Ligand Binding and Activation of Rat Nicotinic a 4 b 2 Receptors Stably Expressed in HEK 293 Cells

HEK293 cells were stably transfected with rat neuronal nicotinic a4 and b2 subunits. Binding of tritiated cytisine and nicotine to cell homogenates revealed the presence of a single class of high-affinity sites (dissociation constants 0.1 nM and 0.4 nM, respectively). Activation of nicotinic receptors was studied using whole-cell patch clamp methods, and acetylcholine, nicotine, dimethylphenylpiperazinium, and cytisine all produced a conductance increase. Responses desensitized to prolonged applications, at both positive and negative membrane potentials. The conductance was strongly rectifying, and outward currents were essentially absent. Responses were maximal at about 2 mM external calcium ion concentration and were reduced by about one-half at either nominally 0 or 10 mM external calcium. Di-hydro-b-erythroidine blocked physiological responses to acetylcholine and nicotine (IC50, 2.5 nM), and reduced cytisine binding in a competitive manner (Ki 20 nM). Physostigmine enhanced the response to low concentrations of acetylcholine or nicotine. The anesthetic steroid (1)-3a-hydroxy-5a-androstane-17b-carbonitrile blocked responses to acetylcholine (IC50, 1.3 mM), but had no effect on cytisine binding at a concentration of 30 mM. The binding properties of the receptors are those expected for rat neuronal nicotinic receptors composed of a4 and b2 subunits. The pharmacological properties indicate that the responsiveness of the receptors may be allosterically enhanced or inhibited. A number of subunits for nicotinic receptors are expressed by cells of the nervous system (reviewed in McGehee and Role, 1995; Brioni et al., 1997), including several subunits most similar to the muscle a subunit (a2 – a9) and others termed b subunits (b2 – b4). A major type of neuronal nicotinic receptor in the mammalian and avian brain is composed of the a4 and b2 subunits (Whiting et al., 1987a; Schoepfer et al., 1988; Flores et al., 1992; Picciotto et al., 1995) and forms a protein with a very high affinity for agonists such as nicotine and cytisine (reviewed in Brioni et al., 1997). The functional role of the neuronal nicotinic a4b2 receptor is not well understood; at present, it seems likely that it may modulate the release of neurotransmitter as a consequence of presynaptic localization (reviewed in McGehee and Role, 1996). Consistent with this idea is the finding that genetically altered mice in which the b2 subunit has been knocked out are grossly normal behaviorally, although they show changes in some forms of learning (Picciotto et al., 1995). It has been found, however, that the numbers of a4b2 receptors are increased by chronic nicotine exposure in animals (Flores et al., 1992), and the numbers of high-affinity nicotine-binding sites are increased in the brains of humans who smoked and decreased in patients with Alzheimer’s disease (reviewed in Brioni et al., 1997). It is also of note that mutations of the human a4 subunit have been identified in families showing autosomal dominant nocturnal frontal lobe epilepsy (Steinlein et al., 1997). As a step toward better understanding of the functional properties of this nicotinic receptor, we have stably expressed rat nicotinic a4 and b2 subunits in human epithelial cells (HEK293) and characterized the binding and functional properties of the receptors. Materials and Methods Unless otherwise noted, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). (1)-3a-Hydroxy-5a-androstane-17bcarbonitrile (1ACN) was a gift from Dr. D. Covey (Washington University School of Medicine, St. Louis, MO). All values are presented and shown in graphs as the arithmetic mean 6 1 S.D. , based on n observations. Production of HEK293 Cells Stably Transfected with Rat Neuronal Nicotinic a4 and b2 Subunits. HEK293 cells (CRL1573: American Tissue Culture Collection, Gaithersburg MD) were maintained in a mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 (1:1, also containing 5 mM HEPES), with 10% fetal bovine serum (Hyclone, Logan UT), penicillin (100 u/ml) and streptomycin (100 mg/ml) in a humidified atmosphere containing 5% CO2 This research was supported by National Institutes of Health grants R01 NS22356 and P01 GM47969 to J.H. S. ABBREVIATIONS:1ACN, (1)-3a-hydroxy-5a-androstane-17b-carbonitrile; PBS, phosphate-buffered saline; HB, homogenate buffer; DMSO, dimethyl sulfoxide; dHbE, di-hydro-b-erythroidine; mAb, monoclonal antibody; GABA, g-aminobutric acid. 0026-895X/99/010058-09$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 55:58–66 (1999). 58 at A PE T Jornals on M ay 7, 2017 m oharm . aspeurnals.org D ow nladed from at 37°C. They were transfected by electroporation with expression constructs for the rat a4 and b2 subunits, and transfected cells were initially selected by growth in medium containing G418 (450 mg/ml; GIBCO, Grand Island, NY). Drug-resistant cells were maintained in G418, and then repeatedly immunoselected with monoclonal antibody (mAb) 270, which binds to an epitope on the extracellular surface of the b2 subunit (Whiting et al., 1987b). Selection was performed with magnetic beads with covalently attached sheep antirat antibody (Dynabeads M-450; Dynal, Lake Success, NY). Beads were prepared by mixing 1 ml of purified mAb 270 (1 mg/ml) with 1 ml of resuspended bead solution and 1 ml of phosphate-buffered saline (PBS) ( 140 mM NaCl, 2.7 mM KCl, 9.6 mM PO4, pH 7.4) in a 15-ml sterile conical tube and rocking at 4°C for 30 min. The beads were washed twice by centrifugation and resuspension in PBS, and finally resuspended in 1 ml of PBS. To select the cells, 0.5 ml of a single-cell suspension (4 3 10 cells/ml) and 30 ml of freshly prepared sterile beads were mixed by gentle trituration and then incubated on ice for 15 min. The mixture was diluted by the addition of 10 ml of PBS, and cells were collected using a magnetic stand (Dynal). The beads (with attached cells) were washed a total of six times with PBS and then plated in a 60-mm tissue culture dish in growth medium. After overnight incubation, the dishes were washed twice with growth medium to remove beads. The cells used in these studies had been sequentially selected 6 to 10 times, but had not been cloned. They will be termed HN42 cells. mAb 270 was purified from serumfree growth medium from hybridoma cells (HB-189; ATCC). The cDNAs for the rat neuronal nicotinic subunits were kindly provided by Dr. J. Patrick (Baylor College of Medicine) (rat a4 subunit HYA23–1E(1), Goldman et al., 1987; b2 subunit PCX49(1), Deneris et al., 1988). They were transferred to pcDNA3 (Invitrogen, San Diego, CA) for expression. Binding Assays. Cells were grown in 10-cm culture dishes for 4 to 6 days and harvested by rinsing with PBS, then releasing the cells with PBS containing 5 mM EDTA. Cells from 10 dishes were pooled and pelleted by centrifugation. The pellet was washed once with PBS by resuspension and pelleting. The washed pellet was resuspended and cell number was estimated in a hemocytometer. The suspension was centrifuged, the supernatant removed, and the cell pellet frozen