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Br J Pharmacol. Nov 1999; 128(6): 1291–1299.
PMCID: PMC1571746

Pharmacological similarities between native brain and heterologously expressed α4β2 nicotinic receptors

Abstract

  1. We studied the pharmacological properties of native rat brain and heterologously expressed rat α4β2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-α4 antibody mAb 299.
  2. Immunodepletion with the anti-β2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained β2.
  3. The association and dissociation rate constants for 30 pM ±[3H]-epibatidine binding to α4β2 receptors expressed in oocytes were 0.02±0.01 and 0.03±0.01 min−1 (±standard error, degrees of freedom=7–8) at 20–23°C.
  4. The Hill coefficients for ±[3H]epibatidine binding to the native brain, α4β2 receptors expressed in oocytes, and α4β2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69–0.70 suggesting a heterogeneous receptor population. Fits of the ±[3H]-epibatidine concentration-binding data to a two-site model gave KD s of 8–30 and 560–1,200 pM. The high-affinity sites comprised 73–74% of the native brain and oocyte α4β2 receptor population, 85% of the CV-1 α4β2 receptor population.
  5. The expression of rat α4β2 receptors in CV-1 cells using vaccinia viral infection-transfection resulted in a more homogeneous receptor population (Hill coefficient of 1.0±0.2). Fits of the ±[3H]-epibatidine binding data to a single-site model gave a KD of 40±3 pM.
  6. DHβE (IC50=260–470 nM) and the novel nicotine analogue NDNI (IC50=7–10 μM) inhibited 30 pM±[3H]-epibatidine binding to the native brain and heterologously expressed α4β2 receptors equally well.
  7. The results show that α4β2-containing nicotinic receptors in the rat brain and heterologously expressed rat α4β2 receptors have similar affinities for ±[3H]-epibatidine, DHβE, and NDNI.
Keywords: Neuronal nicotinic acetylcholine receptors, α4, β2, Xenopus oocytes, dihydro-β-erythroidine, epibatidine binding, immunoprecipitation, mAb 299 antibody

Introduction

Several lines of evidence suggest that the predominant mammalian brain nicotinic subtype contains α4 and β2 subunits. First, the α4 and β2 mRNAs are the most abundant and widespread nicotinic subunit mRNAs in the rat brain (Zoli et al., 1995). Second, the α4 subunit forms functional nicotinic receptors with the β2 subunit in heterologous expression systems (reviewed in Sargent, 1993). Third, the α4 and β2 subunits co-assemble into a brain nicotinic receptor (Whiting & Lindstrom, 1987) that accounts for most of the high-affinity rat brain [3H]-cytisine binding (Flores et al., 1991). Fourth, immunohistochemical localization confirms the widespread distribution of the β2 protein in the rat brain (Hill et al., 1993) and immunodepletion with the anti-β2 antibody mAb 270 removes 92% of the [3H]-nicotine binding sites from rat brain extracts (Whiting & Lindstrom, 1986). Fifth and finally, knocking out the β2 gene eliminates virtually all the high-affinity mouse brain [3H]-nicotine binding sites and most of the high-affinity ±[3H]-epibatidine binding sites (Picciotto et al., 1995; Zoli et al., 1998). Similarly, knocking out the α4 gene also removes most of these binding sites (Marubio et al., 1999).

Nevertheless, rat forebrain homogenates appear to contain at least two major classes of ±[3H]-epibatidine binding sites (Houghtling et al., 1995). The KDs for ±[3H]-epibatidine binding to the high- and low-affinity sites are 15±4 pM (mean±standard error (s.e.)) and 360±150 pM at 24°C (Houghtling et al., 1995). Both sites display characteristic neuronal nicotinic pharmacological profiles. The similarity between the high-affinity rat brain KD and the KD for ±[3H]-epibatidine binding to chick α4β2 receptors expressed in M10 cells (4.1±0.3 pM at 24°C) suggests that the high-affinity rat brain sites are α4β2 receptors (Houghtling et al., 1995). The subunit composition of the low-affinity binding site in the rat brain is unclear. The differences between ±[3H]-epibatidine binding to the rat brain receptors and the chick α4β2 receptors expressed in M10 cells are not due to methodological differences because the assay conditions for both receptors were identical (Houghtling et al., 1995).

