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Expression of Nigrostriatal 6-Containing Nicotinic Acetylcholine Receptors Is Selectively Reduced, but Not Eliminated, by 3 Subunit Gene Deletion

Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Medical Pharmacology and Center of Excellence on Neurodegenerative Diseases, University of Milan, Milan, Italy (C.G., M.M., F.C., L.R.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (M.J.M., A.C.C., P.W.); and Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado (J.M.M.)

Received February 9, 2005; accepted March 4, 2005

	Abstract

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mRNAs for the neuronal nicotinic acetylcholine receptor (nAChR) 6 and 3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that 6- or 3-dependent nAChRs bind -conotoxin MII (-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of 3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that 3-null mutation selectively reduced striatal 6* nAChR expression by 76% versus 3^+/+ control. Parallel experiments showed a smaller reduction in both midbrain 3* and 6* nAChRs (34 and 42% versus 3^+/+ control, respectively). Sedimentation coefficient determinations indicated that residual 6* nAChRs in 3^–/– striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified 3* nAChRs demonstrated that almost all wild-type striatal 3* nAChRs also contain 4, 6, and 2 subunits, although a small population of non-3 6* nAChRs is also expressed. 3 subunit incorporation seemed to increase 4 participation in 62* complexes. ¹²⁵I-Epibatidine competition binding studies showed that the -CtxMII affinity of 6* nAChRs from the striata of 3^–/– mice was similar to those isolated from 3^+/+ animals. Together, the results of these experiments show that the 3 subunit is important for the correct assembly, stability and/or transport of 6* nAChRs in dopaminergic neurons and influences their subunit composition. However, 3 subunit expression is not essential for the expression of 6*, high-affinity -CtxMII binding nAChRs.

Distinct subunit composition defines the many neuronal nAChR subtypes, which exhibit diverse functional and pharmacological properties. The subtypes may be divided into two subfamilies. The first comprises heteropentameric nAChRs combining ligand binding subunits (2, 3, 4, and 6) with structural (2 and 4), and sometimes auxiliary 5 and 3), subunits. The second encompasses nAChRs that bind -bungarotoxin (Bgtx) and are generally thought to exclusively contain ligand binding subunits (7, 8, 9, or 10), although other subunits have been implicated by Yu and Role (1998). The second family of nAChRs may be either homopentameric or heteropentameric.

By acting on mesostriatal dopaminergic system nAChRs, nicotine plays an important role in mediating several behavioral effects such as the modulation of locomotor activity, reinforcement, and habit learning (Di Chiara 2000). These effects are thought to be mediated by increased dopamine release in the mesostriatal dopamine system (Picciotto et al., 1998).

Striatal 6-hydroxydopamine-lesioning studies in rats (Zoli et al., 2002) and knockout mice (Champtiaux et al., 2003) indicate that there are two major nAChR subtype populations in the striatum: 62* and 4(non-6)2*. Both of these populations are heterogeneous, differently expressed by the dopaminergic and nondopaminergic neurons, and involved in mediating the release of dopamine from striatal synaptosomes (Champtiaux et al., 2003; Quik et al., 2003; Salminen et al., 2004). Ligand binding and dopamine release studies have indicated that the two major striatal nAChR populations can be distinguished by their differential interaction with -conotoxin MII (-CtxMII), which selectively binds to and blocks the 62* population with high affinity (Zoli et al., 2002; Champtiaux et al., 2003). This 62* nAChR population may be intimately associated with the 3 subunit. In situ hybridization studies have identified the selective colocalization of 6 and 3 mRNAs in dopaminergic neurons (Le Novère et al., 1996; Azam et al., 2002). Furthermore, 3 subunit-null mice exhibit alterations in behaviors that are controlled by nigrostriatal and mesolimbic dopaminergic activity, and lose much of the -CtxMII-sensitive portion of striatal dopamine release (Cui et al., 2003).

This study used a combination of ligand binding, immunoprecipitation, and immunopurification techniques to test whether the 3 and 6 subunits are indeed extensively associated with each other. It also examined the consequences of 3-null mutation on the expression, properties, and composition of striatal and midbrain nAChRs. 3 subunit deletion markedly and selectively reduced 6* nAChR expression in both striatum and midbrain, without altering the residual 62* nAChRs' -CtxMII affinity. The results also indicated that almost all 3 subunits are present in 6* nAChRs, where they seem to promote the formation of a complex 4623 nAChR subtype. However, some wild-type 6* nAChRs are expressed that do not contain 3. Furthermore, the dopamine cell body and -terminal nAChR populations differ. In particular, the midbrain contains a novel (in mammalian brain) 33* nAChR subtype. It seems that 3 expression is not necessary for the expression of all mesostriatal 62* nAChRs, but it is critical for the correct assembly and/or transport of a major subset.

