Glial Regulation of α7-Type Nicotinic Acetylcholine Receptor Expression in Cultured Rat Cortical Neurons

Abstract: Primary embryonic cortical cultures were used as an in vitro model to evaluate the influence of glia on developmental expression of α7-type nicotinic acetylcholine receptors in rat brain. In cells cultured in serum-containing medium without mitotic inhibitors, specific ¹²⁵I-α-bungarotoxin binding to α7-type nicotinic receptors was maximal 4–8 days after plating. Treatment with 5'-fluorodeoxyuridine (80 µ M ) from 1 to 3 days in vitro significantly reduced glial proliferation and concomitantly increased ¹²⁵I-α-bungarotoxin binding, whereas plating onto a glial bed layer decreased binding. There was no significant binding to pure glial cultures. Treatment-induced changes in neuronal binding resulted from alterations in receptor density, with no change in affinity. 5'-Fluorodeoxyuridine treatment also increased cellular expression of α7 receptor mRNA but had no effect on N -[³H]methylscopolamine binding to muscarinic receptors. Glial conditioned medium decreased ¹²⁵I-α-bungarotoxin binding in both control and 5'-fluorodeoxyuridine-treated cultures, suggesting the release of a soluble factor that inhibits α7-type nicotinic receptor expression. An additional mechanism of glial regulation may involve removal of glutamate from the surrounding medium, as added glutamate (200 µ M ) increased ¹²⁵I-α-bungarotoxin binding in astrocyte-poor cultures but not in those that were astrocyte enriched. These results suggest that glia may serve a physiological role in regulating α7-type nicotinic receptors in developing brain.     

α-Bungarotoxin Binding in House Fly Heads and Torpedo Electroplax

Abstract: House fly heads contain a site that binds α-bungarotoxin with high affinity. It is present at about 23 pmol/g of heads and binds α-bungarotoxin (labeled with [³H]pyridoxamine phosphate) reversibly with a K _d of 6 nM. The effects of 48 drugs have been compared on the α-bungarotoxin binding sites of house fly and Torpedo. The pharmacology of the house fly site is similar to that previously reported for neuronal α-bungarotoxin binding sites in both vertebrates and invertebrates and is distinguishable from that of the classic nicotinic neuromuscular acetylcholine receptor, as exemplified by that of Torpedo electroplax. Differences between the house fly site and Torpedo include higher affinities of the Torpedo receptor for decamethonium, hexamethonium, carbamylcholine, and acetyl-β-methylcholine, but lower affinities for nicotine, atropine, and dihydro-β-erythroidine.     

Presence of α7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective α-neurotoxins in histochemistry

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of α7 nAChR with neurotoxins, utilizing α7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated α-bungarotoxin (Alexa-αBgt) was demonstrated in binding to α7-transfected cells inhibited by long-chain α-cobratoxin (CTX), but not short-chain α-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-αBgt labeling of neurons, with no staining for α7 nAChR knockout tissue. It proved that α7 nAChRs are the major αBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-αBgt and non-labeled CTX/NTII that allows specific histochemical detection of α7 nAChR with a spatial resolution at the level of single axon terminals.     

Structure—Function Studies of the Eighth Hydrophobic Domain of a Serotonin Receptor

Abstract : The most prominent structural feature of the G protein-coupled receptor superfamily is their seven hydrophobic domains, which are postulated to form membrane-spanning α helices. Some members of the G protein-coupled receptor family, specifically several serotonin (5-HT) receptors, possess eight hydrophobic domains. The importance of this extra hydrophobic domain, located at the N terminus of the receptor, is unknown. This question was addressed by deleting the extra hydrophobic region from the 5-HT_2C receptor and comparing its function and topology with those of the wild-type receptor. Immunofluorescence microscopy was used to determine the location of the N terminus of the epitope-tagged wild-type and mutant receptors. The N terminus of both receptors was extracellular, suggesting that the extra hydrophobic domain does not change the topology of this receptor and is unlikely to be a membrane-spanning α helix. Radioligand-binding studies in transfected cells and expression studies in Xenopus oocytes demonstrated that seven hydrophobic domains were sufficient for normal function in these assays. Interestingly, the mutant receptor, now containing seven hydrophobic domains, is expressed at higher levels in transfected cells than the wild-type receptor containing eight hydrophobic domains, suggesting that the extra hydrophobic domain does impact the activity of this receptor by regulating its expression.     

Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression

The RIC-3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC-3 (hRIC-3) protein enhances expression of α7 receptors and abolishes expression of α4β2 receptors. In vitro translation of hRIC-3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC-3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for α7 expression enhancement but not for α4β2 inhibition. A combination of increased α7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in α7 membrane expression induced by hRIC-3. Antibodies against hRIC-3 showed its expression in SH-SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC-3 in rat brain localized, in general, in places where α7 nAChRs were found.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.     