Mouse brain synaptosomes also appear to contain two major classes of nicotinic receptors that mediate α-bungarotoxin insensitive ACh-induced 86Rb+ efflux (Marks et al., 1999). The EC50s of these two receptor classes for ACh-induced 86Rb+ efflux are 7.2±0.3 μM ACh (mean±standard error of the mean s.e.m.) and 550±60 μM ACh (Marks et al., 1999). The nicotinic antagonist dihydro-β-erythroidine (DHβE) inhibits 86Rb+ efflux through the more ACh-sensitive receptors ~ten times more potently (IC50 of 0.2 μM DHβE) than it inhibits 86Rb+ efflux through the less ACh-sensitive receptors (IC50 of 3 μM DHβE) (Marks et al., 1999). Both receptor types are distributed throughout the brain and contain β2 nicotinic subunits (Marks et al., 1999). A regional correlation between high-affinity nicotine binding and DHβE-sensitive 86Rb+ efflux in the brain suggests that the DHβE-sensitive receptors are α4β2 nicotinic receptors (Marks et al., 1999). However, the identity of the DHβE-insensitive receptors is unclear.

±[3H]-Epibatidine binding to immunoprecipitated receptors allows us to compare the pharmacological properties of isolated native brain nicotinic receptors containing a known subunit with those of heterologously expressed nicotinic receptor subtypes, under identical experimental conditions and without confounding intracellular factors. To determine whether α4-containing rat brain nicotinic receptors and heterologously expressed rat α4β2 nicotinic receptors exhibit both high- and low-affinity ±[3H]-epibatidine binding, we immunoprecipitated solubilized nicotinic receptors with the anti-α4 antibody mAb 299 (Whiting & Lindstrom, 1988) and measured their ±[3H]-epibatidine concentration-binding reactions. The results show that both receptor types display high- and low-affinity ±[3H]-epibatidine binding. To determine whether the antagonist profile of the high-affinity native receptors matched that of the high-affinity heterologously expressed receptors, we measured the ability of two competitive antagonists (DHβE, N-n-decylnicotinium iodide (NDNI)) to inhibit 30 pM ±[3H]-epibatidine binding to these receptors. Previous studies show that DHβE inhibits high-affinity ±[3H]-epibatidine binding to rat forebrain homogenates (Houghtling et al., 1995). NDNI is a novel nicotine analogue. The results show that the high-affinity native brain and heterologously expressed α4β2 receptors exhibit similar Kis for DHβE and NDNI.

Methods

Solubilization of rat brain receptors

Adult rats were killed by an overdose of pentobarbital sodium or CO2 inhalation according to the guidelines of the University of California Animal Use Committee. After killing the rats, the entire brain was removed and frozen immediately in liquid nitrogen. Homogenates were prepared from the frozen brains using a previous protocol (Gerzanich et al., 1995). The brain homogenates were stored at −80°C in a lysis buffer containing (mM): NaCl 50, Na phosphate buffer 50, EGTA 5, EDTA 5, 2% Triton X-100, and Complete™ protease inhibitor (1 tablet per 40 ml of lysis buffer, Boehringer Mannheim, Indianapolis, IN, U.S.A.). We used a CuSO4 assay (Micro BCA Protein Assay, Pierce, Rockford, IL, U.S.A.) to measure the homogenate total protein concentration.

Oocyte expression

Stage V–VI oocytes were surgically isolated from anaesthetized Xenopus using a previous protocol (Quick & Lester, 1994) in accordance with the guidelines of the University of California Animal Use Committee. Capped rat α4-1 and β2 cRNAs were synthesized in vitro from linearized pBluescript plasmids using the mMessage mMachine RNA transcription kit (Ambion, Austin, TX, U.S.A.). The most probable subunit stoichiometry for α4β2 receptors expressed in Xenopus oocytes is (α4)2(β2)3 (Cooper et al., 1991). Therefore, we injected the α4 and β2 cRNAs in a 2 : 3 (weight/weight) stoichiometric ratio. Each oocyte received 15 ng of α4-1 RNA and 22.5 ng of β2 RNA. The injected oocytes were incubated for 2–3 days in a modified Barth's solution (mM): NaCl 96, HEPES 5, Na-pyruvate 2.5, KCl 2, CaCl2 1.8, MgCl2 1, 2.5 μg ml−1 gentamycin, 5% horse serum, pH 7.4 at 18°C. The oocytes were then solubilized as previously described (Peng et al., 1994; Gerzanich et al., 1995) using the same buffer that we used for the rat brain receptors (see above).

