Acetylcholine Receptor Delta Subunit Research Articles

A Deficiency in Glutamine-Fructose-6-Phosphate Transaminase 1 (Gfpt1) in Skeletal Muscle Results in Reduced Glycosylation of the Delta Subunit of the Nicotinic Acetylcholine Receptor (AChRδ).

The neuromuscular junction (NMJ) is the site where the motor neuron innervates skeletal muscle, enabling muscular contraction. Congenital myasthenic syndromes (CMS) arise when mutations in any of the approximately 35 known causative genes cause impaired neuromuscular transmission at the NMJ, resulting in fatigable muscle weakness. A subset of five of these CMS-causative genes are associated with protein glycosylation. Glutamine-fructose-6-phosphate transaminase 1 (Gfpt1) is the rate-limiting enzyme within the hexosamine biosynthetic pathway (HBP), a metabolic pathway that produces the precursors for glycosylation. We hypothesized that deficiency in Gfpt1 expression results in aberrant or reduced glycosylation, impairing the proper assembly and stability of key NMJ-associated proteins. Using both in vitro and in vivo Gfpt1-deficient models, we determined that the acetylcholine receptor delta subunit (AChRδ) has reduced expression and is hypo-glycosylated. Using laser capture microdissection, NMJs were harvested from Gfpt1 knockout mouse muscle. A lower-molecular-weight species of AChRδ was identified at the NMJ that was not detected in controls. Furthermore, Gfpt1-deficient muscle lysates showed impairment in protein O-GlcNAcylation and sialylation, suggesting that multiple glycan chains are impacted. Other key NMJ-associated proteins, in addition to AChRδ, may also be differentially glycosylated in Gfpt1-deficient muscle.

Bone Marrow‐Derived Mesenchymal Stem Cells Up‐Regulate Acetylcholine Receptor Delta Subunit through NRG/ErbB3‐Mediated Mitogen‐Activated Protein Kinase Pathway

To investigate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on the expression of acetylcholine receptor delta subunit (AChRd), the murine skeletal muscle cell line Sol8 were grown in DMEM with 20% fetal bovine serum added with (conditional medium group) or without (control group) conditional medium of BMSC cells for 48 hours. RT-PCR and Western blot were performed to access the mRNA and protein levels of AChRd in Sol8 cells, respectively. Western blot was used to detect total and phosphorylated protein levels of Ras, Raf-1, Mek1/2, and Erk1/2, respectively. NRG-1 antibody added in conditional medium of BMSCs, si-ErbB3, and four Ras/Raf/MEK/ERK pathway inhibitors (FTS, Sulindac, U0126, and PD98059) were using to investigate the effect of AChRd levels. Our studies indicated that expression of AChRd was significantly enhanced in the conditional medium group when compared with those in control group and phosphorylation of Ras, Raf, Erk1/2 in Sol8 cells was also increased. Although gene silencing for ErbB3 gene, adding of NRG-1 antibody in conditional medium of BMSCs or treatment of Ras/Raf/MEK/ERK pathway inhibitors can down-regulate expression of AChRd and phosphorylation, which suggesting that the Ras/Raf/MEK/ERK pathway may be involved in BMSCs-induced expression of AChRd.

Agrin-induced Activation of Acetylcholine Receptor-bound Src Family Kinases Requires Rapsyn and Correlates with Acetylcholine Receptor Clustering

During neuromuscular synaptogenesis, neurally released agrin induces aggregation and tyrosine phosphorylation of acetylcholine receptors (AChRs) by acting through both the receptor tyrosine kinase MuSK (muscle-specific kinase) and the AChR-associated protein, rapsyn. To elucidate this signaling mechanism, we examined tyrosine phosphorylation of AChR-associated proteins, particularly addressing whether agrin activates Src family kinases bound to the AChR. In C2 myotubes, agrin induced tyrosine phosphorylation of these kinases, of AChR-bound MuSK, and of the AChR beta and delta subunits, as observed in phosphotyrosine immunoblotting experiments. Kinase assays revealed that the activity of AChR-associated Src kinases was increased by agrin, whereas phosphorylation of the total cellular kinase pool was unaffected. In both rapsyn-deficient myotubes and staurosporine-treated C2 myotubes, where AChRs are not clustered, agrin activated MuSK but did not cause either Src family or AChR phosphorylation. In S27 mutant myotubes, which fail to aggregate AChRs, no agrin-induced phosphorylation of AChR-bound Src kinases, MuSK, or AChRs was observed. These results demonstrate first that agrin leads to phosphorylation and activation of AChR-associated Src-related kinases, which requires rapsyn, occurs downstream of MuSK, and causes AChR phosphorylation. Second, this activation intimately correlates with AChR clustering, suggesting that these kinases may play a role in agrin-induced AChR aggregation by forming an AChR-bound signaling cascade.

