Introduction

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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  subunit (2 - 9) and others termed  subunits (2 - 4). A major type of neuronal nicotinic receptor in the mammalian and avian brain is composed of the 4 and 2 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 42 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 2 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 42 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 4 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 4 and 2 subunits in human epithelial cells (HEK293) and characterized the binding and functional properties of the receptors.

	Materials and Methods

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Unless otherwise noted, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). (+)-3-Hydroxy-5-androstane-17-carbonitrile (+ACN) 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 ± 1 S.D., based on n observations.

Production of HEK293 Cells Stably Transfected with Rat Neuronal Nicotinic 4 and 2 Subunits. HEK293 cells (CRL-1573: 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 µg/ml) in a humidified atmosphere containing 5% CO₂ at 37°C. They were transfected by electroporation with expression constructs for the rat 4 and 2 subunits, and transfected cells were initially selected by growth in medium containing G418 (450 µg/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 2 subunit (Whiting et al., 1987b). Selection was performed with magnetic beads with covalently attached sheep anti-rat 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 PO_4, 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 × 10⁷ cells/ml) and 30 µl 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 serum-free growth medium from hybridoma cells (HB-189; ATCC).

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 at 80°C.

Electrophysiology. Cells were plated in 35- or 60-mm tissue culture dishes, and used within 4 days after plating. Recordings were made in an extracellular saline, most often composed of 140 mM NaCl, 1 mM MgCl₂ , 2 mM CaCl₂, 10 mM HEPES, 5 mM KCl, 10 mM glucose, and 1 µM atropine sulfate, pH 7.3. When a different saline was used, the modifications are noted in Results. The internal solution was composed of 4 mM NaCl, 4 mM MgCl₂, 0.5 mM CaCl₂,, 10 mM HEPES, 5 mM EGTA, and 140 mM CsCl, pH 7.3. Standard methods were used to record currents in the whole cell configuration. Records were filtered with a four-pole Bessel filter (Frequency Devices, Haverhill, MA) and directly digitized by a PC clone computer with a TLC1 or Digidata interface (Axon Instruments, Foster City, CA). Drugs were dissolved in external solution and applied through a multiline perfuser (Fletcher and Steinbach, 1996) or a three-tube perfusion system (Maconochie and Knight, 1989). The multiline perfuser provided a solution change around cells attached to the culture substrate with a 10 to 90% time of about 200 ms, and the three-tube perfuser an exchange time of about 50 ms (data not shown, from the time course of changes in holding current when bath solutions with different ion concentrations were applied to cells). In either case, the cell was perfused with extracellular solution continuously between applications of agonists or other drugs.

	Results

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Binding of Nicotine and Cytisine

Specificity of Cytisine Binding. The equilibrium binding was measured of cytisine to homogenates prepared from HEK293 cells stably transfected with rat 4 and 2 nicotinic receptor subunits (HN42 cells). Nonspecific binding was determined from binding to homogenates prepared from the parental HEK293 cells and from binding to homogenates from HN42 cells in the presence of 100 µM nicotine. The two assays for nonspecific binding were similar (Fig. 1) and amounted to a small fraction of the total binding to HN42 cell homogenates. On average, the specific binding by 10 nM cytisine was better than 90% of the total (92% ± 2%; n = 6).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Specificity of cytisine binding. The binding of cytisine was measured to homogenates prepared from HN42 cells (). Nonspecific binding was determined with homogenates prepared from the parental HEK293 cells () and by binding to homogenates from HN42 cells in the presence of 100 µM nicotine (). The two assays for nonspecific binding are very similar at all concentrations of cytisine. The amount bound per milligram of protein was normalized to the maximal amount bound to HN42 cell homogenates in the absence of nicotine. The points show the mean of two independent experiments, each performed as duplicates, and error bars show 1 S. D. Note that both axes are logarithmically scaled.