Adenoviral expression in CV-1 cells

Recombinant adenoviruses encoding the rat nicotinic α4 subunit (Adtetα4), the rat nicotinic β2 subunit (Adtetβ2), and the tetracycline-dependent trans-activator (AdtTA) were prepared as described previously (Neering et al., 1996; Hardy et al., 1997) using a tetracycline-regulated promoter (Gossen & Bujard, 1992), rather than a simple CMV one. Recombinant adenoviruses were purified from infected HEK cells using a CsCl gradient (Jones & Shenk, 1979). The virus titer was determined from the OD260 (1 OD unit=5×1010 plaque-forming units (p.f.u.) in HeLa cells). Ten p.f.u. of each virus (Adtetα4, Adtetβ2, AdtTA) were added per cell to a just confluent CV-1 monolayer to express the α4β2 receptors. After a 24 h incubation, the cells were washed with PBS, frozen at −80°C, and then thawed in the lysis buffer (above). After thawing and lysis, the homogenate was centrifuged at 13,000 r.p.m. The supernatant was removed and frozen at −80°C.

Vaccinia viral expression in CV-1 cells

The rat α4 and β2 subunits cDNAs were excised from pBluescript and subcloned into the vaccinia expression vector pTM1 (Elroy-Stein et al., 1989) to obtain the cDNA plasmids pTM1α4 and pTM1β2. Expression in pTM1 is under the control of a T7 RNA polymerase promoter, aided by cap-independent translation conferred by the encephalomyocarditis virus 5′ untranslated leader sequence (Elroy-Stein et al., 1989). CV-1 cells, grown to 60–70% confluence in 83 cm2 flasks in a 5% CO2 incubator, were infected with the recombinant vaccinia virus VTF7-3 (Fuerst et al., 1986). The cells were infected at a multiplicity of infection of five viral particles per cell for 30 min at 37°C in serum-free MEM medium supplemented with non-essential amino acids and sodium pyruvate. VTF7-3 is a recombinant virus that continuously expresses T7 RNA polymerase in infected cells. After infection, CV-1 cells were washed three times in MEM medium and each flask was transfected with 4 μg of pTM1α4, 4 μg of pTM1β2, and 24 μl lipofectin in serum-free MEM medium. The DNA-lipofectin mixture was allowed to adsorb to the cells for 16 h at 37°C. After this incubation, the CV-1 cells were washed three times and allowed to recover in MEM medium supplemented with 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, streptomycin (100 mg ml−1), and penicillin (100 units ml−1). After recovery, the infected-transfected cells were frozen at −80°C prior to solubilization in the same buffer used for the oocyte and rat brain experiments.

Measurement of ±[3H]-epibatidine binding

±[3H]-epibatidine (specific activity 50–60 Cimmol−1) was purchased from Amersham Life Science, Inc. (Arlington Heights, IL, U.S.A.). We followed previous protocols (Peng et al., 1994; Gerzanich et al., 1995) to measure ±[3H]-epibatidine binding to the immunoprecipitated receptors. The wells of EIA/RIA strip plates (Costar Corning Corp., Cambridge, MA, U.S.A.) were coated with purified mAb 299 (Research Biochemicals, Natick, MA, U.S.A.) by adding 0.5 or 2 μg of mAb 299 in 100 μl of 10 mM Na bicarbonate (pH 8.8) for an overnight incubation at 4°C. We blocked the coated wells with 3% bovine serum albumin in 200 μl of PBS-Tween buffer (100 mM NaCl, 10 mM Na phosphate, 0.05% Tween 20, pH 7.5) for 2 h at 4°C and rinsed the blocked wells three times with PBS-Tween buffer. Aliquot parts (100 μl) of the solubilized receptors in lysis buffer were added to the wells and incubated overnight at 4°C. We rinsed the wells three times with PBS-Tween the following day and added the appropriate ±[3H]-epibatidine concentration in PBS-Tween to each well for 4 h at 20–23°C (Houghtling et al., 1995). To avoid ligand depletion at low ±[3H]-epibatidine concentrations (0.001–0.3 nM), we incubated each well in 2–4 ml of the ±[3H]-epibatidine solution (Houghtling et al., 1995). At higher ±[3H]-epibatidine concentrations ([gt-or-equal, slanted]1 nM), we used an incubation volume of 200 μl. The amount of receptor added to the wells was adjusted so that the depletion of free ±[3H]-epibatidine was <10%. The free ligand concentrations in the ±[3H]-epibatidine concentration-binding experiments were corrected for ligand depletion. After the ±[3H]-epibatidine incubation, the wells were rinsed three times with ice-cold PBS-Tween buffer, placed whole into 1 ml of scintillation fluid, and counted. We measured nonspecific ±[3H]-epibatidine binding three different ways. The first method was to measure ±[3H]-epibatidine binding as described above but in the presence of 100 μM cold (−) nicotine (Houghtling et al., 1995). The second method (used only with the recombinant receptors) was to measure ±[3H]-epibatidine binding to the immunoprecipitated protein from un-injected oocytes or uninfected CV-1 cells. The third method was to measure ±[3H]-epibatidine binding to uncoated, but blocked, wells (Peng et al., 1994). The nonspecific binding measured by these three methods was not significantly different. Thus, ±[3H]-epibatidine binding to the uncoated, blocked wells and background radiation were the main sources of nonspecific binding. The ±[3H]-epibatidine concentration-binding experiments were replicated twice.

