Receptor-rich Membrane Fragments Research Articles

Direct involvement of a lamin-B-related (54 kDa) protein in the association of intermediate filaments with the postsynaptic membrane of the Torpedo marmorata electrocyte.

Mechanisms by which motor innervation induces postsynaptic membrane differentiation and functional compartmentalization of the subneural sarcoplasm in skeletal muscle fibres are still poorly understood. However, transmembrane control of cytoskeletal activities by the nerve terminal may be considered. Here, we examine several properties of a 54 kDa protein, previously identified in the postsynaptic membrane of the Torpedo marmorata electrocyte with anti-lamin B antibodies, in order to study its role in the assembly of the subneural intermediate filament meshwork. Using a ligand blot assay, we show that this protein binds desmin, a type III intermediate filaments protein, at micromolar concentrations. Moreover, purified acetylcholine receptor-rich membrane fragments are able to generate arrays of desmin filaments in vitro. Immunofluorescence experiments indicate that the 54 kDa protein becomes associated with the acetylcholine receptor-rich membrane at an early stage of development of the electrocyte, and that a polarized desmin network develops concomitantly from the postsynaptic membrane. Taken together, these data show that, like karyoskeletal lamin B, the 54 kDa protein is involved in the organization of the subneural intermediate filament meshwork. Control of the assembly of the subneural cytoskeleton by components of the postsynaptic membrane may thus be a prerequisite for the functional compartmentalization of the muscle fibre triggered by motor innervation.

Photoaffinity labeling of the acetylcholine binding sites on the nicotinic receptor by an aryldiazonium derivative

p-(Dimethylamino)benzenediazonium fluoroborate (DDF) behaves, in the dark, as a reversible competitive antagonist of the electrical response of Electrophorus electricus electroplaque to acetylcholine and of the acetylcholine-gated single-channel currents recorded in the C2 mouse cell line. This chemically stable but highly photoreactive compound binds irreversibly to the acetylcholine receptor when irradiated by visible light. In vivo, it irreversibly blocks the postsynaptic response of E. electricus electroplaque to agonists. In vitro, it reduces the alpha-bungarotoxin-binding capacity of acetylcholine receptor rich membrane fragments prepared from Torpedo marmorata electric organ. Once reversibly bound to the T. marmorata acetylcholine receptor, this ligand can be selectively photodecomposed by an energy-transfer reaction involving a tryptophan residue(s) of the protein. By use of reagent concentrations that are below the dissociation constant at equilibrium, up to 60% of the agonist-binding sites are covalently labeled. Under these conditions the alpha subunit of the acetylcholine receptor is preferentially labeled, and this labeling is partially prevented by agonists or competitive antagonists. This protective effect is substantially increased by prior incubation with phencyclidine, a compound known to prevent the binding of DDF at the level of the high-affinity site for noncompetitive blockers [Kotzyba-Hibert, F., Langenbuch-Cachat, J., Jaganathen, J., Goeldner, M. P., & Hirth, C. G. (1985) FEBS Lett. 182, 297-301]. The incorporation of about one molecule of label in an agonist/competitive antagonist protectable manner per alpha-bungarotoxin-binding site suffices to fully block alpha-bungarotoxin binding to the membrane-bound receptor. Thus, DDF behaves as a monovalent photoaffinity label of the acetylcholine-binding site.

Modification of Vmax of canine cardiac Purkinje fibers and of the effects of lidocaine by SC-72-14.

We used standard microelectrode techniques to determine the effects of SC-72-14, a monoclonal antibody to the fast sodium channel, on the transmembrane potentials and Vmax of canine cardiac Purkinje fibers, and to study the interactions of the monoclonal antibody with lidocaine. SC-72-14 was prepared with receptor-rich membrane fragments of homogenized eel electroplax as the antigen. It reduced Vmax in a concentration-dependent fashion, and depressed membrane responsiveness, as well. These effects were attributable specifically to SC-72-14, and not to other substances in the vehicle. At slow rates of stimulation, the actions of SC-72-14 and lidocaine on Vmax equaled the sum of their effects, administered alone. These results are consistent with the interpretation that SC-72-14 and the local anesthetic did not compete for the same binding site. In contrast, at fast stimulation rates, the use-dependent blocking effects of lidocaine were suppressed by SC-72-14. The basis for the interference with the use-dependent action of lidocaine is not readily apparent, but may be the result of SC-72-14-induced changes in channel state, receptor affinity for drug, or the availability of ionized drug molecules. These possibilities all await further testing.

Transmembrane topology of acetylcholine receptor subunits probed with photoreactive phospholipids.

The domains of the acetylcholine receptor subunits that contact the lipid phase were investigated by hydrophobic photolabeling of receptor-rich membrane fragments prepared from Torpedo marmorata and Torpedo californica electric organs. The radioactive arylazido phospholipids used carry a photoreactive group, either at the level of the lipid polar head group (PCI) or at the tip of the aliphatic chain (PCII), and thus probe respectively the "superficial" and "deep" regions of the lipid bilayer. The four subunits of T. marmorata and T. californica acetylcholine receptor reacted with both the PCI and PCII probes and thus are all exposed to the lipid phase. Ligands known to stabilize different conformations of the acetylcholine receptor (nicotinic agonists, snake alpha-toxin, and noncompetitive blockers) did not cause any significant change in the labeling pattern. The acetylcholine receptor associated 43 000-dalton v1 protein did not react with any of the probes. A striking difference in labeling between T. marmorata and T. californica acetylcholine receptors occurred at the level of the alpha-subunit when the superficial PCI probe was used. An approximately 5-fold higher labeling of the alpha-subunit as compared to the beta-, gamma-, and delta-subunits was observed by using receptor-rich membranes from T. marmorata but not from T. californica. The same difference persisted after purification of the labeled receptors from the two species and was restricted to an 8000-dalton C-terminal tryptic peptide. The only mutation observed in this region of the complete alpha-subunit sequence of the two species is the substitution of cysteine-424 in T. marmorata by serine-424 in T. californica.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid kinetics of agonist binding and permeability response analyzed in parallel on acetylcholine receptor rich membranes from Torpedo marmorata.

Excitable acetylcholine receptor rich membrane fragments from Torpedo marmorata have been used to measure, in parallel, (1) the permeability response to the fluorescent cholinergic agonist Dns-C6-Cho (in the 0.1 microM to millimolar concentration range) characterized by both the initial rate of Li+ transport and the rate of channel closure using the rapid-mixing quench-flow technique and (2) the kinetics of interaction of Dns-C6-Cho with the acetylcholine receptor sites using the rapid-mixing stopped-flow technique. Analysis of the kinetics of Dns-C6-Cho binding in the millisecond to minute time scale leads to the identification of at least three conformational states of the acetylcholine receptor: a "low-affinity" one (approximately 50 microM) that can be interconverted in the fraction of a second to a transient state of "intermediate affinity" (approximately 1 microM), followed by the final stabilization, in the second to minute time range, of a state of "high affinity" (approximately 3 nM). Comparison of Dns-C6-Cho binding data with the permeability response to the same agonist demonstrates that the binding to the low-affinity conformation(s) of the acetylcholine receptor sites coincides with the triggering of the permeability increase--or "activation"--and the transitions to the intermediate- and high-affinity states with the two-step process of channel closing--or "desensitization". The data are interpreted in terms of a minimum four-state "allosteric" model for the acetylcholine receptor.

Phospholipid and fatty acid composition of tetrodotoxin receptor-rich membrane fragments from Electrophorus electricus

To study the interaction of voltage-sensitive Na +-channels with membrane lipids, the phospholipid and fatty acid composition of highly purified membrane fragments from the remarkably differentiated plasma membrane of Electrophorus electricus has been analyzed. After density gradient fractionation and carrier free electrophoresis, fractions with up to 30 pmol tetrodotoxin binding/mg protein can be obtained, which may correspond to a 50% pure preparation of the extrasynaptic part of the excitable face. Phospholipid classes and cholesterol are separated by one-dimensional thin-layer chromatography in acidic and alkaline solvent systems. The following mean molar contents are found: 40% phosphatidylcholine, 23% phosphatidylserine, 30% phosphatidylethanolamine and 7% sphingomyelin. In a series of 11 animals, significant deviations from these mean values have been observed. The fatty acid composition of the phospholipids has been determined by gas chromatography. Phosphatidylcholine contains more than 50% 16:0, and about 20% unsaturated fatty acids in the C-18 group. Compared to other plasma membrane fractions, this phospholipid is the least differentiated. By contrast, phosphatidylethanolamine and phosphatidylserine show many characteristics in different membrane fractions, especially in their unsaturated components representing more than 50%. 22:6, as the major constituent in these fractions, accounts for a quarter to a third of all fatty acids in these fractions. 18:0 is the main saturated component in these two phospholipids with abundances of typically a quarter or less of all fatty acids. Knowledge of the lipid composition of these excitable membranes may help to conserve binding and structural properties when analyzing lipid-sensitive Na +-channels in vitro. It is also useful as a guideline for systematic reconstitution studies.

Multiple affinity states for noncompetitive blockers revealed by [3H]phencyclidine binding to acetylcholine receptor rich membrane fragments from Torpedo marmorata.

Multiple affinity states for noncompetitive blockers revealed by [3H]phencyclidine binding to acetylcholine receptor rich membrane fragments from Torpedo marmorata.

Multiple sites of action for noncompetitive blockers on acetylcholine receptor rich membrane fragments from torpedo marmorata.

Multiple sites of action for noncompetitive blockers on acetylcholine receptor rich membrane fragments from torpedo marmorata.

Effects of calcium on the binding of phencyclidine to acetylcholine receptor-rich membrane fragments from Torpedo californica electroplaque.

The influence of calcium on the binding of phencyclidine (PCP) to acetylcholine (ACh) receptor-rich membrane fragments was investigated. Calcium decreased the equilibrium affinity for PCP in the presence, but not in the absence, of the cholinergic agonist carbamylcholine. The effect of calcium was rapidly reversible by EGTA, indicating that it was not attributable to a calcium-activated protease or a phospholipase. Following detergent solubilization of the nicotinic ACh receptor, the calcium effect on PCP remained, suggesting that calcium may interact directly with the receptor to exert its effect. Other divalent cations (Mn2+, La2+, Co2+, Mg2+) had similar effects. A correlate of "desensitization" of the ACh receptor can be observed using PCP binding, and a two-step "desensitization" process can be observed. Calcium seemed to increase the amplitude of a rapid component of receptor "desensitization." The results presented in this paper suggest that calcium may play a role in the modulation of the nicotinic ACh receptor.

Penicillamine‐induced myasthenia gravis

Autoimmune diseases, including myasthenia gravis, occur in patients treated with D-penicillamine. Because D-penicillamine might induce autoantibodies by the mechanism of antigenic alteration, we studied the reaction of D-penicillamine with purified acetylcholine receptor from Torpedo californica. We found that brief exposure to D-penicillamine resulted in its covalent attachment to two receptor subunits, alpha (40,000 daltons) and gamma (59,000 daltons), presumably by reduction and formation of mixed disulfides. Furthermore, D-penicillamine treatment resulted in a dramatic modification of the equilibrium acetylcholine binding properties of both purified receptor and receptor-rich membrane fragments.

Phosphorylation in vitro of membrane fragments from Torpedo marmorata electric organ. Effect on membrane solubilization by detergents.

Acetylcholine receptor-rich membrane fragments purified from Torpedo marmorata electric organ were phosphorylated, in vitro, by endogenous protein kinases. The 40 000-Mr chain, which carries the acetylcholine receptor site, was never labelled; on the other hand, protein bands of apparent molecular weights 43 000, 50 000 and 66 000, which are present in the acetylcholine receptor-rich membranes, were repeatedly phosphorylated. The phosphorylation of these three peptides required the presence of divalent cations, such as Mg2+ or Mn2+, and was, in addition, stimulated up to 3--5-fold by K+. The effect of Na+ ions appeared less specific since Na+ ions reduced the labelling of all the polypeptides susceptible to phosphorylation. Cholinergic agonists and antagonists, local anesthetics and cyclic nucleotides did not affect the phosphorylation of the receptor-rich membranes. Phosphorylation selectively modified the solubilization of several polypeptides by nondenaturing detergents: phosphorylated 43 000-Mr, 50 000-Mr and 66 000-Mr polypeptides were solubilized at lower concentrations of detergent than their non-phosphorylated counterparts. Two-dimensional gels revealed the existence of a charge heterogeneity of the 40 000-Mr and 43 000-Mr chains. The microheterogeneity of the 43 000-Mr chain, but not that of the 40 000-Mr chain, might result from a selective phosphorylation of this particular chain.

Structural details of membrane-bound acetylcholine receptor from Tropedo marmorata.

A projection, at 15- to 20-A resolution, is presented of the structure of the membrane-bound acetylcholine receptor protein from Torpedo marmorata. The projection has its axis perpendicular to the membrane plane; its main contribution originates from a hydrated portion of the protein, which extends from the membrane into the aqueous medium. The structure is distinctly asymmetric, with individual morphological subunits barely resolvable. These results have been obtained by noncrystallographic averaging, using correlation functions, applied to electron micrographs of receptor-rich membrane fragments. The micrographs had been taken with minimal beam exposure in a scanning transmission electron microscope.

Pyrenesulfonyl azide: A covalent probe permitting [formula omitted] desensitization of labeled acetylcholine-rich membrane fragments from [formula omitted

Acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax after covalent labelling at the protein-lipid boundary by nitrenes generated in situ from pyrenesulfonyl azide can bind [ 125I]-α-bungarotoxin . The covalent attachment of 6–8 molecules of the fluorescent probe/receptor molecule also does not perturb the marked effect on the rate of α-bungarotoxin binding to electroplax membranes exerted by their preincubation with carbamylcholine. This phenomenon, which is analogous to pharmacological desensitization of receptors in synaptic junctions, is fully reversible upon removal of carbamylcholine (Quast, V., Schmerlik, M., Lee, T., Witzemann, V., Blanchard, V. and Raftery, M.A. (1978) Biochemistry 17 , 2405–2414). Torpedo electroplax membranes, whether tagged with the covalent probe or freshly isolated, regain the original fast rate of α-bungarotoxin binding upon dilution of carbamylcholine.

Fast Kinetic Studies on the Interaction of a Fluorescent Agonist with the Membrane‐Bound Acetylcholine Receptor from Torpedo marmorata

The fluorescent cholinergic effector, [1‐(5‐dimethylamino naphthalene)sulfonamido] n‐hexanoic acid β‐(N‐trimethylammonium bromide) ethyl ester (Dns‐C6‐Cho) acts as a potent depolarizing agent or agonist on Electrophorus electricus electroplaque (maximal depolarization 65–70 mV). In vitro, it increases the efflux of 22Na+ from receptor‐rich membrane fragments prepared from Torpedo marmorata electric organ. The apparent dissociation constant determined with the two systems is close to 1 μM. Preincubation of the receptor‐rich microsacs with Dns‐C6‐Cho results in a decrease of the amplitude of the permeability response to carbamylcholine. Like the physiological transmitter, acetylcholine, Dns‐C6‐Cho triggers the two characteristic reactions of ‘activation’ and ‘desensitization’.The equilibrium dissociation constant of Dns‐C6‐Cho measured by following the decrease of the initial velocity of binding of Naja nigricollisα‐[3H]toxin to receptor‐rich microsacs is close to 0.01 μM after prolonged incubation. Using the same method, it is found that Dns‐C6‐Cho triggers, in the range of a few minutes, a time‐dependent increased of affinity.The interaction of Dns‐C6‐Cho with the receptor‐rich membranes can be monitored under conditions of energy transfer from proteins (λex= 290 nm) and results in an approximately 10‐fold increase of fluorescence intensity at the maximal emission wavelength (λem= 557 nm). Rapid mixing of a suspension of membrane fragments with Dns‐C6‐Cho gives rise to changes of fluorescence intensity which can be recorded in the time range of seconds to milliseconds. The signals disappear after pre‐incubation of the membrane fragments with saturating levels of N. nigricollisα‐toxin and therefore are due to the interaction of Dns‐C6‐Cho with the receptor site. Three major relaxation processes are observed. (a) A ‘rapid’ one, in the millisecond range, is characterized by a linear dependence of its apparent rate constant with Dns‐C6‐Cho concentration. It is analysed in terms of Dns‐C6‐Cho binding to receptor sites spontaneously present in a high‐affinity state for agonists (Kd∼ 3 nM). These sites represent approximately 20% of the total population of receptor sites and exist prior to agonist binding in the membrane at rest. (b) An ‘intermediate’ relaxation process, in the second–millisecond range, shows a linear dependence of the apparent rate constant with Dns‐C6‐Cho concentration and is accounted for by a bimolecular binding reaction between Dns‐C6‐Cho and acetylcholine receptor sites in a state (or states) of lower affinity (∼ 1 μM). (c) Finally, a ‘slow’ relaxation process, in the seconds range, is characterised by an apparent rate of an interconversion between low‐affinity and high‐affinity states of the receptor.A model with two pre‐existing and interconvertible affinity states for the receptor qualitatively and quantitatively accounts for the data, and the corresponding rate constants have been determined; the model also accounts for the complex kinetics monitored upon addition of a non‐fluorescent effector to a suspension of receptor‐rich membrane fragments equilibrated with Dns‐C6‐Cho; the possible occurrence of a third state with intermediate affinity is discussed.The slow interