, yielding a Hill value of 0.51, which is much smaller than the values of 1.4 for nicotine or 1.3 for choline action at human 7-nAChRs reported in this study or the Hill values of about 1.3 for nicotine or ACh action at overexpressed rat 7-nAChRs (Puchacz et al., 1994a). Attempts to use a two-site model with Hill numbers either determined during the iterative process or fixed at 1.3 did not provide a better fit to the dose-response profile for ACh action at human 7-nAChRs in SH-EP1 cells, in part because the r² value was 1.0 for the fit to the one-site model already, although two-site fits to the data yielded EC₅₀ values of 150 to 180 µM for a high-affinity site and 4.2 to 7.1 mM for a low-affinity site. The high-affinity site EC₅₀ value is more in line with EC₅₀ determinations from other reports cited above for ACh acting at 7-nAChRs and might have made sense if, perhaps, metabolism of ACh to choline could account for actions at the lower affinity site. However, the current findings of an EC₅₀ for choline action at heterologously expressed human 7-nAChR in SH-EP1 cells of 850 µM discounts this possibility, assuming that choline acting alone or in the presence of ACh would have the same agonist potency at human 7-nAChRs. The EC₅₀ value of 850 µM for choline action at human 7-nAChR is lower than that reported for the choline EC₅₀ value for action on rat cultured hippocampal neuronal native 7-nAChRs (EC₅₀ = 1.6 mM; Alkondon et al., 1997) and 7-fold lower than that for 7-nAChR' responses of isolated rat superior cervical ganglion neurons (EC₅₀ = 6 mM; Cuevas et al., 2000). Collectively, the current results nevertheless indicate that heterologously expressed human 7-nAChRs in SH-EP1 cells exhibit choline sensitivity characteristic of other 7-nAChRs.

Antagonists Acting on Human Neuronal 7 nAChR. Previous studies have shown that autonomic neuronal or central 7-nAChR exhibit very high affinities for Bgt and MLA. The current studies show, at least for human 7-nAChRs heterologously expressed in SH-EP1 cells, that highest functional inactivation potencies for either of these ligands requires pre-exposure of receptors to the antagonists before agonist challenge. Pretreatment converts functional IC₅₀ values for MLA or Bgt from >10 nM to 1.2 to 1.3 nM. These values are comparable with those from previous reports of EC₅₀ values of 1.6 nM for MLA acting at native, chick 7-nAChR in embryonic sympathetic neurons (Yu and Role, 1998). In the present study, nicotinic responses inhibited by 3 nM MLA fully recovered very quickly (within 5-min washout), whereas only partial recovery from MLA block was reported for human 7-nAChRs heterologously expressed in HEK-293 cells (Gopalakrishnan et al., 1995). Full recovery from Bgt-mediated block occurred within 30 min of toxin removal for nicotinic responses in SH-EP1-h7 cells. Agonist dose-response profiling in the absence of presence of MLA or Bgt was consistent with their actions as competitive antagonists.

Inward Rectification and Ca²⁺ Sensitivity of 7-nAChR-Mediated Currents. Inward rectification is another characteristic of neuronal nicotinic receptor function. Previous studies have indicated that inward rectification occurs for recombinant human 7-nAChRs in HEK-293 cells (Gopalakrishnan et al., 1995) or Xenopus oocytes (Peng et al., 1994), for rat 7-nAChRs in freshly dissociated neurons from neonatal intracardic ganglia (Cuevas and Berg 1998), and for cultured rat hippocampal neurons (Alkondon et al., 1994). In transfected SH-EP1 cells, human 7-nAChR responses to nicotine, ACh, or choline showed inward rectification at positive holding potentials. Peak whole-cell current amplitude of human 7-nAChR responses to nicotine in SH-EP1 cells were suppressed with reduction of external Ca²⁺, suggesting that Ca²⁺ permeability is a feature of human 7-nAChR as it is for 7-nAChR from other species (Castro and Albuquerque, 1995) and/or that external Ca²⁺ positively regulates 7-nAChR channel opening.

In conclusion, heterologously expressed human 7-nAChR in the SH-EP1 human epithelial cell host exhibit distinguishing characteristics such as inward rectification, external Ca²⁺ sensitivity, sensitivity to choline as an agonist and MLA and Bgt as antagonists, rapid channel kinetics, and profound sensitivity of acute desensitization and functional rundown to the composition of recording pipette solution used and replacing the nature cytosol. SH-EP1 cells heterologously, constitutively, and stably expressing human 7-nAChRs from the pCEP4 vector harboring the coding, cDNA sequence for human 7 subunits are an excellent model for studies of receptor structure and function.

	Acknowledgements

 
We are very grateful to Sherry Leonard (University of Colorado, Denver, CO) for the human 7 cDNA used to generate the cell line characterized in this study.

	Footnotes

 
This study was supported in part by National Institutes of Health Grant NS040417, the Robert and Gloria Wallace Foundation, Epi-Hab Phoenix, Inc., Arizona Disease Control Research Commission Grants 9615 and 9930, Council for Tobacco Research Grant 4366, the Marjorie Newsome and Sandra Solheim Aiken funds, and endowment and/or capitalization funds from the Men's and Women's Boards of the Barrow Neurological Foundation and was conducted in part in the Charlotte and Harold Simensky Neurochemistry of Alzheimer's Disease Laboratory. The contents of this report are solely the responsibility of the authors and do not necessarily represent the views of the aforementioned awarding agencies.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.048777.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; Bgt, -bungarotoxin; RT-PCR, reverse transcription-polymerase chain reaction; RT, reverse transcription; bp, base pair(s); PCR, polymerase chain reaction; PBS, phosphate-buffered saline; SSC, standard saline citrate; V_H, holding potential; ACh, acetylcholine; MLA, methyllycaconitine; HEK, human embryonic kidney.

Address Correspondence to: Dr. Jie Wu, Division of Neurology, Barrow Neurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013-4496. E-mail: jwu2{at}chw.edu

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