Disorders Affecting the Acetylcholine Receptor: Myasthenia Gravis and Congenital Myasthenia

Abstract

Myasthenia gravis (MG) is an autoimmune disease in which anti-acetylcholine receptor antibodies (anti-AChR) cause loss of functional endplate AChR by increasing AChR degradation, and by complement-mediated destruction. MG anti-AChR binds to regions on the human AChR which can be defined by monoclonal antibodies (mabs).

Several congenital forms of myasthenia have been described, three of which may directly involve abnormalites of the AChR, including one in which the open-time of the ion channel is prolonged.     

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.     

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.     

Altered patterns ofN-linked glycosylation of theTorpedo acetylcholine receptor expressed inXenopus oocytes

Summary The nicotinic acetylcholine receptor (AChR) fromTorpedo electroplax is an oligomeric transmembrane glycoprotein made up of four highly homologous subunits in a stoichiometry of ₂. The role ofN-linked glycosylation of the AChR has been studied in several cell lines and these studies have suggested that the addition of carbohydrate may be important for receptor expression. WhileXenopus oocytes have proven to be an invaluable tool for studying the AChR, little is known aboutN-linked glycosylation of the oocyte-expressed receptor. The present report demonstrates that the oocyte-expressed AChR is glycosylated and contains the same number of oligosaccharide residues per subunit as the native receptor. However, unlike the nativeTorpedo receptor which contains both high mannose and complex oligosaccharides, the oocyte-expressed AChR contains only high mannose oligosaccharide modifications. However, as has been well documented, theTorpedo AChR expressed in oocytes is fully functional, demonstrating that the precise nature of the oligosaccharide modification is not critical for receptor function.The role of the oligosaccharide component of the AChR in receptor function was examined using tunicamycin (TM) to inhibitN-linked protein glycosylation. TM treatment resulted in a 70–80% inhibition of AChR expression in oocytes. Functional, unglycosylated receptors were not expressed; receptors expressed in TM-treated oocytes were functional wild-type, glycosylated AChR, formed only during the initial 12 hr of TM exposure. These data suggest that while glycosylation of the oocyte-expressedTorpedo AChR is required for assembly of subunits into a functional receptor, as has been demonstrated in other cells, oocyte modification of normalTorpedo glycosylation patterns does not affect receptor function or assembly.     

cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA alpha 4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.     

Cellular immune response to acetylcholine receptors in murine experimental autoimmune myasthenia gravis: inhibition with monoclonal anti-I-A antibodies

Gene(s) at the I-A subregion of the murine major histocompatibility complex influence susceptibility to experimental autoimmune myasthenia gravis. C57Bl/6 mice immunized with acetylcholine receptors (AChR) in complete Freund's adjuvant demonstrated cellular and humoral immune responses to AChR. They developed muscle weakness characteristic of myasthenia gravis and demonstrated a reduction in the muscle AChR content. The kinetics of AChR-specific lymphocyte proliferation generally correlate with anti-AChR antibody response. AChR-specific lymphocyte proliferation was also observed in C57Bl/6 splenocytes after secondary immunization with AChR. The in vitro cellular reactivity to AChR in experimental autoimmune myasthenia gravis (EAMG) mice (C57Bl/6) was suppressed by monoclonal anti-I-Ab antibodies directed against private (Ia20) or public (Ia8) specificities, suggesting a critical role for these Ia determinants in the cellular immune response to AChR in murine EAMG.     

Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore.

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.     

Monoclonal Anti-Acetycholine Receptor Antibodies as Probes for Human Acetylcholine-Receptor in Myasthenia Gravis

Abstract

Monoclonal antibodies have been shown to bind to five regions on human acetylcholine receptor, each probably consisting of a discrete epitope on the extracellular surface. Two of these regions are equivalent to the ‘main immunogenic region’, and the other three appear to be close to the a-Bungarotoxin binding sites. These antibodies have been used to probe differences in myasthenia gravis anti-acetylcholine receptor antibodies, to locate acetylcholine receptor in thymic tissue, and to look for naturally-occurring anti-idiotype antibodies.

Anti-acetylcholine receptor antibody specificities differ between groups of patients defined by their age of onset, thymic pathology and HLA associations. Anti-AChR synthesised by the thymus in young onset patients has similar specificity to that found in the individual's serum, and may be stimulated by the presence of AChR on thymic myoid cells. However, myoid cells (defined by staining with anti-troponin and anti-myosin antibodies) do not appear to differ between control and myasthenia gravis patients and show no obvious involvement in an immunological reaction.

There was no convincing evidence for the presence of anti-idiotype antibodies in myasthenia gravis patients.     

