©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neurons Can Maintain Multiple Classes of Nicotinic Acetylcholine Receptors Distinguished by Different Subunit Compositions (*)

(Received for publication, November 17, 1994)

William G. Conroy Darwin K. Berg (§)

From the Department of Biology, University of California, San Diego, La Jolla, California 92093-0357

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Although 10 genes have been cloned encoding putative subunits of neuronal nicotinic acetylcholine receptors, little is known about the variety or subunit composition of such receptors expressed by individual neurons. Chick ciliary ganglion neurons express five of the known genes and assemble a class of synaptic-type receptors collectively containing gene products from three of them: alpha3, beta4, and alpha5. Using subunit-specific monoclonal antibodies, we show here that all of the synaptic-type acetylcholine receptors having alpha3 also have beta4 subunits and vice versa. In addition, most, if not all, of the alpha5 gene product present in fully assembled receptors is associated with both alpha3 and beta4 subunits. Although the receptors may be homogeneous in these respects, only about 20% of them also contain the fourth gene product, beta2, newly identified in the ganglion; essentially all of the neurons express the beta2 gene. No beta2 subunits are found coassembled with the fifth acetylcholine receptor gene product expressed by the neurons, alpha7, which has been shown previously to comprise a class of abundant, nonsynaptic receptors on the cells. The identification of three acetylcholine receptor subtypes distinguished by subunit composition on the same neurons provokes questions about their individual physiological roles.


INTRODUCTION

Neurotransmitter receptors, because of their central role in mediating chemical synaptic transmission in the nervous system, have been the subject of intensive investigation. Most thoroughly characterized have been ligand-gated ion channels where molecular cloning has isolated entire gene families encoding putative subunits of receptors for each of the known neurotransmitters. The complexity of the families has raised questions both about the rules governing subunit assembly and about the purpose of multiple receptor subtypes responding to the same neurotransmitter. Expression studies in Xenopus oocytes and in transfected cell lines have been used to determine the gene combinations that produce functional receptors and to examine the properties of the receptors that result. The types and subunit composition of native receptors, however, are only beginning to be understood.

Nicotinic acetylcholine receptors (AChRs) (^1)represent a family of ligand-gated ion channels that is widely expressed in the vertebrate nervous system(1) . Ten genes have been cloned to date encoding putative subunits of neuronal AChRs. Seven (alpha2-alpha8) were originally thought to encode ligand-binding subunits, and three (beta2-beta4) were considered more structural in purpose. Increasing evidence now indicates that both classes of gene products can influence the pharmacological properties of the receptors. Expressing the genes in Xenopus oocytes has shown that any one of the alpha2, alpha3, and alpha4 genes in combination with either the beta2 or beta4 gene can produce a functional AChR. More remarkably, the alpha7 and alpha8 genes can do so alone. No function has been revealed by this approach for the alpha5, alpha6, and beta3 gene products.

Physiological analysis reveals complex ACh responses from individual neurons. Several time constants can be required for a proper fit of the desensitization, and several kinds of nicotine-induced single channel events can be detected in the same cell. One explanation is that multiple receptor species may contribute to the response. Recently, AChR transcript analysis has confirmed the expression of as many as six AChR genes in a defined population of neurons such as sympathetic or parasympathetic ganglionic cells(2, 3, 4) . How many receptor subtypes can be produced from the gene products and whether all of the subtypes are found in the same neurons remain unanswered questions.

A system in which these questions can be addressed in some detail is the chick ciliary ganglion. The ganglion contains only two kinds of neurons: choroid neurons, which innervate smooth muscle in the vasculature of the choroid layer, and ciliary neurons, which innervate striated muscle in the iris and ciliary body. All of the neurons are cholinergic, and all receive cholinergic input from preganglionic terminals as their primary source of chemical synaptic transmission. Previous studies have shown that the neurons express two major classes of AChRs. One class, termed mAb 35-AChRs, binds the monoclonal antibody (mAb) 35 but not alpha-bungarotoxin (alpha-Bgt). They are largely concentrated in the synaptic membrane and mediate chemical transmission through the ganglion(5, 6, 7, 8) . The other class, termed alpha-Bgt-AChRs, binds alpha-Bgt but not mAb 35. They are located primarily in nonsynaptic regions and have no identified role in the ganglion though they function as ligand-gated ion channels and are nearly an order of magnitude more abundant than mAb 35-AChRs(9, 10, 11) .

Of the 10 known neuronal AChR genes, RNase protections demonstrate that five are expressed in ciliary ganglion neurons: alpha3, alpha5, alpha7, beta2, and beta4(3) . Analysis of subunit composition using subunit-specific mAbs has shown that mAb 35-AChRs collectively contain the alpha3, alpha5, and beta4 gene products but not the alpha7 gene product; the opposite is true of alpha-Bgt-AChRs(12, 13) . Although some mAb 35-AChRs were known to contain all three gene products (alpha3, alpha5, and beta4), it was not clear whether the receptors are homogeneous, particularly with respect to beta4 composition(13) . More importantly, no information was available on the possible distribution of beta2 subunits among AChR subtypes in the ganglion. Those issues are resolved here.