	Materials and Methods

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Antibody Production and Characterization
The polyclonal antibodies (Abs) used were subunit-specific, produced in rabbit against peptides derived from the C-terminal (COOH) or intracytoplasmic loop (CYT) regions of the rat (R), human (H), or mouse (M) subunit sequences, and affinity purified as described previously (Zoli et al., 2002). Most of the Abs were the same as those described previously (Zoli et al., 2002, Champtiaux et al., 2003; Moretti et al., 2004). Given the central role of 3 in this investigation, we generated an antiserum specifically directed against a mouse 3 subunit cytoplasmic peptide (M-CYT), DGTESKGTVRGKFPGKKKQTPTSD, to replace the rat-directed antiserum used previously. These experiments critically depend on Ab specificity and immunoprecipitation efficacy, both of which were carefully checked here or previously (Zoli et al., 2002; Champtiaux et al., 2003; Moretti et al., 2004) in control experiments using tissue obtained from relevant nAChR subunit null mutant animals and/or heterologously expressed nAChRs. Most importantly, the CYT- and COOH-directed 3 Abs failed to immunoprecipitate significant amounts (less than 1%) of [³H]Epi labeled receptors from 3^–/– mouse superior colliculus, confirming their specificity. In 3^+/+ superior colliculus, however, both Abs were effective, although the CYT-directed Ab was slightly more so than the COOH-directed Ab (28.0 ± 0.9% of sites immunoprecipitated versus 21.2 ± 1.2%, respectively; n = 4). For this reason, we used the anti-3 CYT Ab exclusively where possible. It is important to note that although the Abs used here have been tested and shown to have high efficacy, few Ab preparations achieve absolutely complete recovery of their targets. Therefore, all immunoprecipitation values should be treated as close, but potentially slightly low, determinations of the proportion of target subunits in the nAChR populations investigated.

Animals
Mice modified to contain a null mutation in the 3 nAChR subunit gene (Cui et al., 2003) were bred at the Institute for Behavioral Genetics (University of Colorado, Boulder, CO). All mice used in this study were maintained on the original mixed C57BL/6J/129SvEv/Tac background. Mice were housed five per cage, and the vivarium was maintained on a 12-h light/dark cycle (lights on from 7:00 AM to 7:00 PM). Mice were given free access to food and water. Mice were genotyped by Polymerase chain reaction, using DNA extracted from tail clippings obtained at approximately 40 days of age. All procedures used in this study were approved by the Animal Care and Utilization Committee of the University of Colorado, Boulder. All mice used in this study were between 60 and 120 days of age.

Preparation of Membranes and 2% Triton X-100 Extracts from Striatum and Midbrain
The tissues were dissected, immediately frozen on dry ice, and stored at –80°C for later use. In every experiment, the tissues from striatum (0.15–0.25 g) or midbrain (0.15–0.25 g) were homogenized in 10 ml of 50 mM sodium phosphate, pH 7.4, 1 M NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM phenylmethylsulfonyl fluoride (PMSF) with a Potter homogenizer. The homogenates were then diluted further in the same buffer and centrifuged for 1.5 h at 60,000g.

The procedures of homogenization, dilution, and centrifugation of the total membranes were performed twice, after which the pellets were collected, rapidly rinsed with 50 mM Tris-HCl, pH 7, 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, and 2 mM PMSF, and then resuspended in the same buffer containing a mixture of 20 µg/ml of each of the following protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin. Triton X-100 to a final concentration of 2% was added to the washed membranes, which were extracted for 2 h at 4°C.

The extracts were then centrifuged for 1.5 h at 60,000g, recovered, and an aliquot of the resultant supernatants was collected for protein measurement using the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard.

Binding Assay and Pharmacological Experiments
(±)-[³H]Epibatine (Epi; specific activity, 56–60 Ci/mmol) and ¹²⁵I-Epi (specific activity, 2200 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston MA); ¹²⁵I-Bgtx (specific activity, 214 Ci/mmol) was purchased from Amersham Biosciences Inc. (Piscataway, NJ); nonradioactive Epi was from RBI/Sigma (Natick, MA); -CtxMII was synthesized as described by Cartier et al. (1996). All other compounds were sourced from Sigma-Aldrich (St. Louis, MO).

Membranes. [³H]Epibatidine Binding. 2* and 4* nAChRs bind [³H]Epi with picomolar affinity, and 7 receptors bind it with nanomolar affinity (Gerzanich et al., 1995). To ensure that the 7 subtype did not contribute to [³H]Epi binding, tissue extract and immunoprecipitation epibatidine binding experiments were performed in the presence of 2 µM Bgtx, which specifically binds to the 7 subtype and prevents Epi from binding to the subtypes containing this subunit. Binding to membrane homogenates obtained from striatal and midbrain membranes was performed overnight by incubating aliquots of the membrane with [³H]Epi concentrations ranging from 0.005 to 2.5 nM at 4°C. Nonspecific binding (averaging 5–10% of total binding) was determined in parallel by means of incubation in the presence of 100 nM unlabeled Epi. At the end of the incubation, the samples were filtered on a GFC filter soaked in 0.5% polyethylenimine and washed with 15 ml of 10 mM sodium phosphate, pH 7.4, plus 50 mM NaCl, and the filters were counted in a liquid scintillation counter.

