AChR-rich Membrane Research Articles

Immunochemical characterization of anti-acetylcholine receptor antibodies in primary biliary cirrhosis

Although the presence of anti-mitochondrial antibodies is the main characteristic of primary biliary cirrhosis (PBC), other autoantibodies have been described in this disease. This study employs immunoblot methods to test whether the sera of PBC patients also contain antibodies directed against nicotinic acetylcholine receptors (AChR). We show that the majority of patients' sera indeed react with AChR just as sera of myasthenic patients do. In contrast, however, these anti-AChR antibodies do not lead to significant clinical symptoms of myasthenia. In all cases studied, PBC sera recognized a protein with the molecular weight of the alpha-chain of acetylcholine receptor (40 kDa). In addition, with both liver mitochondria and AChR-rich membranes as antigens, PBC sera reacted with proteins with apparent molecular weights around 68 kDa and the same pI values. This protein is not present in purified AChR preparations. These data suggest structural, if not functional, relationships between membrane components of liver mitochondria and muscle endplates.

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.

Protein methylase II in five taxa from three phyla

1. 1. Protein methylase II (protein O-methyltransferase, EC 2.1.1.24) was found in Dictyostelium discoideum amoebae, Astacus leptodactylus axonal, Locusta migratoroides neuronal, Torpedo marmorata electroplaque and Bos bovis stratial tissue and compared in both the soluble and particulate fractions. 2. 2. The intrinsic decay data of the methyl groups transferred onto proteins from Dictyostelium and Torpedo tissues were virtually identical. 3. 3. The short term kinetics of the methyl group transfer of all fractions and of all taxa investigated were non-linear and multiphasic. The particulate fractions displayed transient peaks at 1 min, 3 min, or both after the start of the reaction. 4. 4. The methyl group transfer was stimulated by the neurotoxins veratridine (VTx) and inhibited by veratridine plus tetrodotoxin (TTx) (axonal membrane vesicles of Astacus), stimulated transiently and in a biphasic manner by carbamoylcholine and phospholipase A 2 (AChR-rich membrane vesicles of Torpedo), and stimulated transiently and biphasically by the adequate chemotactic stimulus cAMP (aggregation competent amoebae of Dictyostelium).

Reaction of [3H]meproadifen mustard with membrane-bound Torpedo acetylcholine receptor.

The Torpedo nicotinic acetylcholine receptor (AChR) contains a binding site for aromatic amine noncompetitive antagonists that is distinct from the binding site for agonists and competitive antagonists. To characterize the location and function of this allosteric antagonist site, an alkylating analog of meproadifen has been synthesized, 2-(chloroethylmethylamino)-ethyl-2, 2-diphenylpentanoate HCl (meproadifen mustard). Reaction of [3H]meproadifen mustard with AChR-rich membrane suspensions resulted in specific incorporation of label predominantly into the AChR alpha-subunit with minor incorporation into the beta-subunit. Specific labeling required the presence of high concentration of agonist and was inhibited by reversible noncompetitive antagonists including proadifen, meproadifen, perhydrohistrionicotoxin (HTX), and tetracaine when present at concentrations consistent with the binding affinity of these compounds for the allosteric antagonist site. No specific alkylation of the AChR alpha-subunit was detected in the absence of agonist, or in the presence of the partial agonist phenyltrimethylammonium or the competitive antagonists, d-tubocurarine, gallamine triethiodide, or decamethonium. Reaction with 35 microM meproadifen mustard for 70 min in the presence of carbamylcholine produced no alteration in the concentration of [3H]ACh-binding sites, but decreased by 38 +/- 4% the number of allosteric antagonist sites as measured by [3H]HTX binding. This decrease was not observed when the alkylation reaction was blocked by the presence of HTX. These results lead us to conclude that meproadifen mustard alkylates the allosteric antagonist site in the Torpedo AChR and that part of that site is associated with the AChR alpha-subunit.

A p60 v-src-related tyrosine kinase in the acetylcholine receptor-rich membranes of Narke japonica: Association and dissociation of phosphatidylinositol kinase activity

We have isolated a tyrosine-specific protein kinase from the acetylcholine receptor (AChR)-rich membranes of the electric ray Narke japonica. The enzyme is immunologically related to p60v-src, the product of the transforming gene of Rous sarcoma virus. A substantial phosphatidylinositol (PI) kinase activity was associated with this enzyme when it was purified through tyrosine-agarose affinity chromatography used previously for the purification of p60v-src. However, by subsequent chromatography on casein-agarose, most of the associated PI kinase activity was separated from the tyrosine kinase activity. The results suggest that the tyrosine-specific protein kinase in the AChR-rich membranes of N. japonica has no intrinsic PI kinase activity.