EXPERIMENTAL PROCEDURES

mAbs

mAb 35, which was raised against electric organ AChR and recognizes the alpha1 gene product in muscle and electric organ AChRs(14) , was purified from a hybridoma cell line obtained from Dr. Jon Lindstrom (University of Pennsylvania). mAb 35 also recognizes some classes of chicken neuronal AChRs (13, 15, 16) and has been shown to cross-react with the AChR alpha5 gene product(12) . mAbs 268, 270, 313, and 318 were also generously provided by Dr. J. Lindstrom and were available as concentrated stocks obtained from hybridoma supernatants by ultrafiltration followed by ammonium sulfate fractionation as described previously(17) . mAbs 268 and 270 were raised against chicken brain AChRs (17) and have been shown to recognize the alpha5 gene product and the beta2 gene product, respectively(12, 13, 18, 19) . mAbs 313 and 318 were raised against fusion proteins containing putative cytoplasmic domains of the chicken alpha3 and alpha7 gene products, respectively(20, 21) .

mAb A3-1, which is specific for the alpha3 gene product, and mAbs B4-1 and B4-2, which are specific for the beta4 gene product, were raised against fusion proteins containing putative cytoplasmic domains of their respective gene products and have been described previously (13) . Unless otherwise indicated these mAbs were diluted from ascites fluid for use or were purified using protein G-Sepharose 4 Fast Flow (Pharmacia Biotech Inc.). Purified mAbs 35, 313, 318, A3-1, B4-1, and B4-2, and normal IgG were individually coupled to Actigel (Sterogene Bioseparations) at 3-6 mg/ml according to the manufacturer's specifications.

mAb B2-1, which has not been described previously, was raised against a fusion protein encoding a portion of the beta2 gene product which includes amino acids 326-396(13) . The fusion protein was expressed in bacteria, purified, and used as an immunogen for mAb production as described previously(12) . Hybridomas were screened for their ability to precipitate AChRs in chick brain extracts that were labeled with I-mAb 270. mAb B2-1 was found to be an IgG1 and was used as diluted ascites fluid and was purified and coupled to Actigel as described above.

Solid Phase Immunoprecipitation Assays

On embryonic days 17-18 chick ciliary ganglia were dissected and frozen at -70 °C until used. Ganglia were homogenized in 50 mM sodium phosphate, pH 7.4, containing 1% (v/v) Triton X-100 (Pierce) and the following protease inhibitors: iodoacetamide (0.4 mM), benzamidine (5 mM), phosphoramidon (5 µg/ml), soybean trypsin inhibitor (10 µg/ml), leupeptin (10 µg/ml), pepstatin A (20 µg/ml), EDTA (5 mM), EGTA (5 mM), aprotinin (2 µg/ml), phenylmethylsulfonyl fluoride (1 mM). Insoluble material was removed by centrifugation for 15 min in a microcentrifuge at 4 °C.

Solid phase immunoprecipitation assays were used to quantify AChRs present in detergent extracts or sucrose gradient fractions by tethering the receptors in Immulon 2 Removawells (Dynatech Laboratories, Inc.) with subunit-specific mAbs and using a radiolabeled mAb to determine the amount of receptor bound. The wells were coated with mAb by first incubating the wells overnight at 4 °C on a shaker with affinity purified rabbit anti-mouse antibodies (Jackson ImmunoResearch Inc.) at a concentration of 20 µg/ml in 0.15 M sodium chloride and 0.01 M sodium phosphate, pH 7.4 (phosphate-buffered saline, PBS) containing 0.02% azide. The wells were then washed three times with PBS containing 0.5% Triton X-100 (PBS-TX) and incubated on a shaker overnight at 4 °C with 50 µl of anti-AChR mAb diluted in buffer. To analyze AChRs containing alpha3 or beta4 subunits, mAbs A3-1 and B4-1 were combined and used at dilutions of 1:200 to 1:400 from ascites fluid. After incubating for 6 h at 37 °C or overnight at 4 °C, the wells were rinsed three times with PBS-TX and incubated overnight with the detergent extracts or gradient samples. The samples were then removed, and the wells were washed four times with PBS-TX. Normal rat serum plus normal mouse serum at 2% (v/v) each in PBS-TX were incubated in the wells for 30 min at 37 °C followed by incubation with 5 nMI-mAb 35 in 2% (v/v) normal rat and normal mouse serum solution for 2 h at 37 °C. Unbound I-mAb 35 was removed with four washes of PBS-TX, and bound radioactivity was determined by gamma counting individual wells. Nonspecific binding was determined by including an excess of unlabeled mAb 35 (1 µM) with the labeled mAb 35 or by substituting buffer for the sample and was subtracted in all cases. Combining mAbs A3-1 and B4-1 for tethering receptors from sucrose gradient fractions yielded the same results as using either of the mAbs individually.

AChRs containing beta2 subunits were assayed by adsorbing the receptors to anti-beta2 mAb-Actigel and measuring the amount bound with radiolabeled mAb. Ganglion extracts or sucrose gradient fractions were incubated with 10 µl of mAb B2-1-Actigel and 5 nMI-mAb 35 in the presence and absence of 1 µM unlabeled mAb 35 in a total volume of 80 µl. After an overnight incubation at 4 °C on a shaker, the mAb-Actigel was centrifuged and washed four times with PBS-TX. The resin was recovered and counted in a gamma counter to quantify bound I-mAb 35. AChRs containing beta4 subunits were assayed using mAb B4-1-Actigel. Nonspecific immunoprecipitation was assessed by substituting normal rat IgG-Actigel for the mAb-Actigels. A similar immunoprecipitation assay was used to test for beta2 subunits in alpha-Bgt-AChRs, substituting 10 nMI-alpha-Bgt for the labeled mAb 35 and using 100 µM nicotine to determine nonspecific binding. The resin was incubated, washed, and counted as above. mAb 318-Actigel was substituted for the B2-1-Actigel to quantify the alpha7-containing alpha-Bgt-AChRs as a positive control.

Immunoblot Analysis

Ciliary ganglion AChRs immunopurified from detergent extracts or sucrose gradient fractions were analyzed on immunoblots by solubilizing the material in SDS sample buffer containing 5% beta-mercaptoethanol, subjecting the sample to SDS-polyacrylamide gel electrophoresis on a 9% polyacrylamide gel, and electroblotting the fractionated components onto nitrocellulose as described previously(22) . The blots were blocked with 3% (w/v) Carnation nonfat dry milk in PBS containing 0.1% Tween 20, incubated overnight at 4 °C with mAbs diluted in the same solution, washed in PBS containing 0.05% Tween 20, and then incubated for 2 h at room temperature with horseradish peroxidase coupled to goat anti-rat IgG (Jackson ImmunoResearch Inc.) to detect bound mAbs. Signals were visualized by enhanced chemiluminescence (Amersham). Dilutions of mAbs from concentrated stocks (cs) or ascites fluid (af) were as follows: mAb 268, 1:1000 (cs); mAb B2-1, 1:200 (af); mAb 313, 1:1000 (cs); mAb B4-2, 1:500 (af); and mAb 319, 1:200 (cs). Molecular mass markers for the blots included phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa) (Bio-Rad, low range).