¹²⁵I--Bungarotoxin binding. Saturation experiments were performed by incubating midbrain and striatal membranes overnight with 0.01 to 10 nM ¹²⁵I-Bgtx at 20°C. For ¹²⁵I-Bgtx, 2 mg/ml bovine serum albumin was added to the suspension buffer. Specific radioligand binding was defined as total binding minus nonspecific binding determined in the presence of 1 µM nonradioactive Bgtx.

[³H]Epibatidine binding to solubilized receptor. Triton X-100 extracts were preincubated with 2 µM Bgtx for 3 h and then labeled with 2 nM [³H]Epi. Tissue extract binding was performed using DE52 ion-exchange resin (Whatman, Maidstone, UK) as described previously (Vailati et al., 2000).

Immunoprecipitation of [³H]Epibatidine-Labeled Receptors by Anti-subunit–specific Antibodies
Membrane preparations were extracted with 2% Triton X-100 (1 h; 22°C). Extracts were preincubated with 2 µM Bgtx, labeled with 2 nM [³H]Epi and then incubated overnight with a saturating concentration of affinity purified Abs (20–30 µg). The immunoprecipitate was recovered by incubating the samples with beads containing bound anti-rabbit goat IgG (Technogenetics, Milan, Italy). The level of Ab immunoprecipitation was expressed as the percentage of [³H]Epi-labeled receptors immunoprecipitated by the antibodies (taking the amount present in the Triton X-100 extract solution before immunoprecipitation as 100%) or as femtomoles of immunoprecipitated receptors per milligram of protein.

Striatal 3* Population Immunopurification and Analysis
For each immunopurification, striatal membranes of 20 to 30 mice (0.4–0.5 g) were prepared as described above. The membranes (12 ml) were then extracted by addition of 2% Triton X-100 as described above and centrifuged. Extracts (14–15 ml) were incubated three times with 5 ml of Sepharose-4B with bound anti-3 CYT Abs to remove the 3 subunit-containing receptors (3* population). This 3* population was eluted from column by means of incubation with 100 µM M-CYT peptide, and the flow-through of the 3 column was then analyzed for the subunit content of the remaining nAChRs. Analysis of the purified 3* population's subunit content was performed by immunoprecipitation using subunit-specific Abs, as described above, after labeling with 2 nM [³H]Epi.

Pharmacological Experiments on Immunoimmobilized Subtypes
Affinity-purified anti-6 or anti-2 Abs (10 µg/ml in 50 mM phosphate buffer, pH 7.5) were bound to microwells (Maxi-Sorp; Nunc, Naperville, IL) by means of overnight incubation at 4°C. The following day, the wells were washed to remove the excess of unbound Abs and then incubated overnight at 4°C with 200 µl of 2% Triton X-100 striatal membrane extract prepared from the 3^+/+ and 3^–/– genotypes (containing 10–30 fmol of ¹²⁵I-Epi binding sites). After incubation, the wells were washed and immobilized receptors quantified using ¹²⁵I-Epi binding.

Immobilized nAChRs were incubated overnight at 4°C with 200 µl of ¹²⁵I-Epi at concentrations ranging from 0.005 to 1 nM. All of the incubations were performed in a buffer containing 50 mM Tris-HCl, pH 7, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 2 mg/ml bovine serum albumin, and 0.05% Tween 20. Specifically labeled ligand binding was defined as total binding minus the binding in the presence of 100 nM unlabeled Epi. Epibatidine and -CtxMII inhibition of ¹²⁵I-Epi binding to the immobilized nAChRs was measured by preincubating the indicated concentrations of the compounds for 30 min at room temperature, followed by overnight incubation with 0.05 nM ¹²⁵I-Epi. After incubation, the wells were washed seven times with ice-cold phosphate-buffered saline containing 0.05% Tween 20, and bound radioactivity was recovered by incubation with 200 µl of 2 N NaOH for 2 h. Bound radioactivity was then determined by counting.