Nicotinic acetylcholine receptors in health and disease

Nicotinic acetylcholine receptors (AChRs) are a family of acetylcholine-gated cation channels that form the predominant excitatory neurotransmitter receptors on muscles and nerves in the peripheral nervous system. AChRs are also expressed on neurons in lower amounts throughout the central nervous system. AChRs are even being reported on unexpected cell types such as keratinocytes. Structures of these AChRs are being determined with increasing precision, but functions of some orphan subunits are just beginning to be established. Functional roles for postsynaptic AChRs in muscle are well known, but in neurons the post-, peri-, extra-, and presynaptic roles of AChRs are just being revealed. Pathogenic roles of AChRs are being discovered in many diseases involving mechanisms ranging from mutations, to autoimmune responses, to the unknown; involving cell types ranging from muscles, to neurons, to keratinocytes; and involving signs and symptoms ranging from muscle weakness to epilepsy, to neurodegenerative disease, to psychiatric disease, to nicotine addiction. Awareness of AChR involvement in some of these diseases has provoked new interests in development of therapeutic agonists for specific AChR subtypes and the use of expressed cloned AChR subunits as possible immunotherapeutic agents. Highlights of recent developments in these areas will be briefly reviewed.     

C5 gene influences the development of murine myasthenia gravis

The influence of the C5 gene and C5 deficiency on murine experimental autoimmune myasthenia gravis (EAMG) susceptibility was evaluated. Two co-isogenic strains, B10.D2/nSn (C5 sufficient) and B10.D2/oSn (C5 deficient), which are genetically identical except for the C5 gene locus, were immunized with acetylcholine receptors (AChR) in CFA to induce myasthenia gravis. Both strains had equivalent concentration of serum autoantibodies to muscle AChR and antibodies bound to muscle AChR. C5-sufficient B10.D2/nSn, but not C5-deficient B10.D2/oSn, demonstrated increased incidence of clinical disease and death and lost significant amounts of muscle AChR. Therefore, C5 deficiency in B10.D2/oSn prevented EAMG. C5 gene, which codes for C component C5, may influence EAMG pathogenesis through activation of the terminal lytic C sequence (C5 to C9) required for muscle AChR destruction, which is the primary pathology.     

Efficient Expression of Functional (α6β2)2β3 AChRs in Xenopus Oocytes from Free Subunits Using Slightly Modified α6 Subunits

Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)₂β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)₂β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.     

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.     

Determinants of Phencyclidine Potency on the Nicotinic Acetylcholine Receptors from Muscle and Electric Organ

1.Phencyclidine (PCP) is an inhibitor of the nicotinic acetylcholine receptor (AChR) with characteristics of an open-channel blocker. The location of PCP binding site on the AChR molecule is unknown.2.PCP inhibits the AChR from electric organ with a higher potency than muscle AChR. To find the molecular basis of this difference, we expressed the two native and six hybrid receptors, and two receptors containing mutated mouse subunits in Xenopus laevis oocytes. The inhibition of ACh-induced current in these receptors by PCP was studied using whole-cell voltage-clamp. All hybrid receptors generated robust ACh-induced currents, while incomplete receptors (-less or -less) did not.3.PCP potency was higher on hybrids containing Torpedo and subunits regardless of the and subunit origin. A mouse subunit containing the asparagine 6 to the serine mutation in the M2 segment conferred a high sensitivity to PCP.4.These results support the conclusion that the amino acid residues at the position 6 of the M2 segments contribute to the PCP potency difference between Torpedo and mouse receptors.5.Another noncompetitive inhibitor of the AChR, the cembranoid eupalmerin acetate (EUAC), also inhibited the electric organ receptor with a somewhat higher potency than muscle AChR. However, the IC₅₀ values for EUAC inhibition of hybrid receptors did not follow the pattern observed for PCP. Therefore, these two inhibitors interact differently with the AChR molecule.     

Assembly and clustering of acetylcholine receptors containing GFP-tagged epsilon or gamma subunits: selective targeting to the neuromuscular junction in vivo.

Acetylcholine receptor (AChR) gamma and epsilon subunits were tagged by green fluorescent protein (GFP) to analyse assembly and targeting in live muscle fibers at the neuromuscular junction. N- or C-terminal fusion polypeptides showed no fluorescence upon transfection of HEK cells. When GFP was inserted into the cytoplasmic loop connecting putative transmembrane regions M3 and M4, the gamma/GFP and epsilon/GFP subunits were fluorescent and formed together with the alpha, beta, and delta subunits GFP-tagged AChR complexes that were integrated into the plasma membrane. As the AChR were also clustered by rapsyn, the results indicate that the cytoplasmatic domains of the gamma and epsilon subunits may not be required for assembly and rapsyn-dependent clustering. The gamma/GFP and epsilon/GFP subunit-containing receptors were expressed in X. laevis oocytes and have affinities for acetylcholine similar to that of the wild-type receptors. Direct gene transfer into single muscle fibers reveals that gamma/GFP or epsilon/GFP polypeptides are expressed at the site of injection and are transported within the endoplasmatic reticulum. When reaching subsynaptic regions, both gamma/GFP or epsilon/GFP subunits compete with endogenous epsilon subunits to assemble GFP-tagged receptors, which are selectively targeted to the postsynaptic membrane.     