Affinity of Cytisine and Nicotine Binding. Specific binding of cytisine and nicotine was saturable (Fig. 2). The curves through the data show fits of eq. 1:

(1)

The apparent dissociation constants (K_h) are about 0.1 nM for cytisine (0.12 ± 0.05; n = 3) and 0.4 nM for nicotine (0.40 ± 0.01; n = 2), whereas the Hill coefficients (N_h) are close to 1 (0.97 ± 0.14 for cytisine and 0.99 ± 0.00 for nicotine).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Affinity of cytisine and nicotine binding. The specific binding of cytisine ( and solid line) and nicotine ( and dotted line) to homogenates of HN42 cells saturates at high ligand concentrations. Nonspecific binding was determined with homogenates prepared from the parental HEK293 cells, and has been subtracted from the data shown. The curves show fits of eq. 1 to the data, and the horizontal dotted line shows half-maximal binding to indicate the Kh. The data are means of two or three separate binding experiments, each performed in duplicate. The data have been normalized to the binding measured at 10 nM of either ligand and then renormalized to the maximal fit binding. Parameters for the fit curves are given in the text.

Inhibition of Cytisine Binding. dHE inhibited the binding of cytisine (Fig. 3). The curves through the data show fits of eq. 2:

(2)

As shown in Fig. 3, the concentration of dHE required to reduce the amount bound by one half (IC₅₀) was larger at 3 nM cytisine than at 0.03 nM cytisine. The Hill slope was close to 1 in each case.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Inhibition of cytisine binding by dHE. The ability of increasing concentrations of dHE to inhibit binding of 0.03 nM cytisine ( and dotted line) or 3 nM cytisine ( and solid line) is shown. The amount bound is normalized to the binding in the absence of dHE. The curves show fits of eq. 2 to the data. The parameters fit were IC50: 656 ± 201 nM (3 nM cytisine) and 23 ± 8 nM (0.03 nM cytisine; n = 2 in each case). The Hill coefficients were: 0.75 ± 0.17 (3 nM cytisine) and 0.84 ± 0.09 (0.03 nM cytisine).

(3)

View larger version (15K): [in this window] [in a new window]   Fig. 4.   The steroid +ACN has no effect on cytisine binding but blocks acetylcholine-elicited currents. The binding of cytisine (0.03 nM cytisine) is shown at various concentrations of the steroid analog +ACN (, mean of two experiments). +ACN was dissolved in DMSO before addition to the binding buffer; the final concentration of DMSO was 14 mM (0.1% V/V) for all concentrations of +ACN. The binding data were normalized to the binding seen in the presence of this concentration of DMSO, but no +ACN. The  show the relative response to 1 µM acetylcholine in the presence of +ACN (data from 3-5 cells), and the solid line shows the fit of eq. 2 to the data (IC50 1.3 µM, Nh 2.4).

Physiological Responses

Activation and Desensitization by Nicotinic Agonists. HN42 cells responded to applications of nicotinic agonists (nicotine, acetylcholine, dimethylphenylpiperazinium, cytisine) with a conductance increase (Fig. 5). The concentration-response relationship was examined for acetylcholine. As shown in Fig. 5 (top panel), the relationship showed relatively low affinity and Hill slope. When fit with eq. 1, the estimate for K_h was 80 µM and for N_h was 0.7. 

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Concentration-response relationship for activation of currents. The upper panel shows responses to applications of acetylcholine, normalized to the response in the same cell to 10 µM acetylcholine. The solid line shows the fit of eq. 1 to the data (Kh 80 µM, Nh 0.7, maximal relative response 6.1). The lower panel shows responses to other agonists, again normalized to the response to 10 µM acetylcholine (abbreviations: NIC, nicotine; DMPP, dimethylphenylpiperazinium; CYT, cytisine). The solid line repeats the curve from the upper panel to summarize the concentration-response relationship for acetylcholine. Symbols show the means of 3 to 17 values (±1 S.D., when larger than the symbol size).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Desensitization to prolonged applications of 100 µM acetylcholine. The upper panel shows a response to a 2-s application of 100 µM acetylcholine, made to a cell held at 100 mV. Overlaid on the data trace is the sum of two exponentials, declining to a baseline of zero (that is, it was assumed that eventually the desensitization would have been complete). The fitted values are: fast decaying component time constant 170 ms, slow component time constant 2700 ms, and fraction fast 0.8. Overall, immediately after breaking into the cell, the values were: fast component time constant 183 ± 52 ms, slow component time constant 2040 ± 720 ms, and fraction fast 0.7 ± 0.08 (n = 10). The lower panel shows three superimposed responses. In one (thin line), the cell was held at 100 mV throughout the response. In the others (thicker lines), the cell was held at +50 mV for the initial 250 ms or 1000 ms of the application of 100 µM acetylcholine. The responses on jumping to 100 mV are clearly larger than the control response; when the cell was held at +50 mV for 250 ms, the response immediately on jumping to 100 mV was about 1.4 times control, whereas when the cell was held at +50 for 1000 ms, it was about 1.9. Overall, after 250 ms the response was increased to 1.5 ± 0.3 times control (n = 6), whereas after 1000 ms it was 1.8 ± 0.3 (n = 15).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Current-voltage relationship for nicotinic responses. Acetylcholine was applied to cells held at potentials between 100 mV and +50 mV (symbols; seven or eight cells), or alternatively the membrane potential was changed by a linear ramp between 100 mV and +100 mV (solid line shows mean for data from 12 cells, dotted lines show ± 1 S.D.). Note that there is no indication of outward current even at potentials between +50 and +100 mV. The amplitudes are normalized to the response at 50 mV.