Kinetic measurements

The forward rate constant for ±[3H]-epibatidine binding to the α4β2 receptors expressed in oocytes at 20–23°C was measured by placing wells containing the immunoprecipitated receptors in 4 ml of 0.03 nM ±[3H]-epibatidine for times ranging from 1–300 min. The forward binding reaction was stopped by adding an excess (1 mM) of cold (−)-nicotine. After stopping the reaction, we rinsed the wells three times with ice-cold PBS-Tween and counted the bound ±[3H]-epibatidine. The backward rate constant for ±[3H]-epibatidine binding to the α4β2 receptors at 20–23°C was measured by incubating wells containing the immunoprecipitated receptors in 4 ml of 0.03 mM ±[3H]-epibatidine for 4 h. After the 4 h incubation, we stopped the reaction by adding 1 mM cold (−)-nicotine to the wells. At times ranging from 1–600 min after adding the nicotine, the wells were rinsed three times with ice-cold PBS-Tween and counted. Nonspecific binding was measured by adding 1 mM cold (−)-nicotine to the 0.03 nM ±[3H]-epibatidine incubations.

Inhibition of ±[3H]-epibatidine binding

NDNI was synthesized according to the method of Crooks et al. (1995). The IC50 of DHβE and NDNI for inhibiting ±[3H]-epibatidine binding was estimated by fitting the inhibitor concentration-binding data to the following equation:

equation image

where [I] was the inhibitor concentration, B was the amount of bound ±[3H]-epibatidine, and Bmax was the maximum bound ±[3H]-epibatidine. The errors reported for the fitted parameters (nH, KD, IC50, Bmax, kf, kb, etc.) are s.e.s. The degrees of freedom (d.f.) associated with these errors are the number of points in the graph (number of different concentration or time measurements) minus two. The Ki for inhibition was calculated from the following equation,

equation image

where 0.03 nM was the ±[3H]-epibatidine concentration used to measure the IC50. To calculate the s.e. for the Ki, we used the following approximation,

equation image

This approximation assumes that 0.03 nM was >> the KD for the high-affinity ±[3H]-epibatidine binding sites. We calculated the variance of the product (KDIC50) using a previously derived formula for the variance of the product of two independent random variables (Mood et al., 1974). To avoid radioligand depletion, the maximum number of counts bound per well in the inhibitor experiments was kept to 300–500 c.p.m. This level of binding yielded an adequate signal : noise ratio because the maximum number of counts bound per well was still 7–15 times larger than the typical amount of nonspecific binding. The inhibitor experiments were replicated 2–4 times.

Immunodepletion with mAb 270

Dr Kenneth Dorshkind (University of California, Los Angeles, CA, U.S.A.) provided the mAb 270 ascites used for the immunodepletion experiments. Further antibody purification was unnecessary because the ascites was obtained from severe combined immunodeficient (SCID) mice. Comparisons between the amount of receptor bound to wells coated with 2 μg of mAb 299 and wells coated with 2 μl of mAb 270 ascites suggest that the mAb 270 ascites contained 1–2 mg antibody ml−1 (assuming similar immunoprecipitation efficiencies for both antibodies). To immunodeplete the β2-containing nicotinic receptors, the rat brain extracts were subjected to three serial overnight incubations with mAb 270 at 4°C (25–50 μg antibody (μl of rat brain extract added)−1) before immunoprecipitation onto the EIA/RIA strip plates. After each overnight incubation, 100 μl of extract was rotated with 50 μl of a 20% slurry of Protein G sepharose beads (Pharmacia, Piscataway, NJ, U.S.A.) for 3 h at 4°C to collect the immune complexes. The sample was centrifuged and the supernatant was collected after incubation with the beads. After the third mAb 270 pre-incubation, we immunoprecipitated the remaining receptors onto EIA/RIA strip plates and measured ±[3H]-epibatidine binding. To control for non-specific protein loss, aliquot parts of the rat brain extract were processed exactly as above but without mAb 270.