Immunopurification and immunodepletion of AChRs

Ciliary ganglion extracts prepared as described above were incubated either with 20 µl of mAb-Actigel (experimental sample) or with an equivalent amount of mouse IgG-Actigel or rat IgG-Actigel (negative control). In all cases, the mAb-resins had been prepared by treatment with sodium citrate, pH 3.0, in 0.1% Triton X-100 as described previously(15) . For immunopurifications, the resin was centrifuged and washed three times with PBS-TX, three times with PBS-TX containing 1 M sodium chloride, 5 mM EDTA, and 5 mM EGTA, and two more times with PBS-TX. Bound material was eluted with two 30-µl aliquots of 0.1 M sodium citrate, pH 3, in 0.1% Triton X-100 and then immediately neutralized by adding 1 M Tris, pH 8, and 1 N NaOH. Eluates were cleared of contaminating mAbs by incubating with 5 µl of protein G-Sepharose for 10 min, recovering the supernatant after centrifugation, diluting them appropriately with concentrated SDS sample buffer, and subjecting them to SDS-polyacrylamide gel electrophoresis/immunoblot analysis as described above. For immunodepletions, mAb-Actigel or rat IgG-Actigel was incubated with extract at 4 °C either for 6-8 h when the recovered samples were to be analyzed by sucrose gradient sedimentation or overnight when the recovered samples were to be analyzed for residual mAb 35 binding sites. The Actigels with bound material were removed by centrifugation.

Sucrose Gradient Analysis

Velocity sedimentation analysis of mAb 35-AChRs was performed by layering 125-µl aliquots (20 ganglia/aliquot) onto 5-ml linear sucrose gradients (5-20% sucrose in PBS-TX) and centrifuging for 70 min in a VTi-65.2 rotor (Beckman) at 65,000 rpm and 4 °C. Fractions were collected (16-20/gradient) and assayed for mAb 35-AChRs in the solid phase immunoprecipitation assay or used for immunoblot analysis by diluting immediately with concentrated SDS-polyacrylamide gel electrophoresis sample buffer. Typically, 50 µl of each fraction was used to quantify alpha3- and/or beta4-containing mAb 35-AChRs, 200 µl for beta2-containing AChRs, and 15 µl for immunoblot analysis. When extracts were to be immunodepleted prior to sucrose gradient sedimentation, they were incubated with a combination of mAb B4-1-Actigel and mAb B4-2-Actigel to remove beta4-containing components or with mAb A3-1-Actigel and mAb 313-Actigel to remove alpha3-containing components as described above. In some experiments 5 µl of protein G-Sepharose containing bound mAb B4-2 was also included with the mAb B4-1-Actigel.

Aliquots of Torpedo electric organ extract, prepared as described previously (23) and containing the AChR 13 s dimer and the 9.5 s monomer were included in all sucrose gradients for calibration. In separate gradients, Torpedo AChRs were centrifuged together with catalase (11.1 s), human IgG (7.2 s), bovine hemoglobin (4.3 s), and soybean trypsin inhibitor (2.3 s) to generate standard curves for sedimentation velocity.

Immunocytochemistry

Ciliary ganglia were dissected and immediately immersed in 0.1 M sodium phosphate, pH 7.4, containing 25% sucrose. After 1-2 h, the ganglia were embedded in optimum cutting temperature compound, frozen, and stored at -20 °C overnight. Frozen sections of 10 µm were cut with a cryostat at -18 °C and mounted on Vectabond (Vector Laboratories) coated slides and air dried for 1-2 h at room temperature. The sections were treated with avidin D (Blocking Kit, Vector Laboratories) for 15 min and incubated with 10% goat serum in PBS containing 0.1 M glycine, pH 7.4 (PBS-Gly), for 30 min. The sections were rinsed with PBS and incubated for 2 h with either mAb 35, mAb 270, or normal rat IgG at 3 µg/ml in PBS-Gly containing biotin (Blocking Kit, Vector Laboratories) and 20% goat serum. The sections were then washed in PBS and incubated for 1 h with biotinylated-goat anti-rat IgG (Jackson ImmunoResearch) at 1:2,000 in PBS-Gly containing 20% goat serum. After washing in PBS, the sections were incubated for 30 min with an avidin-biotinylated horseradish peroxidase complex (Vectastain Elite ABC Kit, Vector Laboratories) and then washed in PBS and developed in diaminobenzidine and H(2)O(2) (DAB Substrate Kit, Vector Laboratories). The sections were mounted in Aqua-mount (Lerner Laboratories) and viewed by bright-field microscopy.

Materials

White Leghorn embryonated chick eggs were obtained locally and maintained at 39 °C in a humidified incubator as described(24) . mAb 35 was purified and radioiodinated as described previously(10) . alpha-Bgt was purified from Bungarus multicinctus venom (5) and radioiodinated to a specific activity of 3-7 times 10 cpm/mol using chloramine T. Torpedo electric organ tissue was obtained from Dr. Palmer Taylor (University of California, San Diego). Other compounds were obtained from Sigma unless otherwise indicated.