Sucrose Gradient Centrifugation
Linear 5 to 20% sucrose gradients in phosphate-buffered saline plus 1 mM PMSF and 0.1% Triton X-100 were prepared using a Buckler gradient maker (Fort Lee, NJ) and stored for 4 h at 4°C before use. The volume of each gradient was 12 ml. Then, 500 µl of 2% Triton X-100 extracts obtained from Torpedo californica electric organ (0.5–1 g, labeled with 6 nM ¹²⁵I-Bgtx) and 2% Triton X-100 extracts of the striatum or midbrain of 3^+/+ or 3^–/– mice were loaded on the gradients and centrifuged for 14 h at 40,000 rpm in a Beckman SW41. Fractions of 0.5 ml were collected from the top of the gradient and directly counted on a gamma counter (in the case of the T. californica gradients) or added to the affinity-purified anti-6 or anti-2 Abs bound to microwells, and processed as described for the pharmacological experiments.

Data Analysis. The experimental data obtained from the saturation binding experiments performed on immunoimmobilized subtypes were analyzed by means of a nonlinear least square procedure using the LIGAND program as described by Munson and Rodbard (1980). The calculated binding parameters were obtained by simultaneously fitting three independent experiments.

The selection of the best fitting (i.e., one- versus two-site model) and evaluation of the statistical significance of the parameters (i.e., comparison of the binding parameters of the two groups) were based on the F-test for the "extra sum of square" principle. A p value of <0.05 was considered statistically significant (Munson and Rodbard, 1980).

The K_i values of -CtxMII and epibatidine inhibition binding were also determined by means of the LIGAND program using the data obtained from three independent competition experiments and compared by means of the F-test as described above.

	Results

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nAChR Expression in the Striatum and Midbrain of 3 Genotypes. To determine the effect of 3 nAChR gene deletion on overall nAChR expression in the mesostriatal pathway, we performed ligand binding studies using membranes prepared from 3 wild-type (3^+/+), heterozygote (3^+/–), and knockout (3^–/–) mice.

[³H]Epibatidine Binding nAChRs. [³H]Epi binding was measured in striatum and midbrain membranes obtained from each 3 genotype. The membranes were preincubated with 2 µM Bgtx to block epibatidine binding at Btx-sensitive sites. Binding was conducted using a saturating concentration of [³H]Epi (2 nM).

The density of striatal [³H]Epi binding nAChRs was 98.7 ± 4.0, 90.2 ± 2.7, and 93.0 ± 7.0 fmol/mg of protein (mean ± S.E.M. of three experiments) in 3^+/+, 3^+/–, and 3^–/– mice, respectively. These values were not statistically different from each other (Fig. 1A). The density of midbrain [³H]Epi binding nAChRs was higher than in the striatum, being 143.9 ± 5.6, 152.1 ± 6.2 and 137.6 ± 7.6 fmol/mg of protein (mean ± S.E.M.; n = 3) in 3^+/+, 3^+/–, and 3^–/– mice, respectively. Again, no statistically significant differences were seen between the genotypes (Fig. 1C). Both the [³H]Epi binding site densities, and the lack of effect of the 3-null mutation on them are very similar to the results reported by Cui et al. (2003).

View larger version (31K): [in this window] [in a new window]   Fig. 1. Expression of [3H]Epi (A) and 125I-Bgtx (B) binding nAChRs in striatum membranes from each 3 genotype (3+/+, 3+/–, and 3–/–). The membrane homogenates (M) or 2% Triton X-100 extracts (E) were prepared as described under Materials and Methods The reported values are expressed as femtomoles of specific labeled [3H]Epi and 125I-Bgtx binding sites per milligram of protein and are the mean values ± S.E.M. of five experiments performed in triplicate for the different genotypes. Expression of [3H]Epi (C) and 125I-Bgtx (D) binding nAChRs in midbrain membranes from each 3 genotype (3+/+, 3+/–, and 3–/–). The binding experiments were performed as described for A and B.

¹²⁵I--Bungarotoxin Binding nAChRs.

Subunit Composition of Striatal [³H]Epibatidine nAChRs. The results mentioned above demonstrate that deletion of the 3 subunit has no measurable effect on the overall amount of nAChR expression. However, as outlined under Introduction, several distinct nAChR subtypes have been reported in the mesostriatal dopamine pathway. To quantify the relative contribution of each nicotinic subunit to [³H]Epi binding in the striatum, we performed quantitative immunoprecipitation experiments using subunit-specific antibodies and [³H]Epi-labeled nAChRs. The results, expressed as femtomoles of immunoprecipitated nAChRs per milligram of protein are the mean values of three or four separate experiments for each subunit, in each genotype (Fig. 2A).