Serial Triton X-100 extraction

To determine whether a single detergent extraction removed all of the Triton X-100-soluble nicotinic receptors from the rat brain homogenates, we performed two extractions in series. In the first extraction, rat brain tissue was solubilized with 2% Triton X-100 and centrifuged as described above. We then measured 30 nM ±[3H]-epibatidine binding to the initial supernatant, re-solubilized the initial pellet in lysis buffer, centrifuged the mixture, and measured ±[3H]-epibatidine binding to the supernatant from the re-solubilized pellet. The supernatant from the re-solubilized pellet contained 11% of the total ±[3H]-epibatidine bound to the combined extracts from the initial homogenate and re-solubilized pellet. Therefore, a single detergent extraction removed 89% of the Triton X-100-soluble receptors from the homogenates.

MAb 299 immunoprecipitation efficiency

We used serial immunodepletions to measure the efficiency of mAb 299 immunoprecipitation. The Triton X-100 extract from oocytes expressing α4β2 receptors was divided into seven groups of aliquot parts (four aliquot parts per group). Group 1 was incubated once overnight in mAb-299-coated EIA/RIA strip plate wells (2 μg antibody per well) and then the ±[3H]-epibatidine bound to the wells, after a 40 min incubation in a saturating concentration of ±[3H]-epibatidine (30 nM), was measured. The six remaining groups were divided into experimentals (groups 2–4 and controls (groups 5–7). Groups 2–4 were pre-incubated 1–3 times in mAb-299-coated wells before a final overnight incubation in mAb-299-coated wells. Groups 5–7 were similarly pre-incubated 1–3 times in blocked, uncoated wells before a final overnight incubation in mAb-299-coated wells. Nonspecific receptor loss was estimated by dividing ±[3H]-epibatidine bound by groups 5–7 by that bound by group 1. To correct for nonspecific loss, we divided the ±[3H]-epibatidine bound by the experimental groups (2–4) by the fraction of ±[3H]-epibatidine binding in group 1 remaining in the corresponding control groups (5–7). Three serial pre-incubations in the mAb-299-coated wells removed >80% of the ±[3H]-epibatidine binding activity from the extracts (Figure 1A, mAb 299 Immunodepleted). Three serial pre-incubations in blocked, uncoated wells removed ~20% of the ±[3H]-epibatidine binding activity from the extracts (Figure 1A, Control). After correcting for nonspecific receptor loss, the amount of ±[3H]-epibatidine bound to the mAb-299-precipitated receptors declined exponentially as a function of the number of mAb 299 pre-incubations (Figure 1B). Each pre-incubation removed 26% of the ±[3H]-epibatidine binding activity. Thus, the mAb 299 immunoprecipitation efficiency under our experimental conditions was 26%.

Figure 1
A single incubation in a mAb-299-coated EIA/RIA well immunoprecipitated 26% of the mAb-299-precipitable receptors in the oocyte extract. (A) The amount of ±[3H]-epibatidine bound to the mAb-299-coated wells after 0–3 ...

Results

Kinetics of ±[3H]-epibatidine binding to α4β2 receptors

±[3H]-Epibatidine association to (Figure 2A) and dissociation from (Figure 2B) α4β2 receptors expressed in Xenopus oocytes followed a single-exponential time course. We used a concentration of 30 pM ±[3H]-epibatidine for these experiments because our preliminary experiments suggested that it should produce 40–50% receptor occupancy. The association rate constant (kf) for 30 pM ±[3H]-epibatidine was 0.02±0.01 min−1 (±s.e., d.f.=7) at 20–23°C. The dissociation rate constant (kb) for the same ±[3H]-epibatidine concentration was 0.03±0.01 min−1 (d.f.=8). The receptor occupancy is given by kf(kf+kb)−1. Therefore, the kinetic data predict that 30 pM ±[3H]-epibatidine should produce 40% receptor occupancy.

Figure 2
Time course of 30 pM ±[3H]-epibatidine binding to (A) and dissociation from (B) mAb-299-precipitated rat α4β2 nicotinic receptors expressed in Xenopus oocytes. The error bars are ±s.e.mean (n=4 ...

