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Nicotinic Cholinergic Receptors in the Rat Retina: Simple and Mixed Heteromeric Subtypes

Department of Pharmacology, Georgetown University School of Medicine, Washington, DC (A.M.M., B.C.C., R.P.Y., Y.X., B.B.W., K.J.K.); and Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M.)

Received February 25, 2005; accepted August 29, 2005

	Abstract

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Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in the rat retina to determine the heteromeric subtypes. We detected seven nicotinic receptor subunit mRNA transcripts, 2–4, 6, and 2–4, with RNase protection assays. The density of heteromeric nAChR binding sites is 3 times higher in the retina than in the cerebral cortex. Moreover, the density of the sites in the retina measured with [³H]epibatidine ([³H]EB) is 30% higher than with ¹²⁵I-3-(2(S)-azetidinylmethoxy)pyridine (A-85380) and more than twice that measured with [³H]cytisine or [³H](-)nicotine. These data suggest that the retina expresses multiple subtypes of nAChRs, including a large fraction of receptors containing the 2 subunit and a smaller fraction containing the 4 subunit. Consistent with this, in binding competition studies, nicotinic ligands fit a model for two affinity classes of binding sites, with the higher affinity sites representing 70 to 80% of the nAChRs in the retina. To determine the specific subtypes of nAChRs in the rat retina, we used subunit-specific antibodies in immunoprecipitation assays. Immunoprecipitation of [³H]EB-labeled nAChRs with antibodies specific to the 2 and 4 subunits indicated that 80% of the receptors contained 2 subunits and 25% contained 4 receptors, consistent with the binding pharmacology results. Sequential immunoprecipitation assays indicated that the rat retina contains multiple subtypes of nAChRs. The majority of the receptors measured seemed to be simple heteromeric subtypes, composed of a single type of and a single type of subunit; but a significant fraction are mixed heteromeric subtypes, composed of two or more and/or subunits.

nAChRs exist as subtypes composed of different combinations of and subunits. Vertebrate nervous systems express nine and three nAChR subunits, and the mammalian retina expresses mRNA transcripts for most of them, including 2, 3, 4, 6, 7, 2, 3, and 4 (Hoover and Goldman, 1992; Britto et al., 1994; Zoli et al., 1995; Moretti et al., 2004). All of the nAChR subtypes conduct Na⁺, K⁺, and Ca²⁺, but the different subtypes have distinguishing biophysical properties such as channel conductances and rates of desensitization and resensitization that could markedly affect signaling in the pathways in which they function. In addition, the receptor subtypes display differences in pharmacological characteristics that in some cases allow subtypes to be distinguished by the affinity and efficacy of certain drugs.

nAChRs mediate signals related to different aspects of retina development and physiology, including those that influence neurite outgrowth from ganglion cells (Lipton et al., 1988) and those that coordinate increases in cytosolic calcium in amacrine and ganglion cells (Wong et al., 1995). In addition, these receptors seem to play a critical role in the proper development of visual pathways. For example, the precise projections of the optic nerves from the retinal ganglion cells to their specific targets in the lateral geniculate nuclei and the superior colliculi during development depend on spontaneous bursts of action potentials from the ganglion cells called retinal waves (Meister et al., 1991; Feller et al., 1996). These retinal waves depend on cholinergic transmission from amacrine cells to ganglion cells mediated by nAChRs (Feller et al., 1996; Bansal et al., 2000; Feller, 2002). Moreover, in mice lacking the 2 subunit (2 knockout mice), retinal waves are absent (Bansal et al., 2000), and the segregation of the axonal projections to eye-specific layers is markedly altered (Hwang et al., 2000).

The presence of several nAChR subunit mRNA transcripts (Hoover and Goldman, 1992; Britto et al., 1994; Zoli et al., 1995) and subunit proteins (Swanson et al., 1987; Britto et al., 1994) suggests the presence of more than one nAChR subtype in the mammalian retina. In fact, Gotti and colleagues (Moretti et al., 2004), using subunit-selective antibodies to immunoprecipitate [³H]epibatidine-labeled receptors, identified multiple nAChR subtypes in the rat retina. The studies reported here also address the question of which heteromeric nAChR subtypes are in rat retina by using sequential immunoprecipitation assays with subunit-specific antibodies to determine the associations of the subunits that comprise putative subtypes. Our data confirm that the rat retina contains multiple nAChR subtypes, including both simple heteromeric subtypes, composed of a single type of and a single type of subunit, and mixed heteromeric subtypes, composed of two or more and/or two or more subunits; however, some differences from the exact subtypes reported by Moretti et al. (2004) are noted.