View larger version (28K): [in this window] [in a new window]   Fig. 2. A, immunoprecipitation analysis of the subunit content of the [3H]Epi nAChRs expressed in striatum labeled with 2 nM [3H]Epi. Immunoprecipitation was carried out as described under Materials and Methods using saturating concentrations (20–30 µg) of anti-subunit Abs. The amount immunoprecipitated by each antibody was subtracted from the value obtained in control samples containing an identical concentration of normal rabbit IgG, and the results obtained with each Ab are expressed as femtomoles of immunoprecipitated, labeled [3H]Epi nAChR per milligram of protein. Results are the mean values ± S.E.M. of four to five experiments performed in duplicate for each antibody. Statistical analyses were made using Student's paired t test. The significance of the difference from controls was *, p < 0.05; **, p < 0.01; and ***, p < 0.001. B, immunoprecipitation analysis of the subunit content of the [3H]Epi nAChRs expressed in midbrain labeled with 2 nM [3H]Epi. The reported values, (expressed as in A) are the mean values ± S.E.M. of three to four experiments performed in triplicate.

Interestingly, 3^+/– striatum expressed a population of nAChR subtypes that was indistinguishable from 3^+/+ striatum. The lack of difference between nAChR populations isolated from 3^+/+ and 3^+/– mice (seen here, and in midbrain preparations, see below) presumably indicates that even though 3 mRNA expression in the heterozygotes is significantly reduced (Cui et al., 2003), the residual mRNA transcription drives sufficient subunit protein production to allow receptor assembly similar to that of wild-type mice.

Reassuringly, 3* nAChRs were not detected in 3^–/– striatum, confirming the specificity of the new antiserum. 3 subunit-null mutation greatly reduced the expression of striatal 6* nAChRs (from 16.6 ± 2.0 fmol/mg of protein in 3^+/+ to 4.0 ± 1.2 fmol/mg of protein in 3^–/–). In contrast, deletion of the 3 subunit did not significantly affect expression of striatal 4*, 5*, or 2* nAChRs. The main finding in the striatum of 3^–/– mice is therefore a dramatic (76%) decrease in 6* nAChRs.

Subunit Composition of [³H]Epibatidine Binding nAChRs in Midbrain. We also studied the subunit composition of the [³H]Epi binding nAChRs in midbrain and expressed the results as femtomoles of immunoprecipitated nAChRs per milligram of protein (mean values of three or four separate experiments for each subunit in each genotype) (Fig. 2B).

We found that the [³H]Epi binding nAChRs expressed in midbrain of 3^+/+ mice are much more heterogeneous than those expressed in the striatum, with almost all possible subunits being expressed at variable levels. The majority of sites could be precipitated by the 2 (75.4 ± 3.5%) and 4 (68.9 ± 4.9%) antisera, whereas the other antisera were much less efficacious: 2 (4.3 ± 0.8%), 3 (10.8 ± 0.8%), 5 (6. 1 ± 0.9%), 6 (9.3 ± 1.0%), 3 (10.2 ± 1.5%), and 4 (7.9 ± 3.0%). Similar to the situation in striatum, 3^+/– midbrain provided almost identical results to those obtained from 3^+/+ tissue.

However, the composition of nAChRs in 3^–/– midbrain was different from that in 3^+/+ and 3^+/– striatum. Again, the main finding in 3^–/– midbrain was a strong reduction in 6* nAChRs (41%; from 11.6 ± 0.9 to 6.8 ± 0.6 fmol/mg of protein) this time accompanied by a smaller, but still significant, decline in 3* nAChRs (34%; from 15.3 ± 1.1 in WT to 10.1 ± 0.7 fmol/mg protein). No significant effects of 3 gene deletion on the expression of nAChRs containing any other subunit were observed.

Immunopurification of 3* nAChR Subtypes from Striatum. To identify the subunits assembled with the 3 subunit, we immunodepleted the striatal extract of 3* nAChRs using an affinity column with bound anti-3 CYT Abs. Selective immunodepletion was confirmed by the decrease in 3* nAChRs from 18% in the total striatal extract to 1% in the flow-through of the 3 column.

To identify the subunit composition of the captured 3* nAChRs, they were eluted from the affinity column using an excess of the 3 CYT peptide, labeled with [³H]Epi and then immunoprecipitated with subunit specific antisera. As shown in Fig. 3, the anti-4, 6, 2, and 3 COOH antisera immunoprecipitated 66.6 ± 0.8, 79.3 ± 9.1, 93.9 ± 2.5, and 61.0 ± 6.0% of the purified [³H]Epi-labeled 3* nAChRs, respectively. The anti-2, 3, 5, and 4 antisera were essentially ineffective, immunoprecipitating 2.0 ± 1.4, 1.1 ± 0.9, 2.7 ± 2.2, and 2.1 ± 2.2% of the purified 3* nAChRs, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Immunoprecipitation analysis of the subunit content of purified 3* nAChRs. The extracts prepared from 3+/+ striatum were incubated on an affinity column with bound anti-3 M-CYT Abs (see Materials and Methods) to bind the 3* population, which was eluted from the column by means of incubation with the 3M-CYT peptide. Recovered nAChRs were labeled with 2 nM [3H]Epi and then immunoprecipitated by the indicated subunit-specific Abs. Immunoprecipitation was carried out as described for Fig. 2A. The amount immunoprecipitated by each antibody was subtracted from the value obtained in control samples containing an identical concentration of normal rabbit IgG, and the results are expressed as the percentage of total [3H]Epi binding present in the solution before immunoprecipitation. Each data point is the mean value ± S.E.M. of two determinations performed in triplicate using two Abs directed against two separate epitopes of the same subunit (except for 3).

