A differential scanning calorimetry study of acetylcholine receptor-rich membranes from Torpedo californica

Various acetylcholine receptor-rich membrane preparations from Torpedo californica electroplax tissue were examined using the techniques of differential scanning calorimetry coupled with gel electrophoretic analysis of heat-denaturing material and functional assays following passage through discrete transitions. In unfractionated membranes, four irreversible calorimetric transitions were observed, one of which (Td = 59 degrees C) could be assigned to a complete loss of acetylcholine receptor function. A second lower temperature transition apparently corresponds to loss of certain peripheral membrane proteins including the Mr = 43,000 polypeptide and the acetylcholinesterase activity. Membrane preparations highly enriched in acetylcholine receptor polypeptides contained a major transition at 59 degrees C which could be shown to be sensitive to the presence of added ligands of the acetylcholine receptor, supporting its assignment to structural alterations of the receptor protein or its arrangement in the membrane.     

The acetylcholine receptor as part of a protein complex in receptor-enriched membrane fragments from Torpedo californica electric tissue

The acetylcholine receptor from Torpedo californica electric tissue consisting of polypeptide chains of molecular weight 42000 (+/- 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270000 (+/- 30000). Besides the receptor subunit (alpha-chain), at least three further classes of polypeptide chains are part of the complex: beta (Mr 48000), gamma (Mr 62000) and delta (Mr 68000). This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent: From the 270000 molecular weight particle the four predominant polypeptide chains of the membrane, alpha, beta, gamma, and delta, can be obtained. The gamma-polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge.     

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.     

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.     

Affinity-labeling of purified acetylcholine receptor from Torpedo californica

The receptor for acetylcholine purified from electric tissue of $\text{Torpedo californica}$ has been assayed both by affinity-alkylation and by neurotoxin binding. The specific activity by the latter method is about twice that by the former. Four major components of apparent molecular weights of 39,000, 48,000, 58,000 and 64,000 are separated by dodecyl sulfate-acrylamide gel electrophoresis. Reduction and affinity-alkylation of the receptor with a tritiated quaternary ammonium maleimide derivative results in the exclusive labeling of the 39,000 dalton subunit. This subunit, it is concluded, contains all or part of the acetylcholine binding site.     

Symmetry and dimensions of membrane-bound nicotinic acetylcholine receptors from Torpedo californica electric tissue: rapid rearrangement to two-dimensional ordered lattices

Computer-aided image-averaging methods are applied to different preparations of membrane-bound nicotinic acetylcholine receptor. Circular harmonic averaging (CHA), a novel, reference-independent averaging method developed by W. Kunath and H. Sack-Kongehl [1989) Ultramicroscopy 27:171-184) allows analyzing images of single molecules of the receptor in its native membrane-bound state. The five subunits of the receptor are clearly resolved. At the resolution obtained (approximately 20 A) no differences were observed with resting and agonist-desensitized receptors. A method is proposed for rapidly arranging the acetylcholine receptors to ordered lattices. Depending on the conditions, tetragonal or hexagonal, two-dimensional lattices can be obtained within 2 to 6 days at 4 degrees C. Analysis by CHA shows that the receptor molecules preserve their gross structure and dimensions in these membranes, but that they are randomly oriented. Both lattices, therefore, do not represent true two-dimensional crystals.     

Monovalent ion effects on acetylcholine receptor from Torpedo californica

The effects of the five Group I monovalent ions, Li, Na, K, Rb, and Cs, on [³H]acetylcholine binding to Triton X-100 solubilized acetylcholine receptor from Torpedo californica electroplax were examined. Acetylcholine binding was not greatly affected by Li or Na, but was inhibited by the other ions in the order Cs > Rb > K. The inhibition by K appeared to occur by a mechanism identical to that for d-tubocurarine inhibition of acetylcholine binding.     

Phospholipase A inhibition of acetylcholine receptor function in Torpedo californica membrane vesicles

A protein isolated from $\text{Naja naja siamensis}$ venom on the basis of its phospholipase A activity inhibits acetylcholine receptor function in post-synaptic membrane vesicles from $\text{Torpedo californica}$. Specifically, the phospholipase A prevents the large increase in sodium efflux that can normally be induced by carbamylcholine, a receptor agonist. The phospholipase A inhibition shows the following properties: 1) it occurs at concentrations 50 times lower than the concentrations required for inhibition by α-neurotoxins; 2) the phospholipase A has no effect on the binding properties of the receptor; 3) the inhibition is abolished by removal of calcium ions; and 4) some phospholipid hydrolysis accompanies inhibition. It is suggested that the phospholipase A acts enzymatically to uncouple ligand binding from ion permeability in the receptor containing membrane vesicles.     