	Materials and Methods

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Materials. Frozen retinas and brains from adult Sprague-Dawley rats were purchased from Zivic-Miller Laboratories (Portersville, PA). [³H]Epibatidine ([³H]EB), ¹²⁵I-A-85380, [³H](-)nicotine, and [³H]cytisine were purchased or were gifts from PerkinElmer Life and Analytical Sciences (Boston, MA). Dihydro--erythroidine was from Sigma/RBI (Natick, MA). An A-85380 analog, HFI-55 was synthesized in collaboration with Drs. John Musachio (National Institute of Mental Health, Bethesda, MD) and Hong Fan (The Johns Hopkins University, Baltimore, MD) and will be described in greater detail in a future article. [-³²P]CTP and [-³²P]ATP were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Electrophoresis reagents were purchased from Bio-Rad (Melville, NY). Protein G Sepharose beads were purchased from GE Healthcare. Protein A (Pansorbin) and normal rabbit serum (NRS) were purchased from Calbiochem (San Diego, CA). Nicotine tartrate, cytisine, A85380 [GenBank] , and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Rabbit antisera directed at a bacterially expressed fusion protein containing partial sequences of the cytoplasmic domains of nAChR 2, 4, 3, and 4 subunits were kind gifts from Drs. Scott Rogers and Lorise Gahring (University of Utah, Salt Lake City, UT). These antisera have been described previously (Flores et al., 1992; Rogers et al., 1992). An antibody directed at a peptide sequence of the rat nAChR 3 subunit was affinity-purified from rabbit serum. This antibody has been described previously (Yeh et al., 2001). In addition to these antibodies, a peptide antibody directed at the 6 subunit (R. Yasuda and B. Wolfe, unpublished data) was used. A monoclonal antibody (mAb 270) to the chick 2 subunit was made from hybridoma stocks (American Type Culture Collection, Manassas, VA). This mAb was originally developed and characterized by Whiting and Lindstrom (1987). Table 1 provides references to all of these antibodies and antisera. In some studies, we also measured nAChRs containing 6 and 4 subunits with antibodies that were generously provided by Dr. Cecilia Gotti (University of Milan, Milan, Italy). These antibodies are described in Moretti et al. (2004). For simplicity, in this article we use the term "antibody" to refer to unpurified antisera and to affinity-purified antisera and monoclonal antibody.

RNA Isolation and RNase Protection Assay. Total cellular RNA was isolated using RNA-STAT-60 (Tel Test B, Friendswood, TX). DNA templates for antisense riboprobes were prepared as described previously (Xiao et al., 1998). Antisense riboprobes for the 2–7 and 2–4 nAChR subunits were generated from DNA templates using T7 RNA polymerase and [-³²P]CTP. The RNase protection assays were carried out using the RPA II kit (Ambion, Austin, TX). Total RNA (20 µg) from the tissue samples was hybridized overnight at 42°C with the subunit riboprobes and a riboprobe for rat glyceraldehyde-3-phosphate dehydrogenase, which was used as an internal and loading control. After hybridization, nonprotected fragments were digested with a combination of RNase A and RNase T1 for 30 min at 37°C. The numbers of bases of the full-length probes and the protected fragments of the probe, respectively, were as follows: 2, 416 and 332; 3, 306 and 230; 4, 496 and 408; 5, 411 and 380; 6, 462 and 396; 7, 450 and 376; 2, 322 and 263; 3, 430 and 394; 4, 252 and 170; and glyceraldehyde-3-phosphate dehydrogenase, 204 and 135. The protected probe fragments were separated by electrophoresis on a 6% denaturing polyacrylamide gel, and the fragments were visualized by X-ray filming or phosphorimaging.