±[3H]-Epibatidine binding to mAb-299-precipitated native brain receptors

To measure the ±[3H]-epibatidine-concentration binding relation of the mAb-299-precipitated rat brain receptors, we incubated the immunoprecipitated receptors in 0.001–30 nM ±[3H]-epibatidine for 4 h at 20–23°C (Figure 3). This incubation time was ~eight times longer than the α4β2 ±[3H]-epibatidine dissociation time constant (33 min). Similar to that (nH=0.73±0.03) previously reported for ±[3H]-epibatidine binding to rat forebrain homogenates (Houghtling et al., 1995), the Hill coefficient (0.69±0.05, d.f.=6) for ±[3H]-epibatidine binding to the mAb-299-precipitated native brain receptors was less than unity, suggesting receptor heterogeneity. Therefore, we fit the ±[3H]-epibatidine concentration-binding data to a two independent binding site model (Figure 3). The resulting KDs (11±4 pM; 560±70 pM, d.f.=8) were close to those (15±4 pM, 360±150 pM) reported previously for ±[3H]-epibatidine binding to rat forebrain homogenates (Houghtling et al., 1995). The high-affinity site (Bmax1=32±4 fmol (mg protein)−1) accounted for 74% of the total ±[3H]-epibatidine binding and the low-affinity site (Bmax2=11±4 fmol (mg protein)−1) accounted for 26%. Thus, α4-containing rat brain nicotinic receptors display both high- and low-affinity ±[3H]-epibatidine binding. Based on the two-site fit to the ±[3H]-epibatidine concentration-binding data (Figure 3), 55% of the ±[3H]-epibatidine binding sites should be occupied at a concentration of 30 pM, similar to the 40% occupancy predicted from the oocyte α4β2 kinetic data (above). The ±[3H]-epibatidine bound to mAb-299-precipitated rat brain receptors (2 μg mAb 299 per well) incubated in a saturating (30 nM) ±[3H]-epibatidine concentration was 30±3 fmol ±[3H]-epibatidine (mg protein)−1 (mean±s.e.mean, n=6). Correcting for the mAb 299 immunoprecipitation efficiency (see Methods), the density of mAb-299-precipitable ±[3H]-epibatidine binding sites in the rat brain Triton X-100 extract should be 115±12 fmol ±[3H]-epibatidine (mg protein)−1. This value exceeds the previously measured Bmax (80 fmol (mg protein)−1) for ±[3H]-epibatidine binding to rat forebrain homogenates (Houghtling et al., 1995) and the Bmax (~50 fmol (mg protein)−1) for −[3H]-nicotine binding to non-detergent rat brain membrane extracts (Marks et al., 1986). However, previous results show that Triton X-100 (1%) extraction enriches the density of [3H]-nicotine binding sites per mg of protein in rat brain extracts (Abood et al., 1980). Therefore, rat brain nicotinic receptors represent a greater fraction of the Triton X-100-soluble rat brain proteins than of the total rat brain proteins.

Figure 3
The ±[3H]-epibatidine concentration-binding relation for mAb-299-precipitated rat brain receptors. The symbols are the amount of bound ±[3H]-epibatidine (in fmol (mg protein)−1). The error bars are ...

Most mAb-299-precipitated rat brain receptors contain β2

The β4 subunit is present in the rat brain (Dineley-Miller & Patrick, 1992) and forms functional nicotinic receptors with α4 subunits in Xenopus oocytes (Duvoisin et al., 1989). However, it is far less abundant than the β2 subunit (Zoli et al., 1995). To determine what percentage of mAb-299-precipitated rat brain receptors contain β2, we measured the fraction of maximum ±[3H]-epibatidine binding immunodepleted from rat brain extracts by serial pre-incubations with the anti-β2 antibody mAb 270 (see Methods). We used a saturating ±[3H]-epibatidine concentration (30 nM) for these experiments. Three serial mAb 270 immunodepletions removed 97% of the mAb 270-precipitable ±[3H]-epibatidine receptors from the rat brain extracts and 87% of the mAb-299-precipitable ±[3H]-epibatidine receptors (Figure 4). Thus, 89% (87% (0.97)−1) of the mAb-299-precipitated receptors in the rat brain contained β2.

Figure 4
Three overnight mAb 270 pre-incubations depleted most of the mAb-270- and mAb-299-precipitable ±[3H]-epibatidine binding from the rat brain extracts. The free ±[3H]-epibatidine concentration was 30 n ...

