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.     

Soluble, oligomeric, and ligand-binding extracellular domain of the human alpha7 acetylcholine receptor expressed in yeast: replacement of the hydrophobic cysteine loop by the hydrophilic loop of the ACh-binding protein enhances protein solubility

The N-terminal extracellular domain (ECD; amino acids 1-208) of the neuronal nicotinic acetylcholine receptor (AChR) alpha7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR. This molecule, alpha7-ECD, although able to bind the specific ligand alpha-bungarotoxin, existed mainly in the form of microaggregates. Substitution of Cys-116 in the alpha7-ECD with serine led to a decrease in microaggregate size. A second mutant form, alpha7-ECD(C116S,Cys-loop), was generated in which, in addition to the C116S mutation, the hydrophobic Cys-loop (Cys(128)-Cys(142)) was replaced by the corresponding hydrophilic Cys-loop from the snail glial cell acetylcholine-binding protein. This second mutant protein was water-soluble, expressed at a moderate level (0.5 +/- 0.1 mg/liter), and had a size corresponding approximately to a pentamer as judged by gel filtration and electron microscopy studies. It also bound (125)I-alpha-bungarotoxin with relatively high affinity (K(d) = 57 nm), the binding being inhibited by unlabeled alpha-bungarotoxin, d-tubocurarine, or nicotine (K(i) = 0.8 x 10(-7) m, K(i) = 1 x 10(-5) m, and K(i) = 0.9 x 10(-2) m, respectively). All three constructs were expressed as glycosylated forms, but in vitro deglycosylation reduced the heterogeneity without affecting their ligand binding properties. These results show that alpha7-ECD(C116S,Cys-loop) was expressed in P. pastoris as an oligomer (probably a pentamer) with a near native conformation and that its deglycosylated form seems to be suitable starting material for structural studies on the ligand-binding domain of a neurotransmitter receptor.     

Expression and characterization of soluble forms of the extracellular domains of the beta, gamma and epsilon subunits of the human muscle acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel found in muscles and neurons. Muscle AChR, formed by five homologous subunits (alpha2 beta gamma delta or alpha2 beta gamma epsilon), is the major antigen in the autoimmune disease, myasthenia gravis (MG), in which pathogenic autoantibodies bind to, and inactivate, the AChR. The extracellular domain (ECD) of the human muscle alpha subunit has been heterologously expressed and extensively studied. Our aim was to obtain satisfactory amounts of the ECDs of the non-alpha subunits of human muscle AChR for use as starting material for the determination of the 3D structure of the receptor ECDs and for the characterization of the specificities of antibodies in sera from patients with MG. We expressed the N-terminal ECDs of the beta (amino acids 1-221; beta1-221), gamma (amino acids 1-218; gamma1-218), and epsilon (amino acids 1-219; epsilon1-219) subunits of human muscle AChR in the yeast, Pichia pastoris. beta1-221 was expressed at approximately 2 mg.L(-1) culture, whereas gamma1-218 and epsilon1-219 were expressed at 0.3-0.8 mg.L(-1) culture. All three recombinant polypeptides were glycosylated and soluble; beta1-221 was mainly in an apparently dimeric form, whereas gamma1-218 and epsilon1-219 formed soluble oligomers. CD studies of beta1-221 suggested that it has considerable beta-sheet secondary structure with a proportion of alpha-helix. Conformation-dependent mAbs against the ECDs of the beta or gamma subunits specifically recognized beta1-221 or gamma1-218, respectively, and polyclonal rabbit antiserum raised against purified beta1-221 bound to (125)I-labeled alpha-bungarotoxin-labeled human AChR. Moreover, immobilization of each ECD on Sepharose beads and incubation of the ECD-Sepharose matrices with MG sera caused a significant reduction in the concentrations of autoantibodies in the sera, showing specific binding to the recombinant ECDs. These results suggest that the expressed proteins present some near-native conformational features and are thus suitable for our purposes.     

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.     

Mutant forms of the extracellular domain of the human acetylcholine receptor gamma-subunit with improved solubility and enhanced antigenicity. The importance of the Cys-loop