Glutamine 57 at the Complementary Binding Site Face Is a Key Determinant of Morantel Selectivity for ��7 Nicotinic Receptors

Nicotinic receptors (AChRs) play key roles in synaptic transmission. We explored activation of neuronal α7 and mammalian muscle AChRs by morantel and oxantel. Our results revealed a novel action of morantel as a high efficacy and more potent agonist than ACh of α7 receptors. The EC₅₀ for activation by morantel of both α7 and α7-5HT_3A receptors is 7-fold lower than that determined for ACh. The minimum morantel concentration required to activate α7-5HT_3A channels is 6-fold lower than that of ACh, and activation episodes are more prolonged than in the presence of ACh. By contrast, oxantel is a weak agonist of α7 and α7-5HT_3A, and both drugs are very low efficacy agonists of muscle AChRs. The replacement of Gln⁵⁷ in α7 by glycine, which is found in the equivalent position of the muscle AChR, decreases the efficacy for activation and turns morantel into a partial agonist. The reverse mutation in the muscle AChR (ϵG57Q) increases 7-fold the efficacy of morantel. The mutations do not affect activation by ACh or oxantel, indicating that this position is selective for morantel. In silico studies show that the tetrahydropyrimidinyl group, common to both drugs, is close to Trp¹⁴⁹ of the principal face of the binding site, whereas the other cyclic group is proximal to Gln⁵⁷ of the complementary face in morantel but not in oxantel. Thus, position 57 at the complementary face is a key determinant of the high selectivity of morantel for α7. These results provide new information for further progress in drug design.Nicotinic acetylcholine receptors (AChRs),³ members of the Cys-loop receptor superfamily, are of fundamental importance in synaptic transmission throughout the nervous system in both vertebrates and invertebrates. They are implicated in a wide range of important pathologies and are targets of clinically relevant drugs. AChRs are pentameric proteins composed of highly homologous subunits (1, 2). Subunits are classified as either α, which contain a disulfide bridge formed by two adjacent cysteine residues important for acetylcholine (ACh) binding, or non-α subunits, which lack this motif (3).AChRs assemble from five identical α subunits, forming homomeric receptors, such as neuronal α7 receptors, or from different α and non-α subunits, forming heteromeric receptors, such as the muscle AChR. Human adult muscle AChRs are composed of two α1, one β, one ϵ, and one δ subunits. The five homologous subunits are arranged as barrel staves around a central ion-conducting pore (4). Approximately half of each subunit is extracellular with the remainder comprising transmembrane domains M1–M4 and a large cytoplasmic domain spanning M3 and M4 (4). The neurotransmitter binding sites are formed within the extracellular domain at interfaces between subunits (4, 5). One of the sides, called the principal face, is formed by three discontinuous loops of the α subunit, whereas the complementary face is formed by three discontinuous β-strands of the adjacent subunit. Key residues of the principal face are grouped in regions called loop A (Trp⁸⁶ and Tyr⁹³), loop B (Trp¹⁴⁹ and Gly¹⁵³), and loop C (Tyr¹⁹⁰, Cys¹⁹², Cys¹⁹³, and Tyr¹⁹⁸). The complementary face is formed by residues from α7 or δ or ϵ subunits in the adult muscle AChR. At this face of the muscle AChR, residues are clustered in loop D (Trp⁵⁵), E (Leu¹⁰⁹, Tyr¹¹¹, Tyr¹¹⁷, and Leu¹¹⁹), and F (Asp¹⁷⁴ and Glu¹⁷⁶) (2, 5, 6). Residues of the principal face are highly conserved between α7 and α1 subunits, whereas less conservation is found in residues located at the complementary face (5, 7).The anthelmintic agents levamisole, pyrantel, oxantel, and morantel are full agonists of nematode muscle AChRs, and exert their therapeutic actions by producing muscle paralysis (8). By contrast, levamisole and pyrantel have been shown to be low efficacy agonists of mammalian muscle AChRs (9). A few lines of experimental evidence suggest that these compounds also interact with some types of neuronal AChRs, but instead of acting as agonists, they act as modulators. Morantel and levamisole have been shown to allosterically potentiate responses of α3β2 and α3β4 receptors (10, 11). Thus, the actions of anthelmintic agents seem to be strongly dependent on the AChR subtype. Therefore, these compounds are useful tools for the identification of determinants of drug selectivity, which, in turn, is required for rational design of novel and more specific drugs.We have here determined that, similarly to pyrantel and levamisole (9), morantel and oxantel are low efficacy agonists of mammalian muscle AChRs. However, whereas oxantel is also a weak agonist of α7, morantel is more potent than ACh. By site-directed mutagenesis we determined that position 57, located at the complementary face of the binding site, is involved in the differential selectivity of morantel for α7 and mammalian muscle AChR.Neuronal α7 receptors may be involved in a range of neurological and psychiatric disorders that lead to cognitive impairment, including Alzheimer disease, attention deficit hyperactivity disorder, and schizophrenia (12). Given that its deficit is associated with cognitive impairment in these diseases, enhancement of its activity has recently emerged as a physiological and effective therapeutic strategy. Therefore, the characterization of the novel action of morantel as a potent agonist of α7 together with the identification of the structural basis of this high selectivity become of importance as they provide new information for further progress in drug design.     

The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly.

Ligand-gated ion channels are oligomeric membrane proteins in which homologous subunits specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we used a dominant-negative assay in which truncated subunits of the mouse muscle acetylcholine receptor (AChR) were coexpressed with the four wild-type subunits in transfected COS cells. Fragments of the alpha, delta, and gamma subunits consisting solely of the extracellular N-terminal domain blocked surface expression of the AChR and the formation of alpha delta heterodimers, an early step in the assembly pathway of the AChR. Immunoprecipitation and sucrose gradient sedimentation experiments showed that an N-terminal fragment of the alpha subunit forms a specific complex with the intact delta subunit. Thus the extracellular N-terminal domain of the alpha, delta, and gamma subunits contains the information necessary for specific subunit association.     

Antisera against an acetylcholine receptor alpha 3 fusion protein bind to ganglionic but not to brain nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptor (AChR) subtypes have been defined pharmacologically, immunologically, and by DNA cloning, but the correlations between these approaches are incomplete. Vertebrate neuronal AChRs that have been isolated are composed of structural subunits and ACh-binding subunits. A single kind of subunit can be used in more than one AChR subtype. Monoclonal antibody (mAb) 35 binds to structural subunits of subtypes of AChRs from both chicken brain and ganglia. By using antisera to a unique sequence of alpha 3 ACh-binding subunits expressed in bacteria, we show that ganglionic AChRs contain alpha 3 ACh-binding subunits, whereas the brain AChR subtype that binds mAb 35 does not. Subunit-specific antisera raised against recombinant proteins should be a valuable approach for identifying the subunit composition of receptors in multigene, multisubunit families.     

Design and synthesis of peptides that bind α-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

α-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC₅₀ of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.     

Synthesis of nitrodiazirinyl derivatives of neurotoxin II fromNaja naja oxiana and their interaction with theTorpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) fromNaja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of {2-nitro-4 [3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}acetic acid followed by Chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [¹²⁵I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane bound nicotinic acetylcholine receptor (AChR) fromTorpedo californica. The pattern of labeling the receptor'sα, β, γ, orδ subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series ofp-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.     

Synaptic contacts between embryonic Xenopus neurons and myotubes formed from a rat skeletal muscle cell line

Acetylcholine receptors (AChRs) accumulate at the junctional region during early development. In an attempt to characterize this process of AChR accumulation, we combined embryonic Xenopus neurons with myotubes formed from a rat skeletal muscle cell line. Xenopus neurons in culture are known to induce AChR accumulation in Xenopus muscles [Anderson, M. J., Cohen, M. W., and Zorychta, E. (1977). J. (London), 268, 731–756]. Rat myotubes, however, do not exhibit AChR accumulation in culture even when they are functionally innervated by the fetal rat spinal cord explant [Kidokoro, Y. (1980) Develop. Biol., 78, 231–241]. Establishment of synaptic transmission was examined electrophysiologically by recording synaptic potentials, while the distribution of AChR clusters was visualized using fluorescent α-bungarotoxin. Our results indicate that embryonic Xenopus neurons formed functional synaptic contacts but did not cause AChR accumulation in L6-myotubes. It seems that the ability of a nerve to cause AChR accumulation is separate from that to form the functional synapse. We also found that the mean amplitude of synaptic potentials in L6-myotubes interacted with Xenopus neurons was about half of that in L6-myotubes innervated by fetal rat spinal cord explants. Possible explanations for this finding are discussed.