Receptor Binding. Tissues were homogenized in 50 mM Tris-HCl buffer, pH 7.4 at 24°C, and centrifuged twice at 35,000g for 10 min in fresh buffer. The membrane pellets were resuspended in fresh buffer and added to tubes containing [³H]EB, ¹²⁵I-A-85380, [³H]cytisine, or [³H](-)nicotine with or without competing drugs. Incubations with were carried out in Tris buffer at pH 7.4 for 2 h at 24°C with [³H]EB and ¹²⁵I-A-85380 or at 4°C in Tris buffer at pH 7.0 for 2 h with [³H]cytisine and [³H](-)nicotine. In assays to assess competition by -conotoxin MII, the tissues were preincubated in buffer containing a protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and then incubated with [³H]EB in the presence of both the protease inhibitors and 0.01 mg/ml bovine serum albumin. Bound receptors were separated from free ligand by vacuum filtration over GF/C glass-fiber filters that were prewet with 0.5% polyethyleneimine, and the filters were then counted in a liquid scintillation counter. Nonspecific binding was determined in the presence of 300 µM nicotine, and specific binding was defined as the difference between total binding and nonspecific binding.

Immunoprecipitation. Tissue membrane homogenates were prepared as above for binding assays. The receptors were solubilized by incubating the homogenates in 2% Triton X-100 with gentle rotation for 2 h at room temperature. After centrifuging the mixture at 35,000g for 10 min, aliquots of the clear supernatant (equivalent to 6 mg of original tissue weight) were added to sample tubes containing [³H]EB and either one of the subunit-specific antibodies at a concentration determined in preliminary studies to be optimal for each or an equivalent volume of NRS. The samples were then rotated overnight at 4°C, after which 50 µl of an 6% slurry of Pansorbin (source of Protein A) for the polyclonal antisera or a 50% slurry of Protein G Sepharose beads for the mAb was added, and the rotation of the samples was continued for 1 h. The samples were then centrifuged at 7000g for 5 min, and the supernatants were removed and either filtered over GF/B filters wet with 0.5% polyethyleneimine or placed on ice for later use in sequential immunoprecipitation studies. The remaining tissue pellets were washed once with 1.2 ml of 50 mM Tris-HCl buffer, pH 7.0 at 4°C, dissolved in 0.2 ml of 1 N NaOH and then counted in a scintillation counter. After subtracting the number of counts precipitated in tubes containing NRS, the number of [³H]EB-labeled nAChRs immunoprecipitated by each antibody was compared with the total number of labeled receptors in samples of the solubilized retina membranes measured in filtration binding assays.

Sequential Immunoprecipitation. The immunoprecipitation assay provides important information about the presence of any one subunit in an nAChR, but it does not specify the actual receptor subtype, which is defined by its subunit combination. Even the presence of two or more subunits measured independently in the same tissue preparation does not necessarily specify the receptor subtype because the subunits could each be components of different nAChRs. Therefore, to determine the actual receptor subtype based on subunit composition, we used a sequential immunoprecipitation protocol in which the supernatant remaining after immunoprecipitation with the first antibody (here called the clearing antibody), is subjected to a second round of immunoprecipitation with an antibody against a different subunit (here called the capturing antibody). This procedure or a conceptually similar approach has been used successfully to identify the subunit compositions of nAChR subtypes in the rat forebrain (Flores et al., 1992), trigeminal ganglia (Flores et al., 1996), striatum (Zoli et al., 2002), pineal gland (Hernandez et al., 2004), and retina (Moretti et al., 2004), and in chick ciliary ganglia and brain (Conroy et al., 1992; Conroy and Berg, 1995, 1998) and chick retina (Vailati et al., 2003).

For these sequential immunoprecipitation assays, the clear supernatant remaining after immunoprecipitation with the first antibody or NRS was incubated with a different subunit-selective antibody, and the immunoprecipitation steps with Protein A or Protein G were then repeated, as described above. In control studies, the number of solubilized nAChRs labeled by [³H]EB was not decreased when incubated in the absence of an antibody or NRS over the course of the sequential immunoprecipitation procedure.