Role of the N-terminal α-helix in biogenesis of α7 nicotinic receptors

We studied the role of the α-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in α7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the α-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of α7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (α3β4 and α4β2) and 5-HT_3A receptors also abolished their expression at the membrane. We conclude that the N-terminal α-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.     

N-Linked Glycosylation of the α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate(AMPA)-Selective Glutamate Receptor Channel α2 Subunit Is Essential for the Acquisition of Ligand-Binding Activity

Abstract: The N-linked glycosylation of the α2 subunit of the mouse α-amino-3-hydroxy-5-methylisoxazole-4-propionate(AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N -glycosylation sites. The recombinant receptor proteins were identified by [³⁵S]methionine/[³⁵S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [³H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of ∼102 kDa and a minor species of ∼98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA-binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N -glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N -glycosylation site is required for the formation and maintenance of the GluRα2 subunit protein in an active conformation for ligand binding. Possible roles of N -glycosylation of GluRα2 subunit protein are discussed.     

Subunit Structure of α-Bungarotoxin Binding Component in Mouse Brain

Abstract: The α-bungarotoxin binding component in mouse brain was purified by affinity chromatography with toxin-Sepharose, gel-chromatography on Sepharose 6B, and ion-exchange chromatography with DE52 resin. The iodinated product of the last step produced one major and one minor band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the minor peak was twice as large as that of the major one. The iodinated product could bind α-bungarotoxin, and this binding was inhibited by a nicotinic antagonist, d -tubocurarine, which demonstrated that the iodinated product was a true α-bungarotoxin binding component. The molecular structure of the product was analysed by cross-linking followed by SDS-PAGE. The results fitted the model for an α-bungarotoxin binding component in the mouse brain composed of six identical or very similar subunits of 51,000-52,000. One subunit carrying the binding site for toxin bound one molecule of toxin. This subunit structure of an α-bungarotoxin binding component in the brain is discussed in comparison with that of a nicotinic acetylcholine receptor in the electric organ.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

A mutated transferrin receptor lacking asparagine-linked glycosylation sites shows reduced functionality and an association with binding immunoglobulin protein

The function of the transferrin receptor is to transport iron-bound transferrin into the cell. In order to function properly, this dimeric glycoprotein must be expressed on the cell surface and be able to bind transferrin. Site-directed mutagenesis was performed to abolish the three asparagine-linked glycosylation consensus sequences of the human transferrin receptor. The DNA encoding the mutated transferrin receptor was stably transfected into mouse fibroblasts. This form of the human transferrin receptor shows reduced transferrin binding, reduced intersubunit bond formation, and reduced cell surface expression, indicating that the transferrin receptor which lacks asparagine-linked glycosylation is not fully functional. In addition, the mutated form of the receptor is not processed as quickly. It shows an association with an endoplasmic reticular chaperone protein, binding immunoglobulin protein, leading to the hypothesis that the mutated transferrin receptor experiences increased retention in the endoplasmic reticulum.     

A novel fluorescent α-conotoxin for the study of α7 nicotinic acetylcholine receptors

Homomeric α7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified α9 subunit can also form functional homopentamers as well as α9α10 heteropentamers. Current fluorescent probes for α7 nicotinic ACh receptors are derived from α-bungarotoxin (α-BgTx). However, α-BgTx also binds to α9* and α1* receptors which are coexpressed with α7 in multiple tissues. We used an analog of α-conotoxin ArIB to develop a highly selective fluorescent probe for α7 receptors. This fluorescent α-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked α7 currents in Xenopus laevis oocytes with an IC₅₀ value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated α-BgTx, Cy3-ArIB[V11L;V16A] did not block α9α10 or α1β1δε receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [¹²⁵I]-α-BgTx binding to mouse hippocampal membranes with a K _i value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXα7R1 cells but not KXα3β2R4, KXα3β4R2, or KXα4β2R2 cells. This labeling could be abolished by pre-treatment with α-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for α7 receptors.     

Host Cell-Specific Folding and Assembly of the Neuronal Nicotinic Acetylcholine Receptor α7 Subunit

Abstract: Expression of the cloned neuronal nicotinic acetylcholine receptor (nAChR) α7 subunit in several cultured mammalian cell lines has revealed that the folding, assembly, and subcellular localization of this protein are critically dependent upon the nature of the host cell. In all cell lines that were examined, high levels of α7 protein were detected by metabolic labelling and immunoprecipitation after transfection with the cloned α7 cDNA. In contrast, elevated levels of α-bungarotoxin binding could be detected in only two of the nine cell lines. Both of these "α7-permissive" cell lines [rat phaeochromocytoma (PC12) and human neuroblastoma (SH-SY5Y)] express an endogenous α7 subunit. However, by expression of an epitope-tagged α7 subunit, it has been possible to show that the elevation in surface α-bungarotoxin binding in these two cell lines is due to expression of cDNA-encoded α7. The cell-specific misfolding of the neuronal nAChR α7 subunit is a phenomenon that is not shared by either the hetero-oligomeric muscle nAChR or the homo-oligomeric serotonin receptor 5-HT₃ subunit. Our data also indicate that the cell-specific misfolding cannot be explained by a requirement for the coassembly with other known nAChR subunits and cannot be alleviated by treatments that have been reported to affect the assembly efficiency of other neurotransmitter-gated ion channels.     

Calcium Regulation of Agonist Binding to α7-Type Nicotinic Acetylcholine Receptors in Adult and Fetal Rat Hippocampus

Abstract: Quantitative autoradiography was used to compare the binding properties of α7-type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of ¹²⁵I-α-bungarotoxin (¹²⁵I-α-BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of ¹²⁵I-α-BTX binding, as well as α-cobratoxin and nicotine potency to displace ¹²⁵I-α-BTX, did not change with age. Addition of Ca²⁺ to the assay buffer did not alter ¹²⁵I-α-BTX binding, or α-cobratoxin inhibition of ¹²⁵I-α-BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca²⁺ on agonist affinity was dose-dependent, with an EC₅₀ value of 0.25–0.5 m M . Ca²⁺ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal ¹²⁵I-α-BTX binding sites are largely similar across age. Ca²⁺ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the α7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

Host Cell-Specific Folding of the Neuronal Nicotinic Receptor α8 Subunit

Abstract: Heterologous expression of the neuronal nicotinic acetylcholine receptor α8 subunit in cultured mammalian cell lines has revealed that the correct folding of this protein is dependent on the host cell type. The α8 subunit, which is able to form homo-oligomeric ion channels when expressed in Xenopus oocytes, could be detected in all transfected cell lines by both immunoprecipitation and immunofluorescence microscopy with a monoclonal antibody that recognises a linear epitope. In contrast, the α8 subunit could be detected in some but not in all transfected cell lines with a monoclonal antibody that recognises a conformation-sensitive epitope or by nicotinic radioligand binding. It is interesting that although correctly folded α8 protein could be detected in transfected rat pituitary (GH₄C₁) cells, only misfolded α8 protein could be detected in a large subpopulation of transfectants (transient or clonal stable isolates). We have also found that the protein encoded by a chimaeric cDNA (constructed from the N-terminal region of α8 and the C-terminal domain of the serotonin 5-HT₃ receptor subunit) is expressed efficiently, and in a conformation that binds α-bungarotoxin, in all cell types examined. These results, together with previous expression studies with the homo-oligomeric α7 subunit and hetero-oligomeric nicotinic receptor subunit combinations, suggest that the cell-specific folding described here is a phenomenon that may be characteristic of homo-oligomeric nicotinic receptors.     

CHOLINERGIC SITES IN SKELETAL MUSCLE: INTERACTION WITH CONCANAVALIN A

Abstract– The interaction of normal and denervated skeletal muscle cholinergic sites with the lectin concanavalin A and concanavalin A-Sepharose are detailed. Concanavalin A blocks the binding of ¹²⁵I-α-bungarotoxin to both the high and low affinity sets of cholinergic sites described previously. The characteristics of the block of ¹²⁵I-α-bungarotoxin binding to the high affinity set (acetylcholine receptor) is not competitive. The data suggest that the concanavalin binds multivalently to the macro-molecular complex containing the ACh receptor site and sterically prevents the α-bungarotoxin binding. The interaction of both sets of cholinergic sites with concanavalin A-Sepharose was also studied. The macromolecule(s) containing both the high and low affinity sets of sites bind to the concanavalin A-Sepharose. The data indicate a multivalent association with the affinity resin. Following the affinity procedure, a partial purification in both sets of sites is effected. The equilibrium binding of ¹²⁵I-diiodo-α-bungarotoxin to the preparations from the affinity procedure (both normal and denervated muscle) was examined. The K_D of the α-bungarotoxin binding to the high affinity sets of sites (acetylcholine receptor) in both normal and denervated preparations changes from ∼10⁻⁹mol/l to ∼ 10⁻¹⁰ mol/l following purification. No change in the K_D of the α-bungarotoxin binding to the low affinity set of sites was observed following purification. The ¹²⁵l-α-bungarotoxin binding to the partially purified acetylcholine receptor was blocked by unlabelled α-bungarotoxin, concanavalin A, d-tubocurarine and carbamylcholine.