Isolation of acetylcholine receptor-- -bungarotoxin complexes from Torpedo californica electroplax

Affinity chromatography has been utilized to purify acetylcholine receptors from Torpedo californica electroplax membranes. These have been isolated as their [¹²⁵I]α-bungarotoxin complexes. The major protein subunit found had a molecular weight of 3.5–4.5 × 10⁴ on gel electrophoresis under denaturing conditions. Other components of higher molecular weight were also present in smaller amounts even in the best preparations. This is interpreted as most likely due to either a multiple subunit structure or to more than one type of receptor in the tissue used.     

Disulfide bond cross-linked dimer in acetylcholine receptor from Torpedo californica

Acetylcholine receptor from $\text{Torpedo californica}$ electric tissue occurs in membrane, and is purified, as a mixture of monomer and dimer. Dimer is cross-linked by disulfide bonds involving one of the four polypeptide components of receptor, namely the one of apparent molecular weight of 64,000.     

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.     

Phospholipid chain immobilization and steroid rotational immobilization in acetylcholine receptor-rich membranes from Torpedo marmorata

1. The ESR spectra of both phosphatidylcholine and phosphatidylethanolamine spin labels reveal an immobilized lipid component (tau R greater than or equal to 50 ns), in addition to a fluid component (tau R approximately 1 ns), in acetylcholine receptor-rich membranes prepared from Torpedo marmorata electroplax according to the method of Cohen et al. (Cohen, J.B., Weber, M., Huchet, M. and Changeux, J.-P. (1972) FEBS Lett. 26, 43--27). 2. The ESR spectra of the androstanol spin label display a component corresponding to molecules which are immobilized with respect to rotation about the long molecular axis (tau R greater than or equal to 50 ns), in addition to the fluid lipid bilayer component in which the molecules are rotating rapidly about their long axes (tau R approximately 1 ns). This immobilized component is observed throughout the temperature range 2--22 degrees C, at an approximately constant relative intensity of approx. 45% of the total, which is quantitatively the same as previously observed with fatty acid spin labels.     

Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica

Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.     

Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo californica electroplax.

Pyrene was introduced in acetylcholine receptor (AcChR)-rich membrane preparations of Torpedo californica electroplax. The lifetime of the singlet excited state of pyrene was used to probe the properties of the hydrocarbon regions of the lipid bilayer as well as the possible perturbing effects of cholinomimetic agents on this region. After excitation with a single 15-ns pulse with a Q-switched ruby laser, the lifetime of the pyrene singlet excited state in the membranes was 200 ns. In desensitized membranes the pyrene fluorescence lifetimes remained unchanged when the cholinergic ligands carbamylcholine, d-tubocurarine, decamethonium, and hexamethonium, as well as α-bungarotoxin, were present. By contrast, the lifetime was shortened when local anesthetics were present. In sensitized membranes no changes in the pyrene lifetimes were detected when the membranes were converted from their resting state to a carbamylcholine-induced “desensitized state.” Water-soluble fluorescence quenchers affected the lifetime of pyrene in membranes. The second order rate constants for the pyrene-quencher interaction were used to detect changes in fluidity and/or membrane lipid accessibility to quenchers induced by ligands or anesthetics. No changes were detected in the quenching constants of nitromethane or Tl⁺ in the presence of cholinergic agents (with the exception of d-tubocurarine); on the other hand, a marked decrease in Tl⁺ accessibility was induced by the anesthetics procaine and tetracaine. Fluorescene dynamics measurements indicate that the hydrocarbon core of the bulk lipid in electroplax is not significantly affected by binding cholinergic ligands to membranebound AcChR. However, the hydrophobic region of the membrane is perturbed by both local anesthetics and one cholinergic ligand, d-tubocurarine. Pyrene was also incorporated into lipid vesicles prepared from T. californica electroplax lipids. The fluorescence lifetimes and quenching values of these lifetimes yielded results similar to those obtained with both sensitized and “desensitized” membrane preparations. The d-tubocurarine effect on the Tl⁺ quenching of the pyrene probe is ascribed to direct interaction of d-tubocurarine with the lipids. These findings favor a mechanism in which perturbation of the hydrophobic (lipid) environment of the AcChR in membranes by local anesthetics and even d-tubocurarine may influence the receptor conversion: sensitized state ? desensitized state.     

Active acetylcholine receptor fragment obtained by tryptic digestion of acetylcholine receptor from Torpedo californica.

Tryptic digestion of acetylcholine receptor (AChR) from Torpedo californica did not change the pharmacological specificity and the pathological myasthenic acitivity of the receptor molecule. The product obtained after tryptic digestion was repurified by affinity chromatography on a toxin-Sepharose resin and was designated T-AChR. T-AChR has a sedimentation coefficient of 8.0S and in SDS acrylamide gel electrophoresis shows one major band with a molecular weight of 27,000. Immunological studies reveal that T-AChR binds to anti-AChR antibodies directed only against conformational antigenic determinants.