Statistical Analysis. Data were analyzed using GraphPad Prism 4.0 software package (GraphPad Software, Inc., San Diego, CA). A one-sample t test was used in drug binding competition assays to determine whether the Hill coefficients were different from 1 and in immunoprecipitation assays to determine whether residual values were different from 0. The propagation of error method (Bevington, 1969) was used to calculate the S.E.M. for the difference between groups and for comparing the sum of subtypes to the total number of receptors in the retina. Differences between group means were compared using the Student's t test or one-way analysis of variance followed by Bonferroni's multiple comparison test.

	Results

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nAChR Subunit mRNA Expression Levels in the Retina. We detected mRNA transcripts for seven nAChR subunits in the rat retina, which were the following: 2, 3, 4, 6, 2, 3, and 4 subunits (Fig. 1). Each of these has been found in mammalian retina in previous studies using in situ hybridization and/or RNase protection assays (Hoover and Goldman, 1992; Zoli et al., 1995; Moretti et al., 2004). We did not detect 5 or 7 subunit transcripts in the retina with this method, although we did detect them in parallel studies carried out in rat brain and PC12 cells as positive controls (data not shown).

View larger version (46K): [in this window] [in a new window]   Fig. 1. Expression of nAChR subunit genes in the rat retina. RNase protection assays were carried out as described under Materials and Methods. Total RNA from rat retina was hybridized with a combination of 32P-labeled antisense probes corresponding to the nine rat nAChR subunit genes 2, 3, 4 (lane 1); 4, 5, 2 (lane 2); and 6 (lane 3); 7, 3 (lane 4). All mRNA measurements were repeated at least three times with similar results.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Saturation binding to nAChRs in rat retina: comparison between four different radiolabeled ligands. Receptor binding sites in retina homogenates were measured using [3H]EB, 125I-A-85380, [3H]cytisine, and [3H](-)nicotine as described under Materials and Methods. These curves are representative of two to five independent studies. All of the curves fit best to a model for one binding site, as determined by nonlinear, least-square regression analyses using Prism 3 software. Inset, comparison of [3H]EB binding in rat retina and cerebral cortex. The values for the dissociation constants (Kd) and binding site density (Bmax) from all of the studies are shown in Table 2.

Despite the ability of [³H]EB to label multiple nAChR subtypes in the retina, curve-fitting and nonlinear regression analysis found that its saturation binding curve fit a model for a single class of binding sites. This probably reflects its relatively similar affinity for the binding sites of all of the known heteromeric nAChR subtypes. Because of this characteristic, [³H]EB does not readily discriminate among these subtypes.

Drug Competition for [³H]EB Binding Sites in the Retina. Multiple nAChR subtypes in the retina would be consistent with the multiple nAChR subunit mRNA transcripts expressed there. To examine this further, we carried out competition binding assays with several ligands that can discriminate among some of the subtypes. As shown in Fig. 3, all of these nicotinic ligands competed for the sites labeled by [³H]EB in the retina with shallow competition curves, yielding Hill coefficients significantly less than 1. This is consistent with competition for more than one class of nAChR binding sites; in fact, all of these ligands fit a model for two classes of binding sites (Table 3). The competing ligands shown here all have much higher affinity for nAChRs containing 2 subunits than 4 subunits (Parker et al., 1998; Xiao et al., 1998; Xiao and Kellar, 2004); thus, in general, they can discriminate between these two broad classes of subtypes. The higher-affinity binding site for these competing ligands represented 70 to 80% of the nAChR labeled by [³H]EB in the retina (Table 3), indicating that 2-containing receptors constitute the majority of the nAChRs in the rat retina, which is consistent with the binding site density of ¹²⁵I-A-85380 compared with [³H]EB (Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Competition binding of ligands for nAChRs labeled by [3H]EB in membrane homogenates from rat retina is consistent with multiple nAChR subtypes. Nicotinic ligands competed against 500 pM [3H]EB in binding assays as described under Materials and Methods. In all cases, the competition curves were shallow, with Hill coefficients <1, and fit best to a model for two binding sites. Data shown are representative of three to four independent studies. See Table 3 for summary and analyses of data.

Immunoprecipitation of nAChRs in the Retina. nAChR subtypes are defined by their subunit composition, and although the pharmacological profile can point to possible receptor subtypes or eliminate some, pharmacological studies alone are seldom definitive. Therefore, to begin to determine the nAChR subtypes in the retina, we labeled the receptors with [³H]EB and then carried out immunoprecipitation assays with seven different antibodies, each directed at a specific receptor subunit for which mRNA was detected in the retina.

Before carrying out the immunoprecipitation studies in the retina, we tested the specificity of each of the antibody preparations, which are directed at 2, 3, 4, 6, 2, 3, and 4 subunits. To do this, we determined their ability to immunoprecipitate receptors from rat cerebral cortex, which expresses predominantly 42 nAChRs (Whiting et al., 1987; Flores et al., 1992); pineal gland, which expresses 34 nAChRs virtually exclusively (Hernandez et al., 2004); superior cervical ganglia, which expresses 3-containing receptors (Xu et al., 1999); and superior colliculus and striatum, which express a significant fraction of receptors containing 6 subunits (Whiteaker et al., 2000b; Champtiaux et al., 2002) and 3 subunit mRNA (Cui et al., 2003). In some cases, we also used human embryonic kidney 293 cells stably transfected with different rat nAChR subunit combinations (Xiao et al., 1998; Xiao and Kellar, 2004). As shown in Table 4, the antibody preparations directed at these seven subunits are all highly effective, as measured by the ability of most of them to immunoprecipitate 80 to 100% of the receptors from tissues containing known predominant receptor subtypes with cognate subunits. Our 6 and 3 antibodies immunoprecipitated 25 and 9% of the receptors in the superior colliculus, respectively, one of the few tissues other than the retina itself (Moretti et al., 2004; see below) that express a relatively high percentage of nAChRs which contain these subunits. Moreover, and just as important, all of the antibodies displayed high specificity, recognizing less than 2% of nAChRs that do not contain their cognate subunits (Table 4).

The antibodies directed at the 2, 3, and 4 subunits each immunoprecipitated 33% of the [³H]EB-labeled receptors in the retina, whereas our 6 subunit antibody immunoprecipitated 10% of the receptors (Fig. 4). The antibody directed at the 2 subunit immunoprecipitated 80% of the receptors in the retina, whereas the antibodies directed at the 3 and 4 subunits immunoprecipitated 10% and 25% of the receptors, respectively (Fig. 4).

View larger version (14K): [in this window] [in a new window]   Fig. 4. Immunoprecipitation of nAChRs with antibodies indicates multiple nAChR subunits in the rat retina. Solubilized rat retina homogenates were labeled with 2 nM [3H]EB and were added to tubes containing NRS or one of the subunit-selective antibodies at a previously determined optimal concentration. Protein A (Pansorbin) for the polyclonal antibodies or Protein G Sepharose beads for the monoclonal 2 antibody was used to precipitate the nAChR-antibody complex and after brief centrifugation, the 3H-labeled nAChRs contained in the pellet were counted. The results of the immunoprecipitation studies are expressed as a percentage of the total solubilized nAChRs labeled by [3H]EB in the retina, which was measured in each experiment. The data shown are the mean ± S.E.M. of four to eight separate experiments.

An example of the efficacy of this procedure is shown in Fig. 5. The 2 and 4 antibodies immunoprecipitated, respectively, 80 and 25% of the [³H]EB-labeled nAChRs in retinal homogenates, and combining both antibodies in one assay tube immunoprecipitated essentially all of the [³H]EB-labeled receptors. Moreover, after an initial immunoprecipitation (clearing) of retinal homogenates with the combination of the 2 and 4 antibodies, subsequent immunoprecipitation of the remaining supernatants by the antibodies directed at 2, 3, and 4 subunits was reduced to nearly background levels, indicating that the clearing procedure with the combined 2 and 4 antibodies removed essentially all of the heteromeric receptors from the remaining supernatant. This is consistent with the long-established concept that all heteromeric nAChRs contain an subunit in association with a 2 and/or 4 subunit.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Sequential immunoprecipitation of nAChRs in the rat retina: demonstration that heteromeric nAChRs contain either a 2 or 4 subunit. A, solubilized retinal homogenates were incubated with antibodies to the 2 and 4 subunits either individually or combined. The 2 and 4 subunits individually immunoprecipitated 80% and 25% of the receptors, respectively, whereas incubation with the two antibodies combined immunoprecipitated virtually all of the receptors. B, the supernatant resulting from an initial immunoprecipitation with NRS or the combined 2 and 4 subunit antibodies (the clearing antibodies) was subjected to a second immunoprecipitation with antibodies to the 2, 3, or 4 subunits (the capturing antibodies). As shown, after initial immunoprecipitation with NRS, the antibodies to the three subunits each immunoprecipitated 30 to 35% of the retinal nAChRs from the resultant supernatant; however, after initial immunoprecipitation with the combined antibodies to the subunits, nearly all of the nAChRs containing these subunits were removed from the resultant supernatant, and the antibodies directed at the subunits immunoprecipitated few if any receptors. The percentage of the total number of retinal nAChRs decreased by the initial immunoprecipitation is indicated. Data are the mean ± S.E.M. of three to eight experiments. **, p < 0.01; ***, p < 0.001 values significantly different from the values after NRS.

Associations with the 2 Subunit.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Sequential immunoprecipitation of retinal nAChRs demonstrates associations of the 2 subunit with 2, 3, 4, and 6 subunits. A, retinal homogenates were first immunoprecipitated with NRS or one of the antibodies to the subunits shown (the clearing antibody), and the resulting supernatants were then subjected to a second immunoprecipitation with the 2 antibody (the capturing antibody). B, the sequential immunoprecipitation procedure was carried out in reverse order; that is, the retinal homogenates were first immunoprecipitated with NRS or the 2 antibody, and the resulting supernatants were then subjected to a second immunoprecipitation with one of the antibodies to the subunits. In each case, initial immunoprecipitation with an subunit antibody decreased the immunoprecipitation by the 2 subunit antibody and vice versa (except that the 2/6 sequence was not done, see text). The percentage of the total number of retinal nAChRs decreased by the initial immunoprecipitation is indicated. Data are the mean ± S.E.M. of three experiments. *, p < 0.05; ***, p < 0.001, values significantly different from the values after NRS.

Associations with the 4 Subunit. Similar studies were carried out to examine the association of each of the subunits with 4 subunits. As shown in Fig. 7A, when the retina homogenates were first incubated with normal rabbit serum, subsequent incubation with the 4 antiserum immunoprecipitated 20% of the [³H]EB-labeled nAChRs. Clearing with the 2 or 4 antisera did not significantly decrease the subsequent immunoprecipitation by the 4 antiserum. These data indicate that there are few, if any, retinal nAChR subtypes that include both 2 and 4 subunits or 4 and 4 subunits. In contrast, initial immunoprecipitation with the 3 antibody nearly eliminated subsequent immunoprecipitation of nAChRs by the 4 antiserum. This indicates that nearly all 4 subunits in the retina associate with 3 subunits, forming 34^* nAChR subtypes. It is interesting to note that initial immunoprecipitation with the 6 antiserum decreased the number of receptors subsequently immunoprecipitated by the 4 antiserum to 8% of the total nAChRs in the retina (Fig. 7A). This indicates that 6 and 4 subunits are associated in 11% of the nAChRs in the retina and implies that approximately half of the 34^* receptors are an 364^* mixed heteromeric subtype.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Sequential immunoprecipitation of retinal nAChRs demonstrates associations of the 4 subunit with 3 and 6 subunits. A, retinal homogenates were first immunoprecipitated with NRS or one of the antibodies to the subunits (the clearing antibody), and the resulting supernatants were then subjected to a second immunoprecipitation with the 4 antibody (the capturing antibody). B, the sequential immunoprecipitation procedure was carried out in reverse order; that is, the retinal homogenates were first immunoprecipitated with NRS or the 4 antibody, and the resulting supernatants were then subjected a second immunoprecipitation with one of the subunit antibodies shown. The percentage of the total number of retinal nAChRs decreased by the initial immunoprecipitation is indicated. Data are the mean ± S.E.M. of at least three experiments. *, p < 0.05; ***, p < 0.001, values significantly different from the values after NRS.

Associations with the 3 Subunit. Previous studies have shown that the 6 and 3 subunits are often found together in the same nAChR (Cui et al., 2003; Salminen et al., 2004; Gotti et al., 2005). Consistent with this possibility in the retina, both 6 and 3 subunits were initially found in 10% of the retinal nAChRs (Fig. 4). To directly test whether these two subunits are components of the same receptor we carried out a sequential immunoprecipitation study. As shown in Fig. 8A, initial immunoprecipitation of retinal homogenates with our 6 antiserum cleared virtually all of the receptors that could be immunoprecipitated with the 3 antiserum. To determine whether 3 subunits were also associated with 3 subunits, we carried out a similar study with 3 and 3 antibodies. As shown in Fig. 8B, clearing with the 3 antibody eliminated nearly all of the nAChRs that could be immunoprecipitated by the 3 antiserum. We then tested for associations between 3 and 2 subunits and 3 and 4 subunits. Clearing with the 3 antibody indicated that 9% of the retinal receptors contained both 2 and 3 subunits (Fig. 8C). In contrast, no association between 3 and 4 subunits was detected (Fig. 8D). Because these two subunits are not associated with each other but the 6 subunit was found to be associated with both 3 and 4 subunits (Figs. 7 and 8), it suggests that the 6 subunit is a component of more than one nAChR in the rat retina.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Sequential immunoprecipitation demonstrates associations of the 3 subunit with 6, 3, and 2 subunits in rat retina. Retinal homogenates were first immunoprecipitated with NRS or 6 antibody (A) or 3 antibody (B) (the clearing antibody), and the resulting supernatants were then immunoprecipitated with the 3 antibody (the capturing Ab). In C and D, the homogenates were first immunoprecipitated with NRS or the 3 antibody, followed by immunoprecipitation with either the 2 or 4 antibodies. Associations between 3 and 6, 3 and 3, and 3 and 2 subunits were found, but not between 3 and 4 subunits. Data are the mean ± S.E.M. from three to eight experiments. *, p < 0.01; ***, p < 0.001, values significantly different from the values after NRS.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Sequential immunoprecipitation indicates an association between 2 and 4 subunits and between 2 and 4 subunits in rat retina. Sequential immunoprecipitation studies indicate that 2 and 3 subunits and 3 and 4 subunits are not associated (A), 9% of the retinal receptors contain both 2 and 4 subunits (B), and 13% of retinal nAChRs contain both 2 and 4 subunits (C). Data are mean ± S.E.M. of three to seven experiments. *, p < 0.05; **, p < 0.01, values significantly different from the values after NRS.

Initial immunoprecipitation with the 4 antiserum decreased the amount of receptors subsequently immunoprecipitated by the 2 antibody by 13% (Fig. 9C), indicating that 13% of the receptors in the retina contain both 2 and 4 subunits; moreover, these subunits would have to be associated with at least one subunit to form a [³H]EB binding site. Again, because the 4 subunit does not seem to be associated with either the 2 or 4 subunits (Fig. 7), and we found no association between 3 and 4 subunits, we conclude that 13% of the retina nAChRs that contain both 2 and 4 subunits are associated with 3 and/or 6 subunits.

Further Studies of the 6 and 4 Subunits in the Retina. There are some similarities between the results of the study presented here and a previous study by Gotti and colleagues that also examined nAChRs subtypes in the rat retina (Moretti et al., 2004). For examples, both studies found a large number of subtypes, including several different mixed heteromeric nAChRs; in addition, both studies found that most nAChRs in the rat retina include the 2 subunit. However, there are also some differences between the two studies. In particular, in our initial studies, the percentage of rat retinal nAChRs containing 6 subunits was considerably lower, and the percentage containing 4 subunits was much higher compared with the results reported previously. To try to resolve the differences related to the percentage of nAChRs that contain 6 and 4 subunits, we carried out studies with 6 and 4 antibodies generously provided to us by Dr. Cecilia Gotti (University of Milan). We refer to these antibodies here as the "Milan antibodies". As shown in Fig. 10A, using the Milan 6 antibody in our assay system, we immunoprecipitated 32% of the retinal nAChRs. This is approximately 3 times higher than the results with our 6 antibody and, in fact, is close to the value reported by Moretti et al. (2004). In contrast to this agreement, using the Milan 4 antibody we immunoprecipitated 25% of the retinal nAChRs (Fig. 10A), which is much higher than reported previously (Moretti et al., 2004) but very similar to the value we found with our 4 antibody.

View larger version (16K): [in this window] [in a new window]   Fig. 10. Immunoprecipitation studies with the Milan 6 and 4 antibodies in rat retina. A, nAChRs in rat retina were immunoprecipitated with 6 and 4 antibodies provided by Dr. Cecilia Gotti's laboratory. B, sequential immunoprecipitation with the Milan 6 antibody and the 2 antibody demonstrates that 25% of the nAChRs in the rat retina contain both 6 and 2 subunits. Data are the mean ± S.E.M. of three to five experiments. ***, p < 0.001, value is significantly different from the value after NRS.

In sequential immunoprecipitation studies, clearing with the Milan 6 antibody decreased the retinal nAChRs subsequently immunoprecipitated by the 2 antibody by 25% (Fig. 10B). This agrees closely with the value for 62^* subtypes reported by Moretti et al. (2004) and indicates that most (but probably not all) of the 6 subunits in the rat retina are associated with 2 subunits.

To further investigate the 6-containing receptors in the retina, we measured the ability of -conotoxin MII, which has high affinity for nAChRs with the subunit composition of 62^* (Champtiaux et al., 2002; McIntosh et al., 2004) to compete for retinal nAChRs binding sites labeled with [³H]EB. As shown in Fig. 11, -conotoxin MII competed with high affinity for 17% of the retina receptors labeled with [³H]EB. It is interesting that this value is significantly different from the values for the 62^* subunit association measured with either 6 antibody. This suggests that in addition to the 2 subunit, one or more other subunits influence the ability of -conotoxin MII to bind to the receptor. For example, most 62^* receptors seem to also contain the 3 subunit (Cui et al., 2003; Gotti et al., 2005; Salminen et al., 2005), and binding of -conotoxin MII was substantially decreased in the absence of 3 subunits (Cui et al., 2003; Salminen et al., 2005).

View larger version (13K): [in this window] [in a new window]   Fig. 11. Inhibition of [3H]EB binding by -conotoxin MII in rat retina homogenates. Binding assays were carried out as described under Materials and Methods using 500 pM [3H]EB. The Ki value for -conotoxin MII was 9.9 ± 2.5 nM, assuming a Kd value of 90 pM for [3H]EB (Table 2). The maximum inhibition of binding by -conotoxin MII was 17 ± 2%. Data shown are the mean ± S.E.M. from three experiments.

	Discussion

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Figure 12 summarizes the measured subunit associations that we found in the rat retina and estimates the percentage of seven different nAChR subtypes that can be deduced from these studies. The uncertainty of the measurements of subunit associations in these sequential immunoprecipitation assays was estimated by the propagation of error method, which takes into account the variance for each of the individual measurements from which the percentage of association was derived. From this method, we expressed the percentage of each proposed subtype as a range.

View larger version (27K): [in this window] [in a new window]   Fig. 12. Summary of measured nAChR subunits, subunit associations, and the proposed simple and mixed heteromeric nAChR subtypes in the rat retina determined from the sequential immunoprecipitation data.

Deduced Receptor Subtypes. The measured associations between the 2 and 2 subunits accounted for 20 to 28% of the retinal nAChRs in these studies (Fig. 6). We also found that 9% of the receptors contain both 2 and 4 subunits (Fig. 9B). Because this receptor would require either a 2 or 4 subunit, and we found no associations between the 4 subunit and either the 2 or 4 subunit (Fig. 7), we assigned the 2 subunit to this receptor to yield an 242 subtype, which would represent 9% of the total nAChRs in the retina. The remaining 2-containing receptors are likely to be the simple 22 subtype, which would represent 15% of the retina nAChR receptors.

The associations between the 4 and 2 subunits represented 24 to 29% of the nAChRs in the retina (Fig. 6). Because 9% of the retinal receptors can be accounted for by the 242 subtype (see above) and, again, we found no association between the 4 and 4 subunits, the remaining 42 subunit association probably represents the simple 42 subtype, which would represent 15 to 20% of the nAChRs in the retina.

Approximately 25% of the nAChRs in the rat retina contain 4 subunits; moreover, 13% of the total nAChRs in the retina seem to be a mixed heteromeric subtype containing bo