RESULTS

Coassembly of alpha3 and beta4 Subunits in mAb 35-AChRs

Immunoprecipitations with subunit-specific mAbs demonstrated previously that essentially all ciliary ganglion AChRs capable of binding mAb 35 but not alpha-Bgt contain alpha3 subunits(13) . Only a fraction of the same receptors, however, appeared to contain beta4 subunits when analyzed by immunoprecipitation. The results implied receptor heterogeneity with respect to subunit composition. To examine the issue further here, a sensitive solid phase immunoprecipitation assay was devised for probing subunit composition. In the assay a subunit-specific mAb was used to tether receptors containing a given subunit, and a radiolabeled mAb to a different epitope was used to quantify the number of bound receptors. To avoid contributions from incompletely assembled receptors, ganglion extracts were fractionated by sucrose gradient sedimentation prior to analysis. The 10 s region of the gradient was assumed to contain fully assembled receptors.

The solid phase assay detected a large number of mAb 35-AChRs in the 10 s region of the gradient when ciliary ganglion extracts were mock depleted with IgG-Actigel prior to sucrose gradient sedimentation (Fig. 1). mAbs specific for alpha3 subunits (A3-1) and for beta4 subunits (B4-1) were combined to tether receptors in the assay so that mAb 35-AChRs having either or both kinds of subunits would be scored. Depleting the extracts with mAbs specific for either alpha3 or beta4 subunits prior to sucrose gradient sedimentation removed essentially all of the mAb 35-AChRs that could be detected subsequently in gradient fractions with the solid phase assay. The results demonstrate that mAb 35-AChRs containing alpha3 subunits also contain beta4 subunits and vice versa.


Figure 1: Sucrose density gradient analysis showing that mAb 35-AChRs sedimenting at 10 s contain both alpha3 and beta4 subunits. Ciliary ganglion extracts were fractionated by sucrose gradient sedimentation, and the fractions were assayed for mAb 35-AChRs using the solid phase immunoprecipitation assay with mAbs A3-1 (anti-alpha3) and B4-1 (anti-beta4) combined to tether receptors and I-mAb 35 to quantify them. Depleting the extracts either with anti-beta4 mAbs (triangles) or anti-alpha3 mAbs (circles) prior to sedimentation removed essentially all of the I-mAb 35 binding activity. Extract depleted with normal mouse IgG (squares) served as controls for loss of mAb 35-AChRs through nonspecific adsorption. The gradients were calibrated by including Torpedo AChRs in the IgG-depleted samples; the 13 s dimer and the 9.5 s monomer peaked in fractions 4 and 8, respectively.



Commitment of alpha5 Subunits to mAb 35-AChRs Containing alpha3 and beta4 Subunits

Immunoblot analysis demonstrated previously that at least some mAb 35-AChRs containing alpha3 and beta4 subunits also contain alpha5 subunits(13) . The lack of alpha5-specific mAbs capable of immunoprecipitating solubilized receptors prevented a determination of how many mAb 35-AChRs contain all three kinds of subunits. A different but related question concerns how much of the alpha5 subunit is coassembled with alpha3 and beta4 subunits in mAb 35-AChRs. This can be answered by depleting ganglion extracts with mAbs to alpha3 and beta4 subunits, sedimenting the depleted extracts on sucrose gradients, and using immunoblots to quantify the amount of alpha5 protein remaining in the gradient fractions. In this manner it can be shown that essentially all of the alpha5 subunit in the 10 s region of the gradient is coassembled with both alpha3 and beta4 subunits, indicating that most, if not all, of the fully assembled alpha5 is present in receptors having all three kinds of subunits.

Ciliary ganglion extracts mock depleted with IgG-Actigel were fractionated by sucrose gradient sedimentation, and the fractions were analyzed by probing immunoblots with the alpha5-specific mAb 268 (Fig. 2A; positive control). Substantial amounts of alpha5 protein were detected in the 10 s region of the gradient, implying assembly into pentameric receptors. More than half of the alpha5 protein was recovered as smaller weight material, indicating that a significant amount of the protein, at least in embryonic neurons, is apparently not in fully assembled receptor. Similar patterns were observed for the distributions of alpha3 and beta4 protein on the gradients (data not shown). Immunodepleting the extracts with mAbs specific for either the alpha3 or beta4 subunits removed nearly all of the alpha5 protein present in the 10 s region of the gradient (Fig. 2A; fractions 5-7). Quantifying the results with laser densitometry indicated that more than 80% was removed by the anti-beta4 mAbs and more than 90% by the anti-alpha3 mAbs (Fig. 2B).


Figure 2: Immunoblot analysis showing that alpha5 protein sedimenting at 10 s is associated with alpha3 and beta4 subunits. Panel A, ciliary ganglion extracts were immunodepleted with IgG-Actigel as a positive control (top) or with mAbs A3-1- and 313-Actigels to remove alpha3-containing receptors (middle) or with mAbs B4-1- and B4-2-Actigels to remove beta4-containing receptors (bottom) and then fractionated by sucrose gradient sedimentation. The indicated fractions were analyzed on immunoblots probed with the anti-alpha5 mAb 268 followed by goat anti-rat IgG coupled to horseradish peroxidase and enhanced chemiluminescence. The molecular mass markers (top to bottom) are 97.4, 66.2, 45, and 31 kDa. Panel B, laser densitometry was used to quantify the alpha5 signals obtained in panel A. Control experiments demonstrated that the densitometer signals were within the linear range of the immunoblot assay. The results are taken from the same experiment shown in Fig. 1, permitting direct comparison of receptor and subunit positions on the gradients. Integrating the areas under the curves indicates that the anti-alpha3 and beta4 mAbs each depleted 80-90% of the alpha5 protein in the 10 s region of the gradient, suggesting that essentially all of the alpha5 protein present in receptor is coassembled with both alpha3 and beta4 subunits. Similar results were obtained in two other experiments.