Current-Voltage Relationship and Sensitivity to Extracellular Calcium. The response to agonists was strongly rectifying, such that the outward conductance was essentially zero (Fig. 7). There was no indication of an increase in conductance at large positive membrane potentials (e.g., Ifune and Steinbach, 1990). Others have reported that receptors formed from human (Buisson et al., 1996) and chicken (Whiting et al., 1991) 42 subunits show similar rectification properties.

Physostigmine Potentiates Responses to Low Concentrations of Acetylcholine or Nicotine. It has been reported that the anticholinesterase, physostigmine, can act as a weak agonist at some nicotinic receptors (Pereira et al., 1994) and that 1-methyl-galanthamine can potentiate the response of PC12 cells to low concentrations of acetylcholine (Storch et al., 1995). We found that physostigmine (1 µM) can potentiate responses to both acetylcholine and nicotine. Responses to 1 µM nicotine were potentiated by about 2-fold in the presence of 1 µM physostigmine (2.1 ± 0.9; n = 5). The effect on responses to acetylcholine depended on the concentration of acetylcholine used. Responses to 0.1 µM acetylcholine were potentiated by 1.8-fold (±0.4, n = 3), those to 1 µM acetylcholine by 1.2-fold (1.2 ± 0.2, n = 10) and those to 10 µM acetylcholine by only 1.1-fold (±0.03, n = 3).

Inhibition of Responses by dHE. dHE reduced responses activated by nicotine or acetylcholine. The blocking action of dHE was relatively slow to develop; when dHE was applied simultaneously with an agonist, there was relatively little block. We studied the onset of block using 10 nM dHE preapplied in the absence of agonist (Fig. 8), and found that the onset had a time constant of about 3 s (2.5 ± 0.8 s), with a final level of about 80% block (80 ± 2%, n = 4). A similar value of block after 30-s application of 10 nM dHE was found in tests with 10 nM dHE plus 1 µM nicotine (78 ± 9%, n = 2). If we assume that the block of response can be described by a simple binding isotherm and that the concentration of agonist used is low compared with the affinity, then this would correspond to an apparent affinity for dHE of about 2.5 nM (given 80% block at 10 nM). We then examined the dissociation rate of dHE by applying a long (10-30 s) pulse of a high concentration (100 nM) of dHE to fully block the response, and then washing for different periods before testing with agonist (Fig. 8). When the recovery data were fit with single exponentials, the mean time constant was about 9 s (9.4 ± 5.4 s, n = 11). The data from the onset time course predict a time constant for recovery of about 13 s, assuming that the block is described by a simple binding equilibrium (because the onset rate would be sum of the blocking and unblocking rates). dHE blocked receptor activation by agonists somewhat more potently than it inhibited agonist binding to cell homogenates. This might be the result of the different experimental conditions, or might indicate that dHE binds to desensitized receptors with a lower affinity.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Block of responses by dHE. The upper panel shows the onset of the block of acetylcholine responses by 10 nM dHE (4 cells). dHE was applied for the time shown and then the application was switched to 10 µM acetylcholine plus 10 nM dHE to assay the response. The solid line shows a single exponential decay with the mean time constant and equilibrium block (time constant 2.5 ± 0.4 s, final response 0.20 ± 0.02; four cells). The lower panel shows the recovery of the response after complete block by a 10-s exposure to 100 nM dHE. The response was tested with either 1 µM acetylcholine (five cells) or 1 µM nicotine (six cells). The solid line shows an exponential recovery curve with the mean time constant from the data (time constant 9.4 ± 5.4 s, n = 11). The dotted line shows a recovery curve with the time constant predicted from the onset data (time constant 12.7 s). Note that zero time is offset in both panels.

Inhibition of Responses by +ACN. It has been reported that progesterone inhibits recombinant chicken 42 receptors (Valera et al., 1992) and that anesthetics of various classes inhibit receptors formed from rat (Violet et al., 1997) or chicken (Flood et al., 1997) 42 subunits expressed in Xenopus oocytes. Accordingly, we examined the ability of an anesthetic neuroactive steroid to inhibit responses of these cells. Cells were pre-exposed to +ACN for 30 s before acetylcholine was applied, because preliminary results indicated that the steroids had little effect when applied simultaneously with acetylcholine. As summarized in Fig. 4 (open circles), +ACN blocked responses to 1 µM acetylcholine with an IC₅₀ of about 2 µM and a Hill coefficient of about 2. Because these concentrations of +ACN do not affect cytisine binding (Fig. 4), it appears that the mechanism of inhibition is not competitive. Similarly, the value of the Hill coefficient suggests that the mechanism involves the binding of more than one +ACN molecule to a receptor. DMSO (0.3%; 42 mM) did not affect the amplitude of responses to acetylcholine (relative response 0.99 ± 0.04; n = 3).

	Discussion

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The results indicate that there is a single class of high-affinity sites for cytisine and nicotine in the stably transfected cells, and that both nicotine and cytisine bind to the same site. dHE behaves as a competitive inhibitor for binding of cytisine. dHE also inhibits currents activated by acetylcholine and nicotine at similar concentrations, suggesting that the sites found in equilibrium binding experiments are the same agonist binding sites involved in receptor activation.

The affinities of cytisine and nicotine are comparable to the highest affinity values obtained in previous studies on homogenates from rat brain or spinal cord, whereas the specific activity is about 4-fold higher (Table 1). The affinity for dHE also is comparable to the highest values reported previously (Table 1). The higher apparent affinities we observe may arise from the existence of a homogeneous population of binding sites.

Our studies of receptor activation suggest that nicotine and acetylcholine are equally potent and effective at activating receptors, whereas dimethylphenylpiperazinium appears to be less potent and less effective, and cytisine may be more potent but has a smaller maximal effect. These observations are broadly consistent with the results of previous studies of rat 42 receptors expressed in Xenopus oocytes (Luetje and Patrick, 1991; Fenster et al., 1997). The apparent EC₅₀ for activation by acetylcholine is about 80 µM (previous values range from 3-100 µM) and the apparent Hill slope is about 0.7 (previous values range from 0.8-1.1) (Papke and Heinemann, 1994; Stafford et al., 1994; Violet et al., 1997). However, the shape of the concentration-response curves may have been affected by desensitization. In addition, our data with cytisine or dimethylphenylpiperazinium may be affected by channel blocking activity by these agonists. Hence, full characterization of the activation of these receptors will require further experimentation.

Human 42 receptors have also been stably expressed in HEK293 cells (K177 cells; Gopalakrishnan et al., 1996). The specific binding of cytisine to homogenates prepared from K177 cells is about 1400 fmol/mg protein (3-fold higher than we observed), and the affinities for cytisine (0.2 nM), nicotine (1.1 nM) and dHE (60 nM) are similar (Gopalakrishnan et al., 1996). However, in functional studies, the EC₅₀ for activation by acetylcholine is about 3 µM and the Hill slope 1.2 (Buisson et al., 1996), showing half-maximal activation at significantly lower acetylcholine concentrations than in HN42 cells. In K177 cells, nicotine is slightly more potent (EC₅₀ 1.2 µM) and effective (maximal response about 1.2 times the maximal response to acetylcholine) than is acetylcholine, whereas cytisine is both less potent and less effective (Buisson et al., 1996). All of the agonists tested showed lower EC₅₀ values with the human 42 receptors than our data with rat 42 receptors. This suggests functional differences between the two types of receptor, but additional work with the rat 42 receptors is required before definite conclusions are drawn. In studies of K177 cells, dHE has an IC₅₀ against responses to 1 µM acetylcholine of about 80 nM and methyllycaconitine an IC₅₀ of about 1.5 µM. Both of these values are higher than we found, possibly because the concentration of agonist which we used was further below the EC₅₀ for activation of current. However, the fact that the apparent K_i for inhibition of cytisine binding by dHE is also higher for K177 cells suggests that there might be species differences.

