Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor alpha subunit.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for alpha-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle alpha subunit as a soluble, secretory protein using the yeast Pichia pastoris. By testing a series of truncated fragments of the receptor protein, we show that alpha211, the entire amino-terminal extracellular domain of AChR alpha subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The alpha211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with N-linked glycosylation on SDS-polyacrylamide gel. The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mm ( approximately 30 mg/ml). The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR. In addition, it formed a high affinity complex with alpha-bungarotoxin (k(D) 0.2 nm) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of alpha211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently (15)N-labeled in P. pastoris, and proton cross-peaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy. We conclude that the soluble AChR protein is useful for high resolution structural studies.     

Differential expression of human nicotinic acetylcholine receptor alpha subunit variants in muscle and non-muscle tissues.

The nicotinic acetylcholine receptor (nAChR) is an oligomeric transmembrane glycoprotein consisting of four homologous subunits in stoichiometry of alpha 2, beta (gamma or epsilon). Recently the presence of a novel exon (P3A) in human alpha AChR gene has been reported. Two variants of the human alpha subunit arise from alternate RNA splicing, one with and one without the P3A exon. However, the evolutionary origin of the P3A exon and the regulation of the expression of the two variants in human muscle and non-human tissues is currently unknown. Examination of genomic DNA from various species shows that the P3A exon sequence is present only in hominoids, old world and new world primates species and is absent in the muscle cDNA or genomic DNA from rat, mouse or dog, indicating that P3A exon is evolutionary conserved for at least 50 million years. The P3A+ variant of alpha subunit was found to be constitutively expressed in skeletal muscle, brain, heart, kidney, liver, lung and thymus, while P3A-variant was differentially expressed only in skeletal muscle. Thus it appears that the P3A+ variant is generated by 'default' selection by the splicing machinery, while expression of the P3A- variant is regulated by tissue-specific factors in the skeletal muscle. Mechanisms regulating differential expression of the alpha subunit variants may be pertinent to the pathophysiology of myasthenia gravis.     

Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin- treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits accumulate, and ultimately make functional receptors in AChR- expressing cells.     

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.     

Characterization of the mRNA for mouse muscle acetylcholine receptor alpha subunit by quantitative translation in vitro

The nicotinic acetylcholine receptor of mammalian skeletal muscle is a multisubunit membrane glycoprotein whose synthesis is regulated by developmental and physiological cues. We report here the identification and characterization of the primary translation product of alpha subunit mRNA. The alpha subunit synthesized in rabbit reticulocyte lysate is approximately 2000 larger in apparent molecular weight than the native alpha subunit polypeptide found in acetylcholine receptor. Evidence from peptide maps and the effect of co-translational incubation with dog pancreas microsomes suggests that the in vitro product differs in two ways from native alpha subunit: 1) it is synthesized with an NH2-terminal signal peptide which is removed in vivo, and 2) the in vitro product is not glycosylated. We have characterized the alpha subunit mRNA activity by using a quantitative the membrane-bound polysome fraction. It is poly(A+) and approximately 2000 nucleotides long. Finally, we have shown that in BC3H-1 cells, alpha subunit mRNA is regulated developmentally. We detected a 10-fold increase in the relative abundance of alpha subunit mRNA in cells which had undergone the transition from log phase growth to differentiated myoblast.     

Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor. A model is proposed which accounts for all the experimental data obtained thus far on the transmembrane orientation of the subunit polypeptide chains.     

Linkage disequilibrium study of RFLPs detected at the human muscle nicotinic acetylcholine receptor subunit genes.

We screened DNA from unrelated individuals for RFLPs in the muscle nicotinic acetylcholine receptor (AcChoR) genes. These RFLP markers can be used for genetic linkage and association studies to test the hypothesis that receptor structure or regulation is involved in the development of myasthenia gravis (MG). The cDNAs from four subunits (alpha, beta, gamma, and delta) of the murine muscle AcChoR were used as probes to identify RFLPs in the homologous human genes. Digestion of DNA from 15 unrelated individuals with a set of 10 restriction enzymes revealed 11 RFLPs. At least one RFLP was found for each subunit gene. Eight RFLPs were found at the linked gamma and delta gene loci, six with minor allele frequencies greater than 15%, making that linkage group a very informative marker locus (PIC = .72). PIC values were calculated for the RFLPs from allele and haplotype frequency estimates obtained from a population sample of 53 individuals. The delta gene was assigned by in situ hybridization to region q31----q34 of chromosome 2. All pairs of RFLPs were analyzed for linkage disequilibrium. Of the 16 pairs of RFLPs from the same gene or from the linked gamma and delta genes, 13 pairs showed evidence of disequilibrium that was significant, with P less than .05. The implications of these results are discussed.     