Some of alpha5 protein in the 6-8 s region of the gradient (Fig. 2B, fractions 8-11) was also immunodepleted by mAbs to alpha3 and beta4 subunits, indicating that it was coassembled with one or both of the two kinds of subunits even though the sedimenting species were not fully assembled receptor, judging by their sedimentation rates. The material may represent assembly intermediates or inappropriately folded or assembled protein destined for degradation. Despite its coassembly with alpha3 and/or beta4 protein, alpha5-containing material in this region of the gradient did not score as mAb 35 binding in the solid phase immunoprecipitation assay presumably because the conformation or access of the relevant epitope(s) was not appropriate. The alpha5 protein in the 5 s region does not appear to be assembled with either alpha3 or beta4 protein (Fig. 2B, fractions 12-14) and may be monomeric.

Since the mAbs used for the immunodepletions are subunit-specific(13) , the results permit the conclusion that essentially all of the fully assembled alpha5 protein is present in mAb 35-AChRs containing both alpha3 and beta4 subunits. It is possible the reverse is also true, namely that all ciliary ganglion receptors containing alpha3 and beta4 subunits also contain alpha5, but the mAbs available do not allow the proposition to be tested.

The Association of beta2 Protein with Gene Products Found in mAb 35-AChRs

RNase protection experiments have shown that ciliary ganglion neurons express beta2 mRNA, and the number of transcripts/ganglion are as abundant as those of alpha5 and beta4(3) . Despite this, no beta2 protein has previously been detected in the ganglion. Using mAb 270 and a new mAb (B2-1) that are specific for beta2 protein, it is possible to show not only that the protein is present in the ganglion but also that it is coassembled with alpha3, beta4, and alpha5 subunits in a portion of the mAb 35-AChRs.

mAb 270-Actigel was used to immunopurify beta2 protein from ciliary ganglion extracts. The recovered material was analyzed on immunoblots probed with mAb B2-1 (Fig. 3A). A doublet was detected with the lower band being the more prominent of the two. Similar results were obtained when mAb B2-1-Actigel was substituted for mAb 270-Actigel and the blots were probed with either mAb B2-1 or 270, as well as when mAb 270 was used to probe blots of material immunopurified with mAb 270-Actigel (data not shown). From experiments in which mAb B2-1 was used on immunoblots, the calculated molecular mass for the upper component was 55.5 ± 0.6 kDa (n = 4), and the lower component was 53.7 ± 0.8 kDa (n = 4). In two experiments, only a single band was detected. Whether the doublet arises from partial degradation or differential processing of the beta2 protein is unclear and has not been investigated further. The size of the beta2 protein determined here is slightly larger than the 49-50 kDa reported previously for the beta2 subunit in brain AChRs (18) and in cells transfected with the beta2 gene(25) , but it is close in size to the molecular mass of 54 kDa deduced from the nucleotide sequence of the beta2 gene(26) .


Figure 3: Immunoblots identifying beta2 protein in ciliary ganglion extracts and showing its association with other AChR gene products. Panel A, mAb 270-Actigel was used to adsorb beta2-containing material from ciliary ganglion extracts, and the adsorbed material was eluted and analyzed by immunoblot probed with mAb B2-1. A doublet containing components of about 55 and 53 kDa was detected (lane 2) when the bound antibody was visualized with secondary antibody and chemiluminescence as in Fig. 2. The labeling was specific since it was not detected when the primary mAb was omitted (lane 1); the adsorption to mAb 270-Actigel was specific since it did not occur with IgG-Actigel (lane 3). Panel B, the beta2-containing material recovered from mAb B2-1-Actigel (lanes 5-8) and from mAb 270-Actigel (lanes 9-12) was analyzed on immunoblots probed with mAbs specific for the other AChR gene products known to be expressed in ciliary ganglion neurons. All three gene products previously identified in mAb 35-AChRs (alpha3, beta4, and alpha5) were found to be present in both preparations of beta2-containing material when probed with mAbs 313, B4-2, and 268, respectively. The immunopurifications were specific because the components were not obtained when IgG-Actigel was substituted for the mAb-Actigels (lanes 1-4). No alpha7 protein was detected by mAb 318 in the material immunopurified with anti-beta2 mAbs (lanes 8 and 12). The molecular mass markers are as in Fig. 2.



The components were not obtained when IgG-Actigel was substituted for mAb 270-Actigel in the immunopurification and were not detected when the primary mAb was omitted from the blot analysis (Fig. 3A). mAb 270, which was previously shown to recognize the beta2 gene product specifically(12, 13, 18, 19) , binds to an extracellular epitope, whereas mAb B2-1 is presumed to bind to an intracellular epitope because the mAb was raised against a fusion protein containing the putative cytoplasmic domain of the beta2 gene product. Like mAb 270, mAb B2-1 is specific for the beta2 protein. It recognized the beta2 fusion protein on immunoblots but not fusion proteins corresponding to similar regions of the alpha3, alpha5, or beta4 subunit. In addition, mAb B2-1 recognized the beta2 protein translated in vitro and analyzed by immunoblots but not the full-length alpha3, alpha4, alpha5, or beta4 gene products translated in vitro and analyzed similarly (data not shown). The specificity and the distinct epitopes recognized by the two mAbs provide assurance that the components identified are indeed beta2 protein. Neither mAb B2-1 nor mAb 270 was sufficiently sensitive to detect the small amounts of beta2 protein in ganglion extracts by immunoblot analysis unless the protein was first concentrated by immunopurification.

Coassembly of the beta2 protein with other AChR gene products known to be present in mAb 35-AChRs was revealed by immunoblot analysis. mAbs specific for alpha3 subunits (mAb A3-1), beta4 subunits (mAb B4-2), and alpha5 subunits (mAb 268) each detected their corresponding antigen in material immunopurified either with mAb 270 or mAb B2-1 (Fig. 3B). Since mAbs 270 and B2-1 are specific for the beta2 subunit, the detection of the alpha3, beta4, and alpha5 protein in the immunoprecipitated material can only be attributed to the coassembly of these subunits with beta2 protein. mAb 318 did not detect a component in the immunopurified material, indicating that few, if any, alpha7 subunits are coassembled with beta2 protein. The immunopurifications were specific in that IgG-Actigel did not substitute for mAb 270-Actigel or mAb B2-1-Actigel in recovering the AChR proteins (Fig. 3B).