The muscle nicotinic acetylcholine receptor (AChR) is the prototype of the ligand-gated ion channels (or Cys-loop receptors), formed by 5 homologous subunits (alpha(2)betagammadelta or alpha(2)betagammavarepsilon), and is the major autoantigen in the autoimmune disease, myasthenia gravis. Previously, we expressed the wild-type extracellular domain (ECD) of the gamma-subunit (gammaECD) of the AChR in yeast Pichia pastoris at 0.3-0.8 mg/L, in soluble but microaggregate form, to use as starting material for structural and antigenicity studies. To optimize these characteristics, we constructed and characterized four gammaECD variants: (a) mutants-1 (gammaC61S) and -2 (gammaC106S-C115S), where the non-conserved Cys of gammaECD were replaced by serines, (b) mutant-3 (gammaCysLoop), where the gamma Cys-loop region was substituted by the cognate region of the acetylcholine binding protein (AChBP) and (c) mutant-4 (gammaCysLoop-C106S-C115S), where both the C106S-C115S and Cys-loop mutations were combined. None of mutants-1 and -2 displayed any improvement, while mutant-3 and -4 were mostly in dimeric form and expressed at much higher levels (2.5 mg/L and 3.5 mg/L respectively). All four mutants and wild-type gammaECD were recognized by sera from myasthenic patients, but mutants-3 and -4 exhibited higher efficiency, compared to wild-type or mutants-1 and -2. These results suggest that the substitution of the Cys-loop region of any AChR ECD with the AChBP counterpart leads to AChR ECD of improved conformation, more suitable for structural and therapeutic studies.     

Assembly of mutant subunits of the nicotinic acetylcholine receptor lacking the conserved disulfide loop structure.

Each subunit of the nicotinic acetylcholine receptor (AChR) contains two conserved cysteine residues, which are known to form a disulfide bond, in the N-terminal extracellular domain. The role of this retained structural feature in the biogenesis of the AChR was studied by expressing site-directed mutant alpha and beta subunits together with other normal subunits from Torpedo californica AChR in Xenopus oocytes. Mutation of the cysteines at position 128 or 142 in the alpha subunit, or in the beta subunit, did not prevent subunit assembly. All Cys128 and Cys142 mutants of the alpha and beta subunits were able to associate with coexpressed other normal subunits, although associational efficiency of the mutant alpha subunits with the delta subunit was reduced. Functional studies of the mutant AChR complexes showed that the mutations in the alpha subunit abolished detectable 125I-alpha-bungarotoxin (alpha-BuTX) binding in whole oocytes, whereas the mutations in the beta subunit resulted in decreased total binding of 125I-alpha-BuTX and no detectable surface 125I-alpha-BuTX binding. Additionally, all mutant subunits, when co-expressed with the other normal subunits in oocytes, produced small acetylcholine-activated membrane currents, suggesting incorporation of only small numbers of functional mutant AChRs into the plasma membrane. The functional acetylcholine-gated ion channel formed with mutant alpha subunits, but not mutant beta subunits, could not be blocked by alpha-BuTX. Thus, a disulfide bond between Cys128 and Cys142 of the AChR alpha or beta subunits is not needed for acetylcholine-binding. However, this disulfide bond on the alpha subunit is necessary for formation of the alpha-BuTX-binding site. These results also suggest that the most significant effect caused by disrupting the conserved disulfide loop structure is intracellular retention of most of the assembled AChR complexes.     

Interaction of monoclonal antibodies with alpha-bungarotoxin and (-)-nicotine binding sites in goldfish brain. Identification of putative nicotinic acetylcholine receptor subtypes

Monoclonal antibodies raised against the nicotinic acetylcholine receptor of Electrophorus electricus electroplaque have been used as probes to characterize putative nicotinic acetylcholine receptors in goldfish brain. One monoclonal antibody (mAb), mAb 47, recognized a protein which binds both (-)-[3H]nicotine and 125I-alpha-bungarotoxin with high affinity. Another monoclonal antibody (mAb 172) recognized a protein which binds (-)-[3H]nicotine but not 125I-alpha-bungarotoxin. Both antibodies precipitated a protein(s) (biosynthetically labeled with [35S]methionine) in the absence, but not in the presence, of excess purified nicotinic acetylcholine receptor from Torpedo nobiliana. The dilution of mAb 47 that precipitated half of the maximum amount of 125I-alpha-bungarotoxin binding protein was the same as that which precipitated half of the maximum amount of (-)-[3H]nicotine binding activity. When used in combination, the two antibodies precipitated more (-)-[3H]nicotine radioactivity than either antibody alone. The (-)-[3H]nicotine and 125I-alpha-bungarotoxin binding component-mAb complexes were characterized by sucrose density centrifugation. In the presence of either mAb 172 or 47, the (-)-[3H] nicotine binding component migrated further into the gradient, but only mAb 47 shifted the 125I-alpha-bungarotoxin peak. Incubation of solubilized brain extract with alpha-bungarotoxin-coupled Sepharose reduced the amount of (-)-[3H]nicotine radioactivity precipitated by mAb 47 but not by mAb 172. These data suggest that the antibodies may recognize distinct subtypes of (-)-nicotine binding sites in goldfish brain, one subtype which binds both 125I-alpha-bungarotoxin and (-)-[3H]nicotine and a second subtype which binds only (-)-[3H] nicotine.     

Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits

We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.     

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies

Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.     

Myasthenogenicity of human acetylcholine receptor synthetic alpha-subunit peptide 125-147 does not require intramolecular disulfide cyclization

This study reports the synthesis of a disulfide-looped peptide corresponding to residues 125-147 (Cys 128-Cys 142) of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle, H alpha 125-147 (Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu-Gln- Asn-Cys-Ser-Nle-Lys Leu-Gly), and a nondisulfide-looped analogue, H alpha 125-147(S) (Lys-Ser-Tyr-Ser-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu- Gln-Asn-Cys-Ser-Nle-Lys-Leu-Gly), in which the amino acid Cys 128 was replaced with serine. Both peptides induced antigen-specific helper T cell responses, as evidenced in vitro by lymph node cell proliferation and in vivo by production of anti-AChR antibodies. Rats immunized with 100 micrograms of either synthetic peptide, without conjugation to a carrier, produced anti-peptide antibodies which bound to native AChR in immunoprecipitation assays and induced modulation of membrane-bound AChR from cultured human myotubes. Both peptides also induced electrophysiologic and biochemical signs of experimental autoimmune myasthenia gravis. Thus, region 125-147 of the AChR alpha-subunit is at least partly exposed extracellularly in human muscle and contains one or more autoantigenic sites capable of stimulating T cells and B cells. Disulfide-linkage between residues Cys 128 and Cys 142 is not essential for myasthenogenicity.     

Role of tolerogen conformation in induction of oral tolerance in experimental autoimmune myasthenia gravis

We recently demonstrated that oral or nasal administration of recombinant fragments of the acetylcholine receptor (AChR) prevents the induction of experimental autoimmune myasthenia gravis (EAMG) and suppresses ongoing EAMG in rats. We have now studied the role of spatial conformation of these recombinant fragments in determining their tolerogenicity. Two fragments corresponding to the extracellular domain of the human AChR alpha-subunit and differing in conformation were tested: Halpha1-205 expressed with no fusion partner and Halpha1-210 fused to thioredoxin (Trx), and designated Trx-Halpha1-210. The conformational similarity of the fragments to intact AChR was assessed by their reactivity with alpha-bungarotoxin and with anti-AChR mAbs, specific for conformation-dependent epitopes. Oral administration of the more native fragment, Trx-Halpha1-210, at the acute phase of disease led to exacerbation of EAMG, accompanied by an elevation of AChR-specific humoral and cellular reactivity, increased levels of Th1-type cytokines (IL-2, IL-12), decreased levels of Th2 (IL-10)- or Th3 (TGF-beta)-type cytokines, and higher expression of costimulatory factors (CD28, CTLA4, B7-1, B7-2, CD40L, and CD40). On the other hand, oral administration of the less native fragments Halpha1-205 or denatured Trx-Halpha1-210 suppressed ongoing EAMG and led to opposite changes in the immunological parameters. It thus seems that native conformation of AChR-derived fragments renders them immunogenic and immunopathogenic and therefore not suitable for treatment of myasthenia gravis. Conformation of tolerogens should therefore be given careful attention when considering oral tolerance for treatment of autoimmune diseases.     

Localization of the functional sites on the alpha chain of acetylcholine receptor

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to systematically screen the entire extracellular part (residues 1-210) of the alpha chain of the Torpedo californica acetylcholine receptor (AChR) for the profiles of the continuous regions that are recognized by antibodies against free, or membrane-sequestered, AChR; the regions recognized by AChR-primed T cells; the regions that bind alpha-bungarotoxin and cobratoxin; and an acetylcholine-binding region. Eight continuous antigenic sites were localized in this part of the alpha chain by all of the antisera tested. The sites were independent of the host species from which the antisera were obtained and were also similar to antisera against the isolated pentameric AChR or against the membrane-sequestered AChR. Six regions were found to stimulate AChR-primed T cells (T sites). Three of the T sites coincided with regions recognized by antibodies. At least two T sites had no detectable antibody responses directed to them. Five toxin-binding regions were localized, and may constitute distinct sites or, alternatively, different faces in one (or more) sites. Some of these regions coincided with regions recognized by anti-AChR antibodies. One of the toxin-binding regions bound acetylcholine, and immunization with this peptide induced experimental autoimmune myasthenia gravis.     

Experimental models of myasthenia gravis: lessons in autoimmunity and progress toward better forms of treatment

The nicotinic acetylcholine receptor (AChR) is a large membrane protein found in muscle cells. It is involved in the transformation of acetylcholine packets into a membrane depolarization, which thereby leads to a muscle twitch. This large, complex molecule is the target of the autoimmune attack in myasthenia gravis, and much has been learned in the past decade about myasthenia by the induction of autoimmunity to AChR in experimental animals. Experimental autoimmune myasthenia gravis (EAMG) has been produced in a variety of animals by immunization with AChR or AChR-like material, or by the passive transfer of anti-AChR antibodies or lymphocytes from afflicted animals into normal animals. EAMG is a remarkably faithful model of human myasthenia and has provided much information about how the immune response to AChR progresses and how weakness and damage to the neuromuscular junction ensure. EAMG has also allowed the development of a number of revolutionary forms of treatment in which only the abnormal response to AChR is restrained, and other necessary immune functions are left intact. These advances in treatment are not far from being tested in human myasthenia gravis. The experience gained in applying these concepts in EAMG and human myasthenia will be helpful in developing similar forms of treatment for other autoimmune diseases.     

Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor alpha subunit. Dissecting a potentially myasthenogenic epitope.

The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) alpha subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethylene) rods. mAb cross-reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4-10-residues long were identified. One mAb could bind to either arm on both sides of a beta-turn structure. Five mAb bound to a very-immunogenic cytoplasmic epitope on alpha 373-380 (VICE-alpha). Three of the mAb against VICE-alpha were earlier found to cross-react with non-AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non-myasthenics. Since VICE-alpha has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE-alpha bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICE-alpha-like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti-(AChR) mAb in AChR studies.     

Neuronal Nicotinic Acetylcholine Receptors in Drosophila: Antibodies Against an α-Like and a Non-α-Subunit Recognize the Same High-Affinity α-Bungarotoxin Binding Complex

ALS and ARD proteins are thought to represent a ligand binding and a structural subunit, respectively, of Drosophila nicotinic acetylcholine receptors (nAChRs). Here, antibodies raised against fusion constructs encompassing specific regions of the ALS and ARD proteins were used to investigate a potential association of these two polypeptides. Both ALS and ARD antisera removed 20-30% of the high-affinity binding sites for the nicotinic antagonist 125I-alpha-bungarotoxin (125I-alpha-Btx) from detergent extracts of fly head membranes. Combinations of both types of antisera also precipitated the same fraction of alpha-Btx binding sites, a result suggesting that both polypeptides are components of the previously defined class I 125I-alpha-Btx binding sites in the Drosophila CNS. 125I-alpha-Btx binding to a MS2 polymerase-ALS fusion protein containing the predicted antagonist binding region showed that the ALS protein indeed constitutes the ligand binding subunit of a nicotinic receptor complex. These data are consistent with neuronal nAChRs in Drosophila containing at least two types of subunits, ligand binding and structural ones.     

Production of antibodies to the alpha7-subunit of human acetylcholine nicotinic receptor with the use of immunoactive synthetic peptides

Potential B epitopes and T-helper epitopes in the N-terminal extracellular domain of the alpha7-subunit of human acetylcholine receptor (AChR) were theoretically calculated in order to reveal peptides that can induce the formation of specific antibodies to this domain. Four peptides structurally corresponding to four alpha7-subunit regions containing 16-23 aa and three of their truncated analogues were synthesized. Rabbits were immunized with both free peptides and protein conjugates of their truncated analogues, and a panel of antibodies to various exposed regions of the N-terminal extracellular domain of the AChR alpha7-subunit was obtained. All of the four predicted peptides were shown to induce the production of antipeptide antibodies in free form, without conjugation with any protein carrier. The free peptides and the protein conjugates of truncated analogues induced the formation of almost equal levels of antibodies. Most of the obtained antisera contained antibodies that bind to the recombinant extracellular N-terminal domain of the rat AChR alpha7-subunit and do not react with the analogous domain of the alpha1-subunit of the ray Torpedo californica AChR.     

Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex.

The ard gene of Drosophila melanogaster encodes a structural homologue of vertebrate nicotinic acetylcholine receptors (AChR) and is expressed exclusively in nervous tissue. To study the nature of the ARD protein, antibodies were raised against fusion constructs containing two regions of this polypeptide. One segment is putatively extracellular (amino acids 65-212), the other domain is exposed to the cytoplasm (amino acids 305-444). The ARD antisera obtained served to investigate the physical relationship between the ARD protein and alpha-bungarotoxin (alpha-Btx) binding sites occurring in Drosophila. Two different high-affinity binding sites for [125I]alpha-Btx, a highly potent antagonist of vertebrate muscle AChR, were detected in fly head membranes. Equilibrium binding and kinetic studies revealed Kd values of approximately 0.1 nM (site 1) and approximately 4 nM (site 2). The estimated maximal binding (Bmax) was approximately 240 and 1080 fmol/mg protein respectively. Both sites exhibited a nicotinic-cholinergic pharmacology. Immunoprecipitation experiments with the ARD antisera indicated that the ARD protein is associated with the [125I]alpha-Btx binding site 1 only. These data support the previously postulated hypothesis that the ARD protein is part of an alpha-Btx binding neuronal AChR of Drosophila. Furthermore, they indicate heterogeneity in nicotinic-cholinergic binding sites in the insect nervous system.     

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].     

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.