The striatal nAChRs not captured by the 3 affinity column (i.e., those retained in the flow-through buffer) were also analyzed by immunoprecipitation. Almost all flow-through nAChRs were precipitable by 4 (95%) and 2 (91%) Abs, with lower proportions precipitated by the 5 (15%) and 6 Abs (6%). 2, 3, 3, and 4 Abs were ineffective. These results clearly indicate that after 3* nAChR immunodepletion, the remaining major striatal subtypes are the 42 and 452. Interestingly, the small but measurable recovery of 6 receptors in the flow-through fraction (in the absence of significant 3 leakage from the column, see above) indicates that at least a small proportion (approximately one-quarter) 6* nAChRs in wild-type mice do not contain 3 subunits. Unfortunately, the small size of this population precluded further study on its subunit composition.

Sucrose Gradient Analysis of 6* and 2* nAChRs in the Striatum and Midbrain of 3^+/+ and 3^–/– Mice. Correct assembly of 6* nAChRs in heterologous expression systems is critically dependent on subunit composition (Kuryatov et al., 2000). To ascertain whether 6* subunits were incorporated into correctly assembled pentameric subtypes in 3^–/– mice, the size of detergent-solubilized 6* nAChRs retained in 3^–/– striatum and midbrain was measured using sucrose density gradient centrifugation. After centrifugation, nAChRs within each fraction were captured using anti-6 or anti-2 Ab-coated wells and then quantitated by ¹²⁵I-epibatidine binding. In both tissues, the 6* nAChRs detergent solubilized from 3^–/– mice sedimented as a single species that was slightly larger than T. californica AChR monomers (Fig. 4, A and C), but of the same size as 6* nAChRs present in 3^+/+ mice. These results clearly indicate that, although reduced in number, the 6* nAChRs in 3^–/– striatum and midbrain have a correct pentameric assembly (Fig. 4, A and C). Parallel analysis of the same fractions for the presence of 2* nAChRs revealed that these, too, have the same pentameric conformation. Moreover, expression levels of 2* nAChRs in striatum and midbrain were unaffected by 3 genotype (Fig. 4, B and D), confirming the immunoprecipitation results described previously (Fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 4. Sucrose gradient analysis of 6* (A and C) and 2* (B and D) nAChRs present in striatum or midbrain. Five hundred microliters of 2% Triton X-100 extracts was loaded on to a 5 to 20% (w/v) sucrose gradient in phosphate buffer saline, pH 7.5, 0.1% Triton X-100, and 1 mM PMSF, and centrifuged for 14 h at 40,000 rpm in a Beckman rotor at 4°C. The fractions were collected, added to anti-6or 2 Abs bound to microwells, left for 24 h, and then assayed for 125I-Epi binding as described under Materials and Methods. As a standard, 125I-Bgtx-labeled T. californica AChRs were subjected to sucrose gradient centrifugation in parallel, the fractions were collected and the radioactivity determined by gamma counting. The arrows indicate in each gradient the position of the T. californica monomer and dimer.

Pharmacological Properties of 6* nAChRs in 3^+/+ and 3^–/– Mouse Striatum.

Saturation binding analysis revealed no significant differences in the affinity of ¹²⁵I-Epi binding at 6* nAChRs captured from 3^+/+ and 3^–/– mouse striatum [apparent K_d value of 41 pM (CV 16%) and 37 pM (CV 14%), respectively]. However, as expected, the B_max of the 6* nAChRs immunoimmobilized from 3^–/– mouse striatum was reduced to approximately one-fourth of that obtained using 3^+/+ tissue (Fig. 5A).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Pharmacological characterization of the 6* nAChRs present in the striatum of the 3+/+ and 3–/– genotypes. A, saturation curve of specific 125I-Epi binding to immunoimmobilized 6* nAChRs. B, inhibition by -CtxMII of the binding of 125I-Epi to 6 immunoimmobilized nAChRs. 6* nAChRs present in striatum of the 3+/+ and 3–/– genotypes were immunoimmobilized using anti-6, as described under Materials and Methods. The binding curves were obtained by fitting three separate experiments using the LIGAND program, unless shown, the S.E.M. is in the range of the symbol. In each experiment, each -CtxMII dilution was tested in triplicate. All of the values are expressed in relation to 125I-Epi specific nAChR binding (considered as 100%).