Corroboration that the beta2 gene product is coassembled with the AChR gene products found in mAb 35-AChRs was provided by immunopurifying material with mAb 35-Actigel and probing the immunoblots with subunit-specific mAbs. In addition to alpha3, beta4, and alpha5 protein, which were expected to be present, the antibodies also revealed beta2 protein in the recovered material (Fig. 4). The immunopurification of beta2 protein by mAb 35-Actigel was specific in that it could not be duplicated by IgG-Actigel (Fig. 3A).


Figure 4: Immunoblot analysis showing that mAb 35-purified material includes beta2 protein. Ciliary ganglion extracts were incubated with mAb 35-Actigel, and the adsorbed material was eluted and analyzed on immunoblots probed with no primary mAb (np) as a negative control (lane 1), mAb 313 for alpha3 (lane 2), mAb B4-2 for beta4 (lane 3), mAb 268 for alpha5 (lane 4), and mAb B2-1 for beta2 protein (lane 5). Bound mAbs were detected as in Fig. 2except that the secondary antibody included both anti-rat and anti-mouse IgG. The small components seen in lanes 2-4 most likely represent degradation products; they were detectable only when the immunoblots were deliberately overexposed, as done here to demonstrate beta2 protein. The molecular mass markers are as in Fig. 2.



The Presence of beta2 Subunits in a Subpopulation of mAb 35-AChRs

Sucrose gradient analysis was performed to determine whether the beta2-containing species recognized by mAb 35 had the size expected for fully assembled AChRs. Gradient fractions were measured for such components in the solid phase assay using mAb B2-1 to tether beta2 protein and I-mAb 35 to probe for mAb 35 binding sites. The major peak of such material was discovered in the 10 s region of the gradient, coincident in position with mAb 35-AChRs containing alpha3 and beta4 subunits (Fig. 5).


Figure 5: Sucrose gradient analysis showing that the beta2-containing component recognized by mAb 35 has the size expected for a fully assembled AChR. Ciliary ganglion extracts were fractionated by sucrose gradient sedimentation, and the fractions were assayed for I-mAb 35-binding components tethered in the solid phase assay with the anti-beta2 mAb B2-1 (filled squares) or the anti-beta4 mAb B4-1 (open triangles). To compensate for the small amounts of beta2-containing material present, five times more fraction volume was used with B2-1 in the assays than with B4-1. Nonspecific binding, which has been subtracted from each point, was assessed either by replacing the gradient fraction with buffer in the assay or by including an excess of unlabeled mAb 35 along with the I-mAb 35. The beta2-containing species that binds mAb 35 cosediments with mAb 35-AChRs shown above to contain alpha3 and beta4 subunits coassembled. Similar results were obtained in a second experiment.



The number of beta2-containing AChRs recognized by mAb 35 appeared to be substantially smaller than the number of mAb 35-AChRs containing alpha3 and beta4 subunits, judging from the relative amounts of mAb 35 binding in the sucrose gradient analysis. The reduced amount of mAb 35 binding obtained in the solid phase assay when mAb B2-1 was used to tether receptors could not be attributed to poor antibody affinity because a second pass of the extract through the assay yielded only one-third as much as the first time (see below). To provide additional quantification of the relative amounts of the two kinds of mAb 35-binding components, solid phase assays were carried out in parallel on ciliary ganglion extracts using mAb B4-1 to tether mAb 35-AChRs containing beta4 subunits and mAb B2-1 to tether beta2-containing AChRs. I-mAb 35 was used to measure the number of binding sites retained in each case. By this criterion, beta4-containing mAb 35-AChRs are nearly five times more abundant than beta2-containing AChRs (Fig. 6A). Very little mAb 35 binding was detected in material adsorbed nonspecifically to IgG-Actigel as a negative control.


Figure 6: Immunoprecipitations quantifying the amounts of beta2-containing components binding mAb 35 or alpha-Bgt. Panel A, the amount of beta2-containing material that binds mAb 35 in ciliary ganglion extracts was determined using a solid phase assay with mAb B2-1 to tether the material and I-mAb 35 to measure it. For comparison, mAb B4-1 was used to tether mAb 35-AChRs in similar assays. In both cases nonspecific binding was determined as described in Fig. 5and was subtracted from all points. Normal IgG was substituted for the mAbs to assess nonspecific retention of mAb 35 binding sites in the assay. Values represent the mean ± S.E. from at least six separate experiments and are reported as the number of mAb 35 binding sites tethered per ciliary ganglion (CG). The number of mAb 35 binding sites associated with beta2-containing material is about one-fifth of that associated with beta4-containing mAb 35-AChRs. Panel B, the amount of beta2-containing material that binds alpha-Bgt was determined in a similar manner, substituting I-alpha-Bgt for labeled mAb 35. For comparison, mAb 318 was substituted for mAb B4-1 in the assay to tether alpha7-containing alpha-Bgt-AChRs. Normal IgG was used to assess nonspecific retention of alpha-Bgt-binding material in the solid phase assay. Values represent the mean ± S.E. of three determinations and are reported as the number of alpha-Bgt binding sites tethered per ciliary ganglion. No significant alpha-Bgt binding was detected in beta2-containing material.



No detectable beta2 protein is present in alpha-Bgt-AChRs (Fig. 6B). The number of alpha-Bgt binding sites tethered in the solid phase assay by mAb B2-1 was no greater than that obtained when IgG was substituted for the mAb. In contrast, the number of alpha-Bgt binding sites was substantial when mAb 318 was used to tether alpha7-containing receptors in the assay, as shown previously by other means(13) .