Development of new T-vectors containing the luciferase gene. Easy application for direct cloning of a promoter DNA.

For promoter analyses of genes, it is usually necessary to amplify promoter DNA fragments by polymerase chain reaction (PCR) and clone them into a plasmid containing a reporter gene. In the present study we developed a novel plasmid, pGL2-X, which was constructed through a simple procedure of cloning an XcmI cassette from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene into the multicloning site of pGL2-Basic (Promega) pGL2-X was then converted by XcmI digestion into a T-vector which was named pGL2-T. Unfortunately, however, the firefly luciferase gene in pGL2-Basic contains one XcmI restriction site and therefore one base within the recognition site was silent-mutated. The cloning efficiency of the pGL2-T vector was approximately 63% when tested with a PCR product amplified from a promoter region (-501(-)+24) of the murine acetylcholine receptor delta subunit (AchR delta) gene. In C2C12 muscle cells transiently transfected with pGL2-T containing the AchR delta promoter, transcription of the silent-mutated luciferase gene increased 2.2-fold by neuregulin (EGF domain of heregulin beta 1; 100 ng/mL), a known stimulator of AchR delta expression. This result suggested that the pGL2-T vector was biologically functional. Thus, the present study provides an easy method to construct a variety of T-vectors containing different reporter genes.

Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells.

The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.

Two E-Boxes Are the Focal Point of Muscle-specific Skeletal Muscle Type 1 Na+ Channel Gene Expression

We have characterized a group of cis-regulatory elements that control muscle-specific expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene. These elements are located within a 3. 1-kilobase fragment that encompasses the 5'-flanking region, first exon, and part of the first intron of SkM1. We sequenced the region between -1062 and +311 and determined the start sites of transcription; multiple sites were identified between +1 and +30. The basal promoter (-65/+11) lacks cell-type specificity, while an upstream repressor (-174/-65) confers muscle-specific expression. A positive element (+49/+254) increases muscle-specific expression. Within these broad elements, two E boxes play a pivotal role. One E box at -31/-26 within the promoter, acting in part through its ability to bind the myogenic basic helix-loop-helix proteins, recruits additional factor(s) that bind elsewhere within the SkM1 sequence to control positive expression of the gene. A second E box at -90/-85 within the repressor controls negative regulation of the gene and acts through a different complex of proteins. Several of these cis-regulatory elements share both sequence and functional similarities with cis-regulatory elements of the acetylcholine receptor delta-subunit; the different arrangement of these elements may contribute to unique expression patterns for the two genes.

Muscle-type nicotinic acetylcholine receptor delta subunit determines sensitivity to noncompetitive inhibitors, while gamma subunit regulates divalent permeability

Heterologous expression of nicotinic acetylcholine receptor (nAChR) RNAs in Xenopus oocytes was used to examine the structural basis for pharmacological and physiological differences between muscle-type and neuronal nAChRs. Neuronal nAChRs have a higher permeability to calcium than muscle-type nAChRs and display inward rectification, while muscle-type nAChRs have a linear current–voltage relation. In addition, neuronal nAChRs are more sensitive to inhibition by a class of compounds known as “ganglionic blockers”.It has been shown previously that neuronal-muscle hybrid receptors show increased sensitivity to the use-dependent inhibitor of neuronal nAChRs, BTMPS, based on the presence of a neuronal beta subunit. In this study, we report that omission of gamma subunit RNA has a similar effect. αβδ receptors exhibit prolonged inhibition by BTMPS, show a significant permeability to divalent ions, display inward rectification and are more sensitive to mecamylamine. However, while pharmacological effects are associated with the presence of an additional delta subunit, the physiological changes described seem to be associated with the presence or absence of a gamma subunit. These results suggest that, for nAChRs, as is also the case for non-NMDA ionotropic glutamate receptors, the crucial functional property of limiting calcium permeability can be served by a single subunit.