Responses to high concentrations of acetylcholine desensitized slightly more slowly when cells were held at +50 mV than at 100 mV. This contrasts with a previous study of chicken 4-n1 subunits expressed in Xenopus oocytes, which showed no desensitization at positive membrane potentials (Gross et al., 1991). It is possible that the difference arises from the expression system used. However, the amount of desensitization we observed at +50 mV is also much greater than that reported for rat sympathetic ganglion neurons, which show no desensitization at positive potentials (Mathie et al., 1990). Hence, there are clear differences among subunit combinations in this respect, and are likely to be differences among species as well.

Previous workers have examined the actions of the anticholinesterase agent, physostigmine, on neuronal nicotinic receptors (Pereira et al., 1993, 1994; Storch et al., 1995). Physostigmine, methyl-galanthamine and codeine have been reported to act as weak agonists in activating single channel openings. Activation is not blocked by drugs such as dHE, but can be blocked by a monoclonal antibody which does not prevent binding of -neurotoxin to the muscle nicotinic receptor (FK1). Methyl-galanthamine has also been reported to potentiate the whole-cell responses elicited by acetylcholine applied to PC12 pheochromocytoma cells (Storch et al., 1995). Based on these observations, it has been proposed that physostigmine and some other compounds can activate neuronal nicotinic receptors by binding to a site distinct from the identified acetylcholine-binding sites (Pereira et al., 1993, 1994; Storch et al., 1995). We found that physostigmine potentiates the responses of HN42 cells to low concentrations of nicotine or acetylcholine. Potentiation of nicotine responses indicates that antiesterase activity does not underlie the action. Our results do not directly address the mechanism of potentiation. However, other workers have found that physostigmine is ineffective at preventing cytisine binding to rat brain homogenates (Pabreza et al., 1991; Anderson and Arneric, 1994), which indicates that physostigmine does not interact strongly with the acetylcholine binding site. Although we confirmed a potentiating action of physostigmine on rat 42 type receptors, we found that codeine showed no potentiation on these receptors. This may be a result of different experimental approaches (potentiation versus activation) or different receptor subtypes in the cells.

Neuroactive steroids have been shown to potentiate responses to low concentrations of -aminobutyric acid (GABA) and to directly gate GABA-A receptors (reviewed in Paul and Purdy, 1992). +ACN is a potent anesthetic in tadpoles and mice that both potentiates and activates responses of GABA-A receptors (Wittmer et al., 1996). Potentiation of responses of cultured hippocampal neurons to 2 µM GABA has an EC₅₀ of 1.4 µM, and direct gating of responses has an EC₅₀ of 5 µM (Wittmer et al., 1996). We find, in contrast, that +ACN inhibits responses of the 42 neuronal nicotinic receptors with an IC₅₀ of about 1.3 µM. +ACN is not a competitive antagonist for rat 42 nicotinic receptors because it has no effect on cytisine binding. The concentration dependence of inhibition indicates that there are at least two binding sites on each receptor. We are presently examining the structural requirements for this action of steroid. Other general anesthetic agents, including halogenated volatile agents and the i.v. agent propofol, also inhibit responses of receptors formed from rat (Violet et al., 1997) or chicken (Flood et al., 1997) 42 subunits. It is perhaps not surprising that these agents affect two members of the ligand-gated ion channel family, although it is interesting that they consistently have opposite actions on GABA-A and neuronal nicotinic 42 receptors. However, not all nicotinic receptors are equally affected; responses of neuronal nicotinic 7 (Flood et al., 1997) or muscle nicotinic (Violet et al., 1997) receptors are 10-fold or more less sensitive to the anesthetics tested. Studies of the actions of additional drugs may reveal differences in the binding sites for anesthetics, as well as differences in the functional consequences of binding, for the GABA-A and nicotinic 42 receptors.

In summary, HN42 cells express a single class of high-affinity nicotine-binding sites. The binding properties are those expected from previous studies. Studies of the pharmacological properties of these receptors show that they can be positively or negatively modulated through allosteric sites, which may be important in understanding the role of 42 receptors in the brain.