Are the beta2-containing mAb 35-AChRs a subset of the mAb 35-AChRs containing alpha3 and beta4 subunits? This question was addressed by immunodepleting AChRs with mAbs to either alpha3, beta2, or beta4 subunits and then measuring the number of remaining mAb 35 binding sites in each case which could be tethered by mAb B2-1 or mAb B4-1 in the solid phase assay. IgG was used as a negative control. The anti-beta2 mAb B2-1 was reasonably efficient at removing beta2-containing AChRs, depleting about 70% of the signal detected in the solid phase assay (Fig. 7). The mAb removed little of the beta4-containing receptors, consistent with beta2 subunits being present in only a small fraction of mAb 35-AChRs. Anti-alpha3 mAbs depleted about 80% of both beta2- and beta4-containing receptors defined by mAb 35 binding in the solid phase assay (Fig. 7). This provided strong evidence that the beta2-containing mAb 35-AChRs are a subset of those containing alpha3 and beta4 subunits. The depletion of the beta2-containing AChRs by the anti-beta4 mAb B4-1, however, was variable. In six experiments, the remaining beta2-containing mAb 35-AChRs ranged from 11 to 150% (69 ± 23%; mean ± S.E.) of the IgG-depleted control extracts even when mAb B4-1 efficiently removed 90 ± 3% (n = 4) of the beta4-containing mAb 35-AChRs. The reasons for the variation with B4-1 are not clear. Nonetheless, the fact that most mAb 35-AChRs contain both alpha3 and beta4 subunits and the fact that the anti-alpha3 mAbs are efficient at depleting beta2-containing components indicate that the latter are a subset of mAb 35-AChRs. This interpretation is also consistent with the immunoblot results presented above showing that beta2 is associated with all three gene products known to be present in mAb 35-AChRs. In fact, taken together, the immunoprecipitation and immunoblot analyses strongly suggest that at least a portion of mAb 35-AChRs contains four kinds of subunits coassembled: alpha3, alpha5, beta2, and beta4.


Figure 7: Immunodepletions showing that the beta2-containing material recognized by mAb 35 is a subset of mAb 35-AChRs containing alpha3 and beta4 subunits. Ciliary ganglion extracts were immunodepleted as described in Fig. 2to remove components containing either beta2 (mAb B2-1) or alpha3 (mAbs A3-1 or 313) protein. Nonspecific depletion was assessed by substituting IgG-Actigel for the mAbActigels. The depleted extracts were then measured for the number of remaining I-mAb 35 binding sites that could be tethered with either mAb B2-1 or mAb B4-1 in the solid phase assay. Values are expressed as a percent of those obtained with extracts depleted only by IgG-Actigel (positive control) and represent the mean ± S.E. of six separate experiments for mAb B2-1 and two experiments each for mAbs A3-1 and 313. mAbs A3-1 and 313 immunoprecipitated about 80% of the mAb 35 binding sites associated either with beta2- or beta4-containing components, supporting the contention that beta2-containing components that sediment at 10 s and bind mAb 35 are a subset of the mAb 35-AChRs known to contain alpha3 and beta4 subunits.



Distribution of beta2 Protein among Neurons in the Ciliary Ganglion

The finding that mAb 35-AChRs are heterogeneous with respect to beta2 subunits, i.e. some receptors have them and some do not, raised the question of whether all neurons in the ganglion express the beta2 gene. Conceivably only one-fifth of the neurons in the ganglion express the gene, and all of the mAb 35-AChRs produced by such cells contain beta2 subunits. This possibility was evaluated with immunocytochemistry using the anti-beta2 mAb 270 to probe frozen sections prepared from 18-day embryonic ciliary ganglia. As a control for the efficiency of antibody penetration, sister sections were probed with mAb 35, which has previously been shown to label essentially all of the neurons in the ganglion(24) . The two antibodies produced similar results (Fig. 8). Most, if not all, of the neuronal somata were labeled in both cases, indicating that the heterogeneity of mAb 35-AChRs with respect to beta2 content cannot be explained by the gene product being present in only a subset of the neurons.


Figure 8: Immunocytochemistry with mAb 270 demonstrating the presence in situ of beta2 protein in most ciliary ganglion neurons. Cryostat sections of 18-day embryonic ciliary ganglia were incubated with mAb 270, mAb 35, or normal rat IgG followed by a biotinylated anti-rat IgG antibody and an avidin-biotin-horseradish peroxidase complex. The sections were developed for peroxidase activity and photographed with bright-field microscopy. Comparable numbers of neurons were intensely stained both with mAb 270 (panel A), which recognizes the beta2 subunit, and with mAb 35 (panel B), which has previously been shown to recognize antigen in most ciliary ganglion neurons(24) . Only light background staining was seen when normal IgG was substituted for the mAbs in treating the sections (panel C). Similar results were obtained in adjacent sections as well as sections taken from other regions of the ganglion; a second experiment produced the same outcome. The results indicate that beta2 gene expression is not confined to a small subset of neurons in the ganglion. Calibration bar, 20 µm.




DISCUSSION

The main findings reported here are that a single population of neurons can express at least three classes of AChRs based on subunit composition and that the heterogeneity observed cannot be attributed to differences in gene expression among cells within the population. The results also strongly suggest that some neuronal AChRs may be as complex in subunit composition as muscle and electric organ AChRs, having four types of subunits: alpha3, alpha5, beta2, and beta4. The association of beta2 with beta4 subunits as well as beta2 with both alpha3 and alpha5 subunits together represent new combinations not previously reported for native AChRs.