Binding of monoclonal antibodies against the carboxyl terminal segment of the nicotinic receptor delta subunit suggests an unusual transmembrane disposition of this sequence region.

Monoclonal antibodies (mAbs) specific for the carboxyl terminal region of the delta subunit of Torpedo nicotinic acetylcholine receptor (AChR), derived from mice immunized with AChR or a synthetic carboxyl terminal sequence of the delta subunit (C delta-mAbs), were used to determine the transmembrane disposition of their epitope(s) by immunoelectron microscopy, using AChR-rich postsynaptic membrane fragments from Torpedo electroplax. Some C delta-mAbs recognized only the cytoplasmic side of the membranes, some both sides to a similar extent, and others bound mostly, but not exclusively, to the cytoplasmic side. Binding of C delta-mAbs to the membranes was specifically blocked by synthetic peptides containing the carboxyl terminal region of the delta subunit. Control anti-AChR mAbs specific for the alpha or the delta subunits, whose epitopes have known transmembrane topology, uniquely recognized the expected side of the postsynaptic membrane. Residues involved in C delta-mAb binding were identified using single residue substituted peptide analogues of the sequence delta 481-501. All C delta-mAbs recognized epitopes within the same sequence segment, delta 485-493, at the carboxyl terminal of the AChR delta subunit. These results suggest that the delta subunit of the AChR might have alternative conformations, leading to exposure of the same sequence region on the extracellular or the cytoplasmic surface. Several Pro residues are present in this region. The alternative cis or trans conformation of one or more of them might result in different folding patterns of the carboxyl terminal sequence of the delta subunit, as described for a viral protein [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L., & Harrison, S. C. (1991) Nature 354, 278-284.

Monoclonal Antibodies as Site-Specific Probes for the Acetylcholine-receptor delta-Subunit Tyrosine and Serine Phosphorylation Sites

Phosphorylation of the nicotinic acetylcholine receptor (AChR) has been implicated in the assembly, clustering, regulation of function and degradation rate of the molecule. Torpedo AChR is phosphorylated at eight cytoplasmic residues, four of which are on the delta subunit. We have precisely mapped the epitopes of eleven monoclonal antibodies (mAbs) directed against the Torpedo AChR delta-subunit regions delta 350-396 and delta 484-493, which are therefore exposed on the surface of the intact AChR, and now have four highly specific tools for analysing the role of delta-subunit phosphorylation. More than 160 synthetic peptides attached to polyethylene rods were used for epitope mapping. Four mAbs bound within the region delta 350-380, which contains all of the delta-subunit phosphorylation sites (Ser361, Ser362, Tyr372 and Ser377). Specifically, the epitope for mAb 134 (delta 365-375) contains Tyr372, the epitope(s) for mAbs 139 and 166 (delta 376-381) contains Ser377, while the epitope for mAb 146 (delta 350-359) is close to Ser361 and Ser362 and includes parts of the corresponding phosphorylation consensus sequences. Using peptide analogues with single residue substitutions, Tyr372 was found to be essential for the binding of mAb 134 and Ser377 was found contributing to the binding of mAbs 139 and 166. Finally, tyrosine phosphorylation of Torpedo AChR selectively inhibited binding of mAb 134. These data, and the availability of the defined mAb probes, should facilitate the study of the functional role of single AChR phosphorylation sites.

Monoclonal Antibodies as Site-Specific Probes for the Acetylcholine-receptor delta-Subunit Tyrosine and Serine Phosphorylation Sites

Monoclonal Antibodies as Site-Specific Probes for the Acetylcholine-receptor delta-Subunit Tyrosine and Serine Phosphorylation Sites

Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation.

Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin-induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.

All potential glycosylation sites of the nicotinic acetylcholine receptor delta subunit from Torpedo californica are utilized.

All possible N-glycosylation sites of the delta subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue are utilized. By a combination of microsequencing and mass spectrometry, it was shown that a high-mannose-type oligosaccharide is bound at Asn143 of the delta subunit. The oligosaccharides at positions Asn70 and Asn208 of the delta subunit are probably of the complex type. The utilized glycosylation sites pose restrictions on possible transmembrane folding models of the subunit.

Agonist-independent activation of acetylcholine receptor channels by protein kinase A phosphorylation.

Protein phosphorylation is a ubiquitous and one of the most effective means of regulating protein activity. Receptor phosphorylation is a key event in signal transduction. The question, therefore, that arises is whether this modulatory mechanism might produce functional changes in a membrane receptor in the absence of its naturally occurring ligand. To examine this issue, single-channel properties of purified acetylcholine receptors (AChRs) from Torpedo californica reconstituted in lipid bilayers were studied in the absence of ACh in both unphosphorylated preparations and after in vitro phosphorylation by a purified catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A). Notably, the spontaneous open-channel probability of phosphorylated AChRs is significantly higher than that of unphosphorylated AChRs. Channel activation by protein kinase A is correlated with AChR phosphorylation and is abolished by alpha-bungarotoxin. Analysis of probability distributions of the open dwell times indicates that, similar to unphosphorylated AChR has two distinct open states, short- and long-lived. The frequency of occurrence of the long openings over the short and the magnitude of both time constants increase after phosphorylation, as they do with agonist concentration. Thus, phosphorylation of AChR gamma and delta subunits activates AChR channel opening in the absence of ligand binding. This result is compatible with the notion that protein phosphorylation may effectively act as an intracellular ligand with the phosphorylation sites envisioned as cytoplasmic ligand binding sites.

Synporins--synthetic proteins that emulate the pore structure of biological ionic channels.

A class of proteins that mimic the fundamental pore structure of authentic ionic channels has been designed, synthesized, and characterized. The design is based on our earlier result that a 23-mer peptide with the sequence of the M2 segment of the Torpedo californica acetylcholine receptor delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe -Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms cation-selective channels in lipid bilayers, presumably by self-assembly of conductive oligomers. Accordingly, a tethered parallel tetramer was synthesized with four M2 delta peptides attached to a carrier template--a 9-amino acid backbone with four attachment sites. As expected, the complete 101-residue protein does form channels in lipid bilayers reproducing several features that are characteristic of authentic acetylcholine receptor channels, such as single-channel conductance, cation selectivity, transitions between closed and open states in the millisecond time range, and sensitivity to local anesthetic channel blockers. An analogue protein, in which the serine residue in position 8 is replaced with alanine in each of the four M2 delta 23-mer peptides ([Ala8]M2 delta), also forms channels that, however, exhibit lower single-channel conductance. By contrast, a similar tethered tetramer with M1 delta peptides does not form channels, in accord with expectations. The general validity of this strategy to other channel sequences and oligomer numbers is anticipated. Thus, synporins--a term coined to identify this class of synthetic pore proteins--enrich our armamentarium directed toward the elucidation of structure-function relationships.

Dihydropyridine receptor gene expression is regulated by inhibitors of myogenesis and is relatively insensitive to denervation.

To evaluate developmental and physiological signals that may influence expression of the dihydropyridine-sensitive "slow" Ca2+ channel, we analyzed dihydropyridine receptor (DHPR) mRNA abundance in mouse skeletal muscle. Using synthetic oligonucleotide probes corresponding to the rabbit skeletal muscle DHPR, a 6.5 kb DHPR transcript was identified in postnatal skeletal muscle and differentiated C2 or BC3H1 myocytes, but not cardiac muscle or brain. DHPR gene expression was reversibly suppressed by 0.4 nM transforming growth factor beta-1 or by transfection with a mutant c-H-ras allele, nominal inhibitors of myogenesis that block the appearance of slow channels and DHPR. In contrast, both BC3H1 and C2 myocytes containing the activated ras vector expressed the gene encoding the nicotinic acetylcholine receptor delta subunit, demonstrating that not all muscle-specific genes are extinguished by ras. Denervation stimulated DHPR gene expression less than 0.6-fold, despite 8-fold upregulation of delta-subunit mRNA and reciprocal effects on the skeletal and cardiac alpha-actin genes. Thus, DHPR gene induction is prevented by inhibitors of other muscle-specific genes, whereas, at most, relatively small changes in DHPR mRNA abundance occur during adaptation to denervation.

The sidedness of the COOH terminus of the acetylcholine receptor δ subunit

Abstract The nicotinic acetylcholine receptor from Torpedo sp. occurs as a dimer, disulfide-cross-linked between delta subunits. We determined the sidedness of the COOH terminus of the acetylcholine receptor delta subunit by locating the delta-delta disulfide relative to the membrane and by identifying the Cys residue forming the disulfide. We used receptor-rich native membrane vesicles isolated from Torpedo californica electric tissue and characterized as to orientation and intactness. These vesicles had not been extracted and retained v (43-kDa protein) as a marker of the cytoplasmic surface. Using the reduction of v as an assay of permeability, we showed that two reductants, 2-mercaptoethanesulfonate and reduced glutathione, were relatively impermeant. Both of these reductants reduced the delta-delta disulfide in sealed right-side-out vesicles equally in the presence and absence of saponin, and 2-mercaptoethanesulfonate reduced this disulfide equally in the presence and absence of Triton X-100. By contrast, surfactants enhanced the reduction of dimer in inside-out and sequestered vesicles. We conclude that the disulfide is extracellular. To identify the Cys residue forming the disulfide, we labeled the sulfhydryls both in receptor dimer and in monomer generated by mild reduction of dimer. By high performance liquid chromatography and NH2-terminal sequencing of cyanogen bromide fragments of labeled delta-delta dimer and delta monomer, we found that the penultimate residue, delta-Cys-500, uniquely formed an intersubunit disulfide and that this disulfide was uniquely reduced when receptor dimer was reduced to monomer. Therefore, the delta COOH terminus is extracellular.

Isolation and characterization of the mouse acetylcholine receptor delta subunit gene: identification of a 148-bp cis-acting region that confers myotube-specific expression.

We have isolated the gene encoding the delta subunit of the mouse skeletal muscle acetylcholine receptor (AChR) and have identified a 148-bp cis-acting region that controls cell type-specific and differentiation-dependent gene expression. The 5' flanking region of the delta subunit gene was fused to the protein-coding region of the chloramphenicol acetyltransferase (CAT) gene and gene fusions were transfected into C2 mouse skeletal muscle cells. Both transiently and stably transfected cells were assayed for CAT gene expression. Deletions from the 5' end of the mouse delta gene demonstrate that 148 bp of 5' flanking DNA is sufficient to confer cell type-specific and differentiation-dependent expression: CAT activity is present in transfected myotubes, but not in transfected 3T3 cells or 10T1/2 cells. Moreover, the level of CAT expression in myotubes transfected with constructs containing 148 bp of 5' flanking DNA from the delta subunit gene is identical to that in myotubes transfected with constructs containing 3.2 kb of 5' flanking DNA and similar to expression from the SV-40 early promoter. Increased CAT activity in myotubes is a result of an increased rate of transcription from the delta subunit promoter, since CAT RNA levels are also 35-fold more abundant in myotubes than myoblasts. In contrast, the SV-40 early promoter is similarly active in all cell types. Thus, 148 bp of 5' flanking DNA from the delta subunit gene contains all the information required for cell type-specific and differentiation-dependent expression of the AChR delta subunit.

Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells.

We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta-subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time-course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.

Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit.

Phosphorylation and assembly of nicotinic acetylcholine receptor subunits in cultured chick muscle cells.

The assembly of the nicotinic acetylcholine receptor (AChR), an oligomeric cell surface protein, was studied in cultured muscle cells. To measure this process, the incorporation of metabolically labeled alpha-subunit into oligomeric AChR was monitored in pulse-chase experiments, either by the shift of this subunit from the unassembled (5 S) to the assembled (9 S) position in sucrose density gradients, or by its coprecipitation with antisera specific for the delta-subunit. We have found that AChR assembly is initiated 15-30 min after subunit biosynthesis and is completed within the next 60 min. The alpha-subunit is not overproduced, as all detectable pulse-labeled alpha-subunit can be chased into the oligomeric complex, suggesting that AChR assembly in this system is an efficient process. The rate of AChR assembly is decreased by metabolic inhibitors and by monensin, an ionophore that impairs the Golgi apparatus. We have observed that the gamma- and delta-subunits of AChR are phosphorylated in vivo. The delta-subunit is more highly phosphorylated in the unassembled than in the assembled state, indicating that its phosphorylation precedes assembly and that its dephosphorylation is concomitant with AChR assembly. These findings suggest that subunit assembly occurs in the Golgi apparatus and that phosphorylation/dephosphorylation mechanisms play a role in the control of AChR subunit assembly.