±[3H]-Epibatidine binding to heterologously expressed α4β2 receptors

Similar to the mAb-299-precipitated rat brain receptors (above), the Hill coefficients for ±[3H]-epibatidine binding to the oocyte (nH=0.69±0.03, d.f.=6) and adenoviral-expressed CV-1 α4β2 receptors (0.70±0.02, d.f.=2) were less than unity. Therefore, we fit the ±[3H]-epibatidine concentration-binding data for the oocytes (Figure 5) and adenoviral-infected CV-1 cells (Figure 6A) to a two-site model. The KDs for ±[3H]-epibatidine binding to the oocyte α4β2 receptors were 14±2 pM and 1.2±0.5 nM (d.f.=8). Likewise, the KDs for ±[3H]-epibatidine binding to the adenoviral-infected CV-1 α4β2 receptors were 7±2 pM and 1±2 nM (d.f.=6). These KDs were not significantly different from those of the native brain receptors (11±4 pM, 560±70 pM). Moreover, the high-affinity oocyte site accounted for nearly the same percentage (73%) to total ±[3H]-epibatidine binding sites as the high-affinity site of native brain receptors did (74%). The high-affinity sites in the adenoviral-infected CV-1 cells accounted for a somewhat higher percentage (85%) of the total binding sites. The oocyte equilibrium binding data predicted 50% receptor occupancy in 30 pM ±[3H]-epibatidine, similar to the 40% occupancy rate predicted from the kinetic data (Figure 1A,B). The Hill coefficient (1.0±0.2, d.f.=7) for ±[3H]-epibatidine binding to rat α4β2 receptors expressed in CV-1 cells with vaccinia virus was larger than that for the other expression systems, suggesting a more homogenous receptor population. Therefore, we fitted the vaccinia viral ±[3H]-epibatidine concentration-binding data to a single binding site model. The resulting KD (40±3 pM, d.f.=8) was somewhat larger than the high-affinity KD for native brain receptors (11±4 pM) and α4β2 receptors expressed in oocytes (14±2 pM). This discrepancy could be due to an unresolved population of low-affinity ±[3H]-epibatidine binding sites in the vaccinia infected-transfected cells.

Figure 5
The ±[3H]-epibatidine concentration-binding relation for mAb-299-precipitated rat α4β2 nicotinic receptors expressed in Xenopus oocytes. The symbols are the amount of bound ±[3H]-epibatidine ...
Figure 6
±[3H]-epibatidine concentration-binding relations for mAb-299-precipitated rat α4β2 receptors expressed in CV-1 cells using recombinant adenovirus (A) or vaccinia viral infection/transfection (B) (see Methods for ...

Native and heterologously expressed receptors have similar DHβE and NDNI Kis

To compare the antagonist profiles of the native brain and heterogously expressed α4β2 receptors, we measured the effects of two competitive antagonists (DHβE, NDNI) on 30 pM ±[3H]-epibatidine binding to these receptors (Figures 7 and and8,8, Table 1). Figure 8A shows the structure of the novel antagonist NDNI. At a concentration of 30 pM ±[3H]-epibatidine, we were primarily measuring antagonism of the high-affinity ±[3H]-epibatidine binding sites because the fractional occupancy of the low-affinity sites was only 3–5% at this ±[3H]-epibatidine concentration (assuming a low-affinity KD of 0.6–1 nM). DHβE was a more potent antagonist (IC50=260–470 nM) than NDNI (IC50=7–10 μM) (Table 1) and inhibited ±[3H]-epibatidine binding to the high-affinity site ~ten times more potently than NDNI. The native brain and heterologously expressed α4β2 receptors displayed no significant differences in regard to DHβE (Figure 7) and NDNI (Figure 8B) inhibition of ±[3H]-epibatidine binding (Table 1). However, the DHβE Kis for the native and heterologously expressed receptors (50–130 nM, Table 1) were somewhat larger than that predicted (16 nM) from the inhibition of ±[3H]-epibatidine binding to rat forebrain homogenates by DHβE (Houghtling et al., 1995).

Figure 7
DHβE inhibited 30 pM ±[3H]-epibatidine binding to mAb-299-precipitated rat brain receptors (Rat), rat α4β2 receptors expressed in oocytes (Oocyte), and rat α4β2 receptors expressed ...
Figure 8
(A) Structural formula of N-n-decylnicotinium iodide (NDNI). (B) NDNI inhibited 30 pM ±[3H]-epibatidine binding to mAb-299-precipitated rat brain, rat α4β2 receptors expressed in oocytes, and rat α4β2 ...
Table 1
IC50s and kis for the competitive antagonists

Discussion

Our results show that rat α4β2 nicotinic receptors expressed in Xenopus occytes or CV-1 cells and, native rat brain nicotinic receptors containing α4 and β2 subunits, display similar pharmacological properties in regard to ±[3H]-epibatidine binding and the inhibition of high-affinity ±[3H]-epibatidine binding by the competitive antagonists DHβE and NDNI. Similar to previous studies of rat brain nicotinic receptors (Whiting & Lindstrom, 1987; Flores et al., 1991), our immunodepletion experiments further show that most α4-containing nicotinic receptors in the rat brain also contain β2. Moreover, α4β2 receptors expressed in native brain neurons, oocytes, and CV-1 cells with adenovirus all exhibit both high- and low-affinity ±[3H]-epibatidine binding. Previous studies of chick brain nicotinic receptors immunoprecipitated with mAb 299 show that the pharmacological properties and macromolecular size of these receptors also resemble those of heterologously expressed chick α4β2 nicotinic receptors immunoprecipitated with the anti-β2 antibody mAb 290 (Whiting et al., 1991).

