Interactions of antinicotinic acetylcholine receptor antibodies with rat brain and muscle antigenic determinants

Studies were performed to determine whether antibodies prepared against nicotinic acetylcholine receptors (nAcChoR) from electric tissue are reactive toward nAcChoR-like antigenic determinants in rat brain. Reference experiments involved the use of Torpedo electroplax and rat innervated muscle as tissue controls and an anti-alpha-bungarotoxin antiserum as a probe for curaremimetic neurotoxin binding sites. As evinced by their ability to inhibit immunoprecipitation of Torpedo nAcChoR, brain or muscle membranes specifically interact with polyclonal antisera raised against Electrophorus electroplax nAcChoR. When the extent of polyclonal anti-nAcChoR antibody binding to muscle membranes is measured by protein A binding protocols, receptor-like antigenic determinants and toxin binding sites are found to be present in approximately equal quantities. In contrast, nAcChoR-like antigenic determinants on rat brain membranes are present at concentrations in excess of those of toxin binding sites. The results are consistent with the earlier observation that some antibodies prepared against nAcChoR from peripheral tissues recognize rat brain high-affinity alpha-bungarotoxin binding sites. The results also suggest the existence of nAcChoR-like entities in brain that do not bind toxin with a high affinity.     

Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

Noncompetitive inhibition of acetylcholine receptor-controlled ion translocation was studied in membrane vesicles prepared from both Torpedo californica and Electrophorus electricus electroplax. Ion flux was measured in the millisecond time region by using a spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, and a quench-flow technique using 86Rb+. The rate coefficient of ion flux prior to receptor inactivation (desensitization), JA, was measured at different acetylcholine and inhibitor concentrations, in order to assess which active (nondesensitized) receptor forms bind noncompetitive inhibitors. The degree of inhibition of JA by the inhibitors studied (cocaine, procaine, and phencyclidine) was found to be independent of acetylcholine concentration. The results are consistent with a mechanism in which each compound inhibits by binding to a single site that exists with equal affinity on all active receptor forms. Mechanisms in which the inhibitors bind exclusively to the open-channel form of the receptor are excluded by the data. The same conclusions were reached in cocaine experiments at 0-mV and procaine experiments at -25-mV transmembrane voltage in T. californica vesicles. It had been previously shown that phencyclidine, in addition to decreasing JA (by binding to active receptors), also increases the rate of rapid receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptors (by binding better to inactivated receptor than to active receptor in the closed or open conformations). These effects were not observed with cocaine or procaine. Here it is shown that despite these differential effects on inactivation, cocaine and phencyclidine bind to the same inhibitory site on active receptors (in E. electricus vesicles).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological studies of acetylcholine receptors

Immunochemical techniques for the study of acetylcholine receptors are described. Immunization of rabbits, rats, guinea pigs, and goats with acetylcholine receptor protein purified from Electrophorus electric organ tissue results in muscular weakness and death due to impaired neuromuscular transmission. Serum from immunized animals contains high concentrations of antibodies directed at receptors from the electric organ and low concentrations of antibodies directed at receptors from skeletal muscle. The detailed similarities between the disease of receptor-immunized animals, “experimental autoimmune myasthenia gravis” (EAMG), and myasthenia gravis are compared. Reactions of antisera from animal with EAMG with receptor from Electrophorus and Torpedo are studied. Antireceptor antibodies in these antisera are directed predominantly at determinants other than the acetylcholine-binding site.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

Single-channel current recordings of acetylcholine receptors in electroplax isolated from theElectrophorus electricus main and Sachs' electric organs

Summary Extensive chemical kinetic measurements of acetylcholine receptor-controlled ion translocation in membrane vesicles isolated from the electroplax ofElectrophorus electricus have led to the proposal of a minimum model which accounts for the activation, desensitization, and voltage-dependent inhibition of the receptor by acetylcholine, suberyldicholine, and carbamoylcholine. Comparison of chemical kinetic measurements of the dynamic properties of the acetylcholine receptor in vesicles with the properties of the receptor in cells obtained from the same organ and animal have been hampered by an inability to make the appropriate measurements withElectrophorus electricus electroplax cells. Here we report a method for exposing and cleaning the surface of electroplax cells obtained from both the Main electric organ and the organ of Sachs and the results of single-channel current recordings which have now become possible. The single-channel current recordings were made in the presence of either carbamoylcholine or suberyldicholine, as a function of temperature and transmembrane voltage. Both the channel open times and the single-channel conductance were measured. The data were found to be consistent with the model based on chemical kinetic measurements using receptor-rich membrane vesicles prepared from the Main electric organ ofE. electricus.     

Immunochemical similarities between subunits of acetylcholine receptors from Torpedo, Electrophorus, and mammalian muscle.