Iodo-alpha-conotoxin MI selectively binds the alpha/delta subunit interface of muscle nicotinic acetylcholine receptors

The embryonic mouse muscle nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel formed by alpha1, beta1, delta, and gamma subunits. The receptor contains two ligand binding sites at alpha/delta and alpha/gamma subunit interfaces. [(3)H]Curare preferentially binds the alpha/gamma interface. We describe the synthesis and properties of a high-affinity iodinated ligand that selectively binds the alpha/delta interface. An analogue of alpha-conotoxin MI was synthesized with an iodine attached to Tyr-12 (iodo-alpha-MI). The analogue potently blocks the fetal mouse muscle subtype of nAChR expressed in Xenopus oocytes. It failed, however, to block alpha3beta4, alpha4beta2, or alpha7 nAChRs. Iodo-alpha-MI potently blocks the alpha1beta1delta but not the alpha1beta1gamma subunit combination expressed in Xenopus oocytes indicating selectivity for the alpha/delta subunit interface. Alpha-conotoxin MI was subsequently radioiodinated, and its properties were further evaluated. Saturation experiments indicate that radioiodinated alpha-conotoxin MI binds to TE671 cell homogenates with a Hill slope of 0.95 +/- 0.0094. Kinetic studies indicate that the binding of [(125)I]alpha-conotoxin MI is reversible (k(off) = 0.084 +/- 0.0045 min(-1)); k(on) is 8.5 x 10(7) min(-1) M(-1). The calculated k(d) is 0.98 nM. This potency is approximately 20-fold higher than the unmodified alpha-MI peptide. Unlike [(125)I]alpha-bungarotoxin, [(125)I]alpha-conotoxin MI binding to TE671 cell homogenates is fully displaceable by the small molecule antagonist d-tubocurarine.     

Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.     

Assembly intermediates of the mouse muscle nicotinic acetylcholine receptor in stably transfected fibroblasts

We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma, and alpha and delta subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing alpha and delta subunits are formed in a time-dependent manner. The alpha and gamma- and the alpha and delta-producing transfected cells form complexes that, when labeled with 125I-alpha- bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the alpha and gamma subunits expressed in fibroblasts suggesting that gamma may have more than one binding site for alpha subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of the binding of 125I-alpha- bungarotoxin to intact and detergent-extracted fibroblasts indicate that essentially all of the binding sites are retained in an intracellular pool. The fibroblast delta subunit has the electrophoretic mobility in SDS-PAGE of a precursor that does not contain complex carbohydrates. In addition, alpha gamma and alpha delta complexes had lectin binding properties expected of subunits lacking complex oligosaccharides. Therefore, fibroblasts coexpressing alpha and gamma or alpha and delta subunits produce discrete assembly intermediates that are retained in an intracellular compartment and are not processed by Golgi enzymes.     

The transmembrane topology of the amino terminus of the alpha subunit of the nicotinic acetylcholine receptor

We have investigated the transmembrane topology of the amino-terminal domain of the alpha subunit of the mouse muscle nicotinic acetylcholine receptor synthesized in vitro and in vivo. Using oligonucleotide-directed mutagenesis we introduced new glycosylation consensus sequences at alpha 154 and at alpha 200. For each novel site, additional constructs were made in which the original site at alpha N141 was eliminated. Glycosylation at the new sites, as exhibited in a rabbit reticulocyte cell-free translation system supplemented with canine pancreatic microsomes and in a transient transfection system with COS cells, was taken as evidence of the transmembrane translocation of the new site. Each of the new sites was glycosylated in both systems. In separate experiments we found that an alpha subunit fragment terminating at alpha M207 could be extracted from microsomal membranes with sodium carbonate after in vitro translation, indicating that this fragment is not an integral membrane protein. Our results, taken together with previous experiments, indicate that the amino terminus of the alpha subunit up to at least residue alpha 207 is translocated across the membrane of the endoplasmic reticulum. This topology probably represents the orientation of the amino terminus of the alpha subunit in the assembled receptor.     

The Caenorhabditis elegans unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor alpha subunit

The anthelmintic drug levamisole causes hypercontraction of body wall muscles and lethality in nematode worms. In the nematode Caenorhabditis elegans, a genetic screen for levamisole resistance has identified 12 genes, three of which (unc-38, unc-29, and lev-1) encode nicotinic acetylcholine receptor (nAChR) subunits. Here we describe the molecular and functional characterization of another levamisole-resistant gene, unc-63, encoding a nAChR alpha subunit with a predicted amino acid sequence most similar to that of UNC-38. Like UNC-38 and UNC-29, UNC-63 is expressed in body wall muscles. In addition, UNC-63 is expressed in vulval muscles and neurons. We also show that LEV-1 is expressed in body wall muscle, thus overlapping the cellular localization of UNC-63, UNC-38, and UNC-29 and suggesting possible association in vivo. This is supported by electrophysiological studies on body wall muscle, which demonstrate that a levamisole-sensitive nAChR present at the C. elegans neuromuscular junction requires both UNC-63 and LEV-1 subunits. Thus, at least four subunits, two alpha types (UNC-38 and UNC-63) and two non-alpha types (UNC-29 and LEV-1), can contribute to levamisole-sensitive muscle nAChRs in nematodes.