Calcium- and calmodulin-dependent phosphorylation of diphosphoinositide in acetylcholine receptor-rich membranes from electroplax of Narke japonica.

The phosphorylation of phosphoinositides in the acetylcholine receptor (AChR)-rich membranes from the electroplax of the electric fish Narke japonica has been examined. When the AChR-rich membranes were incubated with [gamma-32P]ATP, 32P was incorporated into only two inositol phospholipids, i.e., tri- and diphosphoinositide (TPI and DPI). Even after the alkali treatment of the membrane, AChR-rich membranes still showed a considerable DPI kinase activity upon addition of exogenous DPI. It is likely that the 32P-incorporation into these lipids was realized by the membrane-bound DPI kinase and phosphatidyl inositol (PI) kinase. Such a membrane-bound DPI kinase was activated by Ca2+ (greater than 10(-6) M), whereas the PI kinase appeared to be inhibited by Ca2+. The effect of Ca2+ on the DPI phosphorylation was further enhanced by the addition of ubiquitous Ca2+-dependent regulator protein calmodulin. Calmodulin antagonists such as chlorpromazine (CPZ), trifluoperazine (TFP), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the phosphorylation of DPI in the AChR-rich membranes. It is suggested that the small pool of TPI in the plasma membrane is replenished by such Ca2+- and calmodulin-dependent DPI kinase responding to the change in the intracellular Ca2+ level.

Complement activation in myasthenia gravis plasma by Torpedo acetylcholine receptor liposomes.

The purpose of this study was to determine if antibodies from patients with myasthenia gravis (MG) react in a biologically relevant manner with membrane bound Torpedo californica acetylcholine receptor (AChR). Antibody-mediated complement activation was measured in plasma from 15 patients with MG and 9 controls following incubation with Torpedo AChR-rich membrane vesicles (AChR liposomes). Significantly more (p less than 0.001) complement activation occurred in the patient samples compared with the controls. Torpedo AChR liposomes are well characterized and have been widely used to study the structure and function of AChR. These results suggest that Torpedo AChR liposomes may also be used in immunologic studies with autoantibodies from patients with MG.

Crosslinking of proteins in acetylcholine receptor-rich membranes: Association between the β-subunit and the 43 kd subsynaptic protein

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four subunits (α, β, γ, δ) of the acetylcholine receptor (AChR) and for polypeptides at 43 kd and 270 kd. Reacton of these membranes with 3H-N-ethylmaleimide ( 3H-NEM) demonstrates that most of the available free sulfhydryls reside on the 43 kd protein. Cross-linking reagents that contain NEM as one reactive group, and N-hydroxysuccinimide as the other, were used to study the topography of the 43 kd protein in AChR-rich membranes. Proteins from cross-linked membranes were resolved by SDS-PAGE and the composition of crosslinked products was determined by Western blots and monoclonal antibodies. A crosslinked product at 110 kd was labeled by a monoclonal antibody to the β-subunit and by a monoclonal antibody to the 43 kd protein, but not by monoclonal antibodies to the α, γ, or δ subunits. The 110 kd crosslink was not produced in the presence of 10 mM lithium diiodosalicylate, which dissociates the 43 kd protein from the membrane. Thus the 43 kd protein is intimately associated with the AChR and in close proximity to the β-subunit.

Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ.

After fixation with glutaraldehyde and impregnation with tannic acid, the membrane that underlies the nerve terminals in Torpedo marmorata electroplaque presents a typical asymmetric triple-layered structure with an unusual thickness; in addition, it is coated with electron-dense material on its inner, cytoplasmic face. Filamentous structures are frequently found attached to these "subsynaptic densities." The organization of the subsynaptic membrane is partly preserved after homogenization of the electric organ and purification of acetylcholine-receptor (AchR)-rich membrane fragments. In vitro treatment at pH 11 and 4 degrees C of these AchR-rich membranes releases an extrinsic protein of 43,000 mol wt and at the same time causes the complete disappearance of the cytoplasmic condensations. Freeze-etching of native membrane fragments discloses remnants of the ribbonlike organization of the AchR rosettes. This organization disappears ater alkaline treatment and is replaced by a network which is not observed after rapid freezing and, therefore, most likely results from the lateral redistribution of the AchR rosettes during condition of slow freezing. A dispersion of the AchR rosettes in the plane of the membrane also occurs after fusion of the pH 11-treated fragments with phospholipid vesicles. These results are interpreted in terms of a structural stabilization and immobilization of the AchR by the 43,000-Mr protein binding to the inner face of the subsynaptic membrane.