Polypeptide chains composing acetylcholine receptors from the electric organs of Torpedo californica and Electrophorus electricus were purified and labeled with 125I. Immunochemical studies with these labeled chains showed that receptor from Electrophorus is composed of three chains corresponding to the alpha, beta, and gamma chains of receptor from Torpedo but lacks a chain corresponding to the delta chain of Torpedo. Experiments suggest that receptor from mammalian muscle contains four groups of antigenic determinants corresponding to all four of the Torpedo chains. Binding of 125I-labeled chains was measured by quantitative immune precipitation and electrophoresis. Antisera to the following immunogens were used: denatured alpha, beta, gamma, and delta chains of Torpedo receptor, native receptor from Torpedo and Electrophorus electric organs and from rat and fetal calf muscle, and human muscle receptor (from autoantisera of patients with myasthenia gravis). The four chains of Torpedo receptor were immunologically distinct from one another and from higher molecular weight chains found in electric organ membranes. Antibodies to these chains reacted very efficiently with native Torpedo receptor, but the reverse was not true. Antibodies to native receptor from Torpedo and Electrophorus reacted slightly with each of the chains of the corresponding receptor. However, cross-reaction between chains and antibodies to any native receptor was most obviuos with the alpha chain of Torpedo or the corresponding alpha' chain of Electrophorus. Antiserum to alpha chains exhibited higher titer aginst receptor from denervated rat muscle. Antibodies from myasthenia gravis patients did not cross-react detectably with 125I-labeled chains from electric organ receptors. Most interspecies cross-reaction occurred at conformationally dependent determinants whose subunit localization could not be determined by reaction with the denatured chains.     

Inhibition of α-bungarotoxin binding to acetylcholine receptors by antisera from animals with experimental autoimmune myasthenia gravis

Conditions are described for an assay that allows the percent inhibition of α-bungarotoxin binding to acetylcholine receptors by antisera and monovalent antigen-binding fragments of antibody molecules (Fab) to be determined. Anti-Torpedo californica acetylcholine-receptor antisera, prepared in New Zealand White rabbits and Lewis rats, were tested for the ability to inhibit [¹²⁵I]-α-bungarotoxin binding to membrane-associated and detergent-solubilized T californica acetylcholine receptors. Similar inhibition studies were performed using rabbit antisera and antigen-binding fragments prepared against each of the four acetylcholine receptor subunits. Antisera and antigen-binding fragments prepared against intact receptor could inhibit a maximum of 50% of the α-bungarotoxin binding to solubilized receptor. The results using monovalent antigen-binding fragments indicated that the inhibition was not due to antibody-mediated aggregation of receptor molecules. Rabbits and rats immunized with receptor denatured by sodium dodecyl sulfate all produced antisera that could bind to nondenatured receptor, but none of these animals developed experimental autoimmune myasthenia gravis. These results suggest that the antigenic determinants present on acetylcholine receptors responsible for induction of experimental auto-immune myasthenia gravis are lost with sodium dodecyl sulfate denaturation. A strong correlation was also observed between the presence of experimental autoimmune myasthenia gravis in rats and rabbits and the ability of the antisera from these animals to inhibit 50% of α-bungarotoxin binding to solubilized acetylcholine receptors.     

Purification and characterization of a nicotinic acetylcholine receptor from chick brain

Immunohistochemical studies have previously shown that both the chick brain and chick ciliary ganglion neurons contain a component which shares antigenic determinants with the main immunogenic region of the nicotinic acetylcholine receptor from electric organ and skeletal muscle. Here we describe the purification and initial characterization of this putative neuronal acetylcholine receptor. The component was purified by monoclonal antibody affinity chromatography. The solubilized component sediments on sucrose gradients as a species slightly larger than Torpedo acetylcholine receptor monomers. It was affinity labeled with bromo[3H]acetylcholine. Labeling was prevented by carbachol, but not by alpha-bungarotoxin. Two subunits could be detected in the affinity-purified component, apparent molecular weights 48 000 and 59 000. The 48 000 molecular weight subunit was bound both by a monoclonal antibody directed against the main immunogenic region of electric organ and skeletal muscle acetylcholine receptor and by antisera raised against the alpha subunit of Torpedo receptor. Evidence suggests that there are two alpha subunits in the brain component. Antisera from rats immunized with the purified brain component exhibited little or no cross-reactivity with Torpedo electric organ or chick muscle acetylcholine receptor. One antiserum did, however, specifically bind to all four subunits of Torpedo receptor. Experiments to be described elsewhere (J. Stollberg et al., unpublished results) show that antisera to the purified brain component specifically inhibit the electrophysiological function of acetylcholine receptors in chick ciliary ganglion neurons without inhibiting the function of acetylcholine receptors in chick muscle cells. All of these properties suggest that this component is a neuronal nicotinic acetylcholine receptor with limited structural homology to muscle nicotinic acetylcholine receptor.     