	Discussion

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nAChR 3 subunit deletion substantially reduces ¹²⁵I--CtxMII binding nAChR expression in SN/VTA dopaminergic projections (Cui et al., 2003). This reduction is accompanied by a diminution of -CtxMII-sensitive striatal synaptosomal [³H]dopamine release (Salminen et al., 2004). The present study applied ligand binding and immunoprecipitation techniques to investigate the consequences of 3 gene deletion on nAChR expression, composition, and pharmacology in this pathway.

This study's most striking result was that nAChR 3 subunit deletion greatly reduces 6* nAChR expression, both in the SN/VTA and their terminal regions. The decrease in 6* nAChR expression in both regions was quantitatively very similar to the decrease in high affinity ¹²⁵I-CtxMII binding in the 3^–/– mice (Cui et al., 2003). This is in stark contrast to the effects on mRNA expression, because 3 subunit-null deletion had no effect on the expression of non-3 nAChR subunit mRNAs, including 6 (Cui et al., 2003). In this case, as with the up-regulation of neuronal nAChRs by long-term nicotine treatment (Marks et al., 1992), it seems that alterations in nAChR expression occur at the level of subunit proteins, rather than subunit mRNAs.

Despite the loss of 6* sites in 3^–/– mice, neither striatal cell membrane nor Triton X-100 extracts exhibited a significant decrease in overall [³H]Epi binding sites after 3 deletion. Furthermore, the immunoprecipitation studies showed no statistically significant decrease in 4* and 2* nAChRs after 3 gene deletion. Using the mouse-directed anti-3 Ab, we immunopurified 3* striatal nAChRs. Most 3* nAChRs contained associated 4, 6, and 2 subunits. Thus, it might be expected that loss of 63* sites would be accompanied by a loss of the accompanying 4 and 2 subunit expression. Although it is possible that minor changes in 4 and 2 expression may have been obscured within the larger population, these results may instead suggest that 42(non-6)* nAChRs replace the lost 4/6* nAChRs. This hypothesis is strongly supported by the recent study of Salminen et al. (2004), who demonstrated that loss of -CtxMII-sensitive striatal [³H]dopamine release in 3^–/– mice is accompanied by a compensatory increase in -CtxMII-resistant function.

Although most 3 subunits in wild-type mice coassemble with 6, approximately one-quarter of striatal 6* nAChRs do not incorporate the 3 subunit. Intriguingly, the total striatal 6* nAChR population in 3^+/+ mice is also approximately 4 times bigger than that of their 3^–/– littermates. Thus, the residual 6* population in 3^–/– mice may be composed of retained non-3, 6* nAChRs, rather than representing a new subtype only expressed after 3 subunit deletion. This non-3, 6* nAChR population has not been identified by previous mouse studies and represents a novel nAChR subtype. The very low expression this residual 6* population in 3^–/– mice precluded a detailed immunoprecipitation study. However, circumstantial evidence suggests that many of these nAChRs also contain 4 subunits, like the larger population found in 3^+/+ mice. Previous studies on mouse striatal 6* receptors indicate that the [³H]epibatidine binding sites associated with this population may be divided into two populations, one with high -CtxMII affinity and one with low affinity (Champtiaux et al., 2003). The high -CtxMII-affinity sites are thought to correspond to 6/2 subunit interfaces. Those with low -CtxMII affinity are likely to arise at 4/2 subunit interfaces, located within 6* nAChR complexes, because low-affinity sites are absent from 6* nAChRs in the striatum of 4^–/– mice (Champtiaux et al., 2003). The fact that, in the present study, 6* nAChRs isolated from 3^–/– mice contain roughly equal proportions of high- and low-affinity -CtxMII binding sites would seem to indicate that much of this population also contains 4/2 subunit interfaces.

Overall, the major effect of 3 gene deletion in both SN/VTA and the striatum was to reduce 6* expression. This further reinforces the concept of a close relationship between the two subunits, as shown by the linkage of 6 and 3 into a gene cluster (Cui et al., 2003), the fact that many 6 nAChRs contain 3 (Champtiaux et al., 2003), and the results described in this article. The specific decrease of 6* nAChRs in 3^–/– mice probably indicates that the 3 subunit is important for the formation of the majority of 62* or 642* pentamers. Decreased 6* nAChR expression could be caused by defects in nAChR assembly, degradation, and/or cell trafficking. Our present approach is not capable of distinguishing definitively between these possibilities, but the much greater decline in cell terminal (striatal) versus cell body (SN/VTA) 6* nAChR expression (76% reduction versus 42%) may support a role for 3 in directing nAChRs to the striatal regions of dopamine neurons. This study's finding that striatum contains higher levels of 63* than dopamine cell bodies further suggests that this nAChR subtype may be selectively addressed to dopaminergic nerve terminals. A trafficking hypothesis is also supported by previously published data (Champtiaux et al., 2003) showing that CtxMII inhibits nicotinic responses more effectively on dopaminergic nerve terminals than on cell bodies, and the results of studies on 6-hydroxydopamine- or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned rodents showing the selective localization of nAChRs containing the 6 and 3 subunits in dopaminergic terminals (Zoli et al., 2002, Champtiaux et al., 2003; Quik et al., 2003). Alternatively, or additionally, nAChR assembly/degradation effects are supported by Kuryatov et al. (2000), who demonstrated that adding the 3 subunit to 6 and 4 subunits greatly improves the yield of functional channels in oocytes.