Nicotinic receptors in rat forebrain homogenates display both high- (KD=15 pM) and low affinity (KD=360 pM) ±[3H]-epibatidine binding (Houghtling et al., 1995). Our experiments show that α4 subunits are present in both types of binding sites. MAb 270 immunodepletion experiments further show that 89% of the mAb-299-precipitated binding sites contain β2. Since the high-affinity sites account for only 74% of the total mAb-299-precipitated rat brain ±[3H]-epibatidine binding sites, at least some low-affinity rat brain receptors ([gt-or-equal, slanted]58%) must contain both α4 and β2 subunits. In support of this hypothesis, we find that α4β2 receptors expressed in Xenopus oocytes and CV-1 cells display both low- and high-affinity ±[3H]-epibatidine binding.

Comparisons between the properties of native and recombinant ganglionic nicotinic receptors (Sivilotti et al., 1997; Lewis et al., 1997) suggest that the host cell can affect the biophysical properties of ganglionic nicotinic receptors. The relative sensitivity of rat nicotinic receptors in the rat superior cervical ganglion (SCG) to different nicotonic agonists (except DMPP) resembles that of rat α3β4 receptors expressed in Xenopus oocytes (Covernton et al., 1994). However, the single-channel conductance and burst duration of the native rat SCG receptors are significantly different (Lewis et al., 1997; Sivilotti et al., 1997). In contrast, mouse fibroblasts expressing rat α3β4 receptors contain one population of nicotinic channels with properties resembling those of the native SCG nicotinic receptor and another with properties resembling those of the rat α3β4 receptors expressed in Xenopus oocytes (Lewis et al., 1997; Sivilotti et al., 1997). Thus, mammalian cell lines, but not Xenopus oocytes, appear to be capable of assembling a population of α3β4 channels with single-channel properties similar to the native ones.

The host cell type appears to affect the properties of brain nicotinic receptors less than those of ganglionic nicotinic receptors. The conductance (49 pS) of rat α4β2 channels expressed in oocytes closely matches that of human α4β2 channels (46 pS) expressed in HEK 293 cells (Buisson et al., 1996; Figl et al., 1998). Moreover, the EC50 and Hill coefficient of rat and human α4β2 ACh concentration-response relations are nearly identical (Buisson et al., 1996; Figl et al., 1998). Our results show that α4β2 nicotinic receptors immunoprecipitated from rat brain neurons, Xenopus oocytes, and CV-1 cells display similar pharmacological properties in regard to ±[3H]-epibatidine binding and the competitive inhibition of ±[3H]-epibatidine binding by DHβE and NDNI. Thus, Xenopus oocytes appear to be capable of synthesizing an α4β2 receptor with pharmacological properties similar to those expressed in rat neurons and mammalian cell lines despite differences in the receptor assembly and synthesis temperature (18°C versus 36 °C) and potential differences in folding proteins.

Heterologous expression provides the only available means of studying the pharmacological and biophysical properties of identified neuronal nicotinic receptor subtypes. The relevance of the results for heterologously expressed receptors depends on how closely the properties of those receptors match the properties of the native receptors. Our results show that rat α4β2 nicotinic receptors expressed in Xenopus oocytes and CV-1 cells display similar pharmacological behaviour in regard to at least three drugs, ±[3H]-epibatidine, DHβE, and NDNI. Further research using this approach will tell us if these similarities extend to other types of competitive antagonists.

Acknowledgments

We thank Dr Kenneth Dorshkind of the University of California, Los Angeles (Los Angeles, U.S.A.) for providing mAb 270 ascites. This research was supported by grants from the American Heart Association, California Affiliate (#96–254), the UC Tobacco-related Disease Program (TRDRP), and the NIH (#RO1DA10934).

Abbreviations

ACh
acetylcholine
AV
adenoviral expression
df
degrees of freedom
CMV
cytomegaloviral
DHβE
dihydro-β-erythroidine
DMPP
dimethylphenylpiperazinium
NDNI
N-n-decylnicotinium iodide
nH
apparent Hill coefficient
SCG
superior cervical ganglion
SCID
severe combined immunodeficient
s.e.
standard error
s.e.m.
standard error of the mean
VV
vaccinia viral infection-transfection

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