Purification of acetylcholine receptors from the muscle of Electrophorus electricus

Muscle from the electric eel Electrophorus electricus contains acetylcholine receptors at 50 times the concentration of normal mammalian muscle and fully one-tenth the concentration of receptors in its electric organ tissue. Receptor is organized much more diffusely over the surface of Electrophorus muscle cells than is the case in normally innervated mammalian skeletal muscle. Receptor was purified from Electrophorus muscle by affinity chromatography on cobra toxin-agarose and found to contain subunits which correspond immunochemically to the alpha, beta, gamma, and delta subunits of receptor from electric organ tissue of Torpedo californica. Receptor purified from Electrophorus muscle appears virtually identical with receptor purified from Electrophorus electric organ tissue.     

THE BINDING OF ACETYLCHOLINE TO SYNAPTIC VESICLES FROM TORPEDO CALIFORNICA ELECTROPLAX

Abstract— The in vitro uptake of exogenous acetylcholine by isolated presynaptic vesicles has been demonstrated in a new system. A preparation of vesicles from Torpedo californica electroplax was developed in which acetylcholinesterase and acetylcholine receptor activity were blocked. The vesicles bound acetylcholine with K_d 1.58 μM, the maximum amount bound being 26 pmol per g of original tissue, or 52 molecules per vesicle. Nicotinic drugs blocked binding, but muscarinic and noncholinergic drugs did not. The relative potency of nicotinic drugs differed greatly from their potency on Torpedo receptor. Sephadex chromatography showed that 26% of the binding was irreversible. The relationship of the binding to acetylcholine uptake and storage was discussed.     

Mapping of surface structures of electrophorus acetylcholine receptor using monoclonal antibodies

Forty monoclonal antibodies to acetylcholine receptor from the electric organs of Electrophorus electricus have been characterized by immunoglobulin isotype, affinity for receptor, and specificity for species, subunit, and determinants within subunits. Using these antibodies, nine immunogenic regions on the receptor molecule were distinguished. Most of these are species specific, and are located on various subunits of the acetylcholine receptor. The least species-specific region forms the "main immunogenic region" (MIR). Most monoclonal antibodies and most antibodies in conventional antisera are directed at this region. The MIR is located on the extracellular surface of the alpha subunits and is homologous to the MIR which we previously described on Torpedo californica receptor. An homologous MIR is also a characteristic feature of receptor from mammalian muscle. The possible immunological and structural significance of the MIR is discussed.     

Inhibition of ion permeability control properties of acetylcholine receptor from Torpedo californica by long-chain fatty acids

The characteristics of fatty acid inhibition of acetylcholine receptor function were examined in membrane vesicles prepared from Torpedo californica electroplax. Inhibition of the carbamylcholine-induced increase in sodium ion permeability was correlated with the bulk melting point of exogenously incorporated fatty acids. Above its melting temperature, a fatty acid could inhibit the large increase in cation permeability normally elicited by agonist binding to receptor. Below its melting temperature, a fatty acid was ineffective. None of the fatty acids altered any of the ligand binding properties of the receptor. Inhibitory fatty acids did not induce changes in membrane fluidity, as determined by electron paramagnetic resonance using spin-labeled fatty acids. The spin-labeled fatty acids also acted as inhibitors, and the extent of inhibition depended largely on the position of the nitroxide group along the fatty acid chain. Addition of noninhibitory fatty acid to the vesicle membranes did not protect the receptor from inhibition by spin-labeled fatty acids. The effects of free fatty acids on acetylcholine receptor function are attributed to the disruptions of protein-lipid interactions.     

Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies

A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.     

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.     

Autoimmune rat T lymphocytes monospecific for acetylcholine receptors: purification and fine specificity

We prepared highly purified acetylcholine receptor (AChR)-specific T lymphocytes from rats with experimental autoimmune myasthenia gravis (EAMG). Inbred rats were primed with AChR frm 3 different sources: from the electric organs of Electrophorus electricus and Torpedo californica and from denervated rat muscle. After 20 to 30 days, lymphocytes from regional lymph nodes (primary cells) were challenged with soluble AChR in vitro. The activated blast cells were isolated by density gradient centrifugation and allowed to revert back to small secondary lymphocytes in the absence of antigen. These secondary anti-AChR cells were highly responsive to the type of AChR with which they had been primed. Their reactivity critically depended on help by syngeneic accessory cells. Anti-Electrophorus AChR primary and secondary cells cross-reacted detectably with rat AChR and vice versa, whereas anti-Torpedo AChR primary and secondary cells did not significantly cross-react with Electrophorus or rat AChR. Secondary T cells strongly reactive against rat AChR could be selected in vitro from Electrophorus AChR-primed populations by using rat AChR as selecting stimulant. These cells responded equally well against Electrophorus and rat AChR and thus include autoreactive T cell clones.     

300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.     

Characterization of Curaremimetic Neurotoxin Binding Sites on Cellular Membrane Fragments Derived from the Rat Pheochromocytoma PC 12

Studies were conducted on the properties of 125I-labeled alpha-bungarotoxin binding sites on cellular membrane fragments derived from the PC12 rat pheochromocytoma. Two classes of specific toxin binding sites are present at approximately equal densities (50 fmol/mg of membrane protein) and are characterized by apparent dissociation constants of 3 and 60 nM. Nicotine and d-tubocurarine are among the most potent inhibitors of high-affinity toxin binding. The affinity of high-affinity toxin binding sites for nicotinic cholinergic agonists is reversibly or irreversibly decreased, respectively, on treatment with dithiothreitol or dithiothreitol and N-ethylmaleimide. The nicotinic receptor affinity reagent bromoacetylcholine irreversibly blocks high-affinity toxin binding to PC12 cell membranes that have been treated with dithiothreitol. Two polyclonal antisera raised against the nicotinic acetylcholine receptor from Electrophorus electricus inhibit high-affinity toxin binding. These detailed studies confirm that curaremimetic neurotoxin binding sites on the PC12 cell line are comparable to toxin binding sites from neural tissues and to nicotinic acetylcholine receptors from the periphery. Because toxin binding sites are recognized by anti-nicotinic receptor antibodies, the possibility remains that they are functionally analogous to nicotinic receptors.     

Two pools of cholesterol in acetylcholine receptor-rich membranes from Torpedo

The acetylcholine receptor (AChR)-containing electroplax membranes from Torpedo californica have a relatively high cholesterol content. Reconstitution studies suggest that this cholesterol may be important in preserving or modulating the function of the acetylcholine receptor-channel complex. We have manipulated cholesterol levels in intact Torpedo AChR-rich membrane fragments using small, unilamellar phosphatidylcholine liposomes. Conditions have been established that allow further subfractionation of sucrose gradient purified Torpedo electroplax membranes into AChR-rich and ATPase-rich populations and that, at the same time, achieve cholesterol depletion without phospholipid back exchange or fusion. The incubation of membranes with excess liposomes could only achieve about a 50% reduction in the molar ratio of cholesterol to phospholipid. In no case was the number of cholesterol molecules per AChR oligomer reduced below 36. The remaining cholesterol could not be depleted either by longer incubations or by multiple, sequential depletions. Cholesterol depletion was accompanied by a significant increase in bulk membrane fluidity as measured by electron spin resonance spectroscopy, but the equilibrium binding parameters of acetylcholine to its receptor were unaltered. This suggests strongly that there exist two pools of cholesterol in the AChR-rich Torpedo electroplax membrane: an easily depleted fraction that influences bulk fluidity, and a tightly-bound fraction perhaps surrounding the AChR oligomer.     

An anti-acetylcholine receptor monoclonal antibody cross-reacts with phosvitin

Rabbit and mouse anti-Torpedo acetylcholine receptor antibodies cross-reacted partially with the highly phosphorylated protein, phosvitin. We have selected an anti-Torpedo acetylcholine receptor monoclonal antibody which binds specifically to phosvitin; this binding is inhibited by acetylcholine receptor. These findings suggest that a phosphorylated amino acid residue may be a part of the determinant on the acetylcholine receptor recognized by this monoclonal antibody.     

Isolation of Membrane Fractions with Sodium Channels Sensitive to Veratridine and tetrodotoxin from the Electric Organ of the Eel Electrophorus electricus

The veratridine/tetrodotoxin-sensitive sodium influx was measured in membrane fractions isolated from the electric organ of Electrophorus electricus. The fractions were characterized, and the main biochemical markers and their acetylcholine receptor content were determined. The innervated and noninnervated faces of the electroplax were separated. The different biochemical criteria used indicate that the pre- and postsynaptic membranes of the innervated face were isolated. Sodium influx increased by veratridine and blocked by tetrodotoxin was found in fractions from the presynaptic membrane. Because some of the vesicles in this fraction are in the inside-out conformation, tetrodotoxin had to be applied to both faces of the vesicles so that sodium influx was blocked completely. The fractions from the innervated face of the electroplax contained sodium channels with sensitivities to tetrodotoxin and veratridine similar to those of fractions from other nerve membrane preparations.