Ciliary ganglion AChRs that bind mAb 35 but not alpha-Bgt contain both alpha3 and beta4 subunits. Previous studies had suggested that the receptors were heterogeneous with respect to beta4 composition, based on the inability of mAb B4-1 to immunoprecipitate all of the mAb 35-AChRs as measured(13) . Those results may have been confounded by the inclusion of assembly intermediates or other components detected in the column assay used to quantify mAb 35-AChRs. The more sensitive and direct solid phase assay used here, together with sucrose gradient fractionation to eliminate small molecular weight material, found that essentially all mAb 35-AChRs containing alpha3 subunit also contained beta4 subunit and vice versa. Available mAbs did not permit a conclusion as to whether all of the mAb 35-AChRs also contain alpha5 subunit. It is clear, however, that most, if not all, of the alpha5 subunit assembled into 10 s receptor is associated with alpha3 and beta4 subunits. Conceivably mAb 35-AChRs are homogeneous with respect to containing alpha3, beta4, and alpha5.

The conclusion that beta2 subunits are present in a portion of ciliary ganglion mAb 35-AChRs is supported by several lines of evidence. Identification of beta2 protein on ciliary ganglion immunoblots was unambiguous since it made use of two beta2-specific mAbs recognizing different epitopes. Immunoprecipitation of beta2 protein using mAbs specific for the beta2 gene product depleted a species that bound mAb 35 and cosedimented with mAb 35-AChRs. It represented about one-fifth of the mAb 35 binding associated with alpha3- and beta4-containing mAb 35-AChRs. Immunoblot analysis of the material immunoprecipitated by beta2specific mAbs revealed all three gene products known to be present in mAb 35-AChRs. Reciprocally, immunopurification of receptors with mAb 35 yielded beta2 protein. The beta2-containing species that binds mAb 35 was efficiently precipitated by anti-alpha3 mAbs as expected if it represents a subset of the mAb 35-AChRs containing alpha3 and beta4 subunits. An anti-beta4 mAb was less reliable in immunoprecipitating the beta2-containing component that binds mAb 35, but there can be little doubt that beta2 subunits are also associated with beta4 subunits in the receptors. Essentially all mAb 35-AChRs that contain alpha3 also contain beta4. Moreover, immunoprecipitation of beta2 protein with specific mAbs coprecipitates beta4 protein.

Much of the beta2 gene product in brain is assembled with alpha4 subunits to make up the major receptor species binding nicotine with high affinity(17, 18, 27) . Analysis of the alpha4 and beta2 gene products expressed either in Xenopus oocytes or in the stably transfected fibroblast cell line M10 indicates that pentameric receptors are produced which contain two alpha4 subunits and three beta2 subunits(19, 25, 28) . In ciliary ganglion neurons little, if any, alpha4 gene product is present(3) . Instead, the results clearly demonstrate that the beta2 subunit is coassembled with alpha3, beta4, and alpha5 subunits in the ganglion, producing almost certainly at least one neuronal AChR as complex as the muscle AChR with its four kinds of subunits. The alternative possibility, namely that the data might be explained by a mixture of several different receptor subtypes having no more than three kinds of subunits each, is difficult to reconcile with the observation that essentially all of the mAb 35-AChRs contain both alpha3 and beta4 subunits, at least some contain alpha5 subunits, and beta2 is coassembled with a portion of each of the three gene products in the mAb 35-AChR pool.

The fact that the vast majority of ciliary ganglion neurons express beta2 protein indicates that the incomplete penetration of beta2 subunits into the mAb 35-AChR population does not represent an all-or-none exclusion of the protein among neurons. It is possible that the beta2 gene product confers special properties on the receptors and therefore is carefully regulated in abundance by the neurons. One such property might be receptor location in the plasma membrane. If the beta2 subunit targets mAb 35-AChRs to a presynaptic location on axon terminals for example, it might explain why relatively few of the receptors are present in dissected ganglia that lack axons although the beta2 transcript is as abundant as beta4 transcripts that contribute subunits to all mAb 35-AChRs in the neurons(3) . The mechanism of receptor targeting in the membrane and the functional significance of possible presynaptic receptors are subjects of considerable interest.

An alternative possibility is that the beta2 subunit imposes regulatory constraints on mAb 35-AChRs such as making them cyclic AMP-dependent. The nicotinic response of ciliary ganglion neurons acquires a partial dependence on cyclic AMP during development both in vivo and in cell culture(29, 30) . The molecular basis for the cyclic AMP effect is unknown. It will be important to determine what proportion of mAb 35-AChRs on the cell surface contains beta2 subunits and whether the proportion changes with conditions that alter the cyclic AMP dependence of the nicotinic response.

Nicotinic responses from neurons can be complex both in kinetics and single channel events(1) . The present results suggest that the complexity may in part reflect heterogeneity in subunit composition among AChRs produced. Ciliary ganglion neurons have alpha-Bgt-AChRs and two classes of mAb 35-AChRs, namely those with and without beta2 subunit. In situ hybridizations and immunohistochemical results presented both here and previously (3, 6, 9, 31) make it clear that most, if not all, neurons in the ganglion express all of the gene products known to comprise the three receptor classes. Accordingly, it is highly likely that all ciliary ganglion neurons, both choroid and ciliary, express each of the three classes. The multiplicity of AChR subtypes distinguished in a single population of neurons calls attention to functional and regulatory features that might be uniquely associated with individual receptor subtypes. Differences in the conditions of activation, receptor location, ion permeability, duration of response, and regulatory control would seem to be among the most likely reasons accounting for comaintenance of multiple receptor species by the same neuron.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants R01 NS12601 and P01 NS25916 and by the California Tobacco-related Disease Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biology, 0357, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0357. Tel.: 619-534-4680; Fax: 619-534-0301; dberg{at}ucsd.edu.

(^1)
The abbreviations used are: AChR(s), nicotinic acetylcholine receptor(s); alpha-Bgt, alpha-bungarotoxin; mAb, monoclonal antibody; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Dr. Jon Lindstrom for generously providing monoclonal antibodies, Dr. Palmer Taylor for kindly providing Torpedo electric organ tissue, Dr. Ann Vernallis for characterization of monoclonal antibody B2-1, and Lynn Ogden for excellent technical assistance. Tissue dissections were performed by Leticia Oliva, Cynthia Brent, Victor Wang, and Silvina Villalobos.


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