Interestingly the midbrain, which includes the SN/VTA cell bodies, expressed a slightly different complement of nAChRs than their terminal regions [an additional 3* (10%) and/or 4* (7%) population, whereas the percentage of nAChRs containing the 6 and 3 subunits was lower (8%) than in striatum (18%)]. The midbrain 3* population was reduced by 33% after 3 deletion, which may imply that 3is incorporated into a portion of midbrain 3* nAChRs, together with 2 and/or 4 subunits. This nAChR subtype has previously been described in chick retinal nAChRs (Vailati et al., 2000) and in heterologous systems (Groot-Kormelink et al., 1998), but it has not before been identified in mammals.

The retention of 6* nAChRs in the striatum of 3^–/– mice (albeit at reduced density) allowed us to test the 3 subunit's influence on 6* nAChRs' -CtxMII affinity. Residual 6* nAChRs without the 3 subunit still bound -CtxMII with high affinity, thus confirming that 3 neither directly nor allosterically affects the high-affinity -CtxMII binding site. This finding is consistent with the idea that the 3 subunit assembles in a position analogous to that of the muscle type 1. In this scenario, 3 facilitates the formation of properly assembled pentamers, without changing the two acetylcholine binding sites at the 42 and/or 62 interfaces. One concern was that, as in Xenopus laevis oocytes (Kuryatov et al., 2000), 6 and 2 may assemble to form large aggregates with high -CtxMII and Epi affinity, but no function. Our sedimentation data indicate that the remaining 6* nAChRs in 3^–/– mice form pentameric assemblies. Salminen et al. (2004) have also demonstrated the retention of a small amount of -CtxMII-sensitive, nAChR-mediated dopamine release in 3^–/– mouse striatum, thus indicating that these are indeed functional nAChRs.

In conclusion, our results demonstrate that the nAChR 3 subunit is not necessary for the high-affinity binding of -CtxMII but it is essential for efficient 6* nAChR assembly and/or selective transport to nerve terminals. It also seems that the 3 subunit encourages formation of unusually complex 4623 subtype nAChRs, which constitute the majority of 6* nAChRs in mesostriatal dopamine neurons. It is noteworthy that comparison with the results of Cui et al. (2003) demonstrates that the 3 subunit's effects are mediated by protein/protein interactions, rather than by a reduction of 6 mRNA synthesis/stability after deletion of the 3 nAChR subunit gene. This subunit protein level, rather than gene expression level interaction, is reminiscent of the situation in agonist-mediated up-regulation (Marks et al., 1992) and may indicate that the former has a predominant role in regulating nAChR regulation. There are indications that loss of 6* nAChR expression is compensated for by increased 42(non-6)* nAChR production. In addition, this study has uncovered evidence for two previously uncharacterized mammalian nAChR subtypes [6(non-3)2* and 33*].

	Footnotes

 
This work was supported in part by grants from the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (MM05152538), European Research Training Network HPRN-CT-2002-00258, Fondo Integrativo Speciale per la Ricerca-Consiglio Nazionale delle Ricerche Neurobiotecnologia 2003, Fondo per gli Investimenti della Ricerca di Base RBNE01NR34, Fondazione Cariplo Grant No. 2002/2010 (to F.C.); Italian Minister of Health RF01.147 and Fondo per gli Investimenti della Ricerca di Base (RBNE01RHZM) 2003 (to C.G.); Colorado Tobacco Research Program 3I-030 (to P.W.); and National Institute on Drug Abuse grant DA12242 (to M.J.M, P.W., and J.M.M.) and National Institute of Mental Health grant MH53631 (to J.M.M.). Production of the 3 subunit-null mutant mice was supported by the National Institute on Drug Abuse animal resources grant DA015663 (to A.C.C.).

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

doi:10.1124/mol.105.011940.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; Bgtx, -bungarotoxin; CntxMII, -conotoxin MII; Ab, polyclonal antibody; CYT, cytoplasmic peptide; PMSF, phenylmethylsulfonyl fluoride; Epi, epibatidine; WT, wild-type; CV, coefficient of variation; SN/VTA, substantia nigra/ventral tegmental area.

Address correspondence to: Dr. Cecilia Gotti, Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Section of Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy. E-mail: c.gotti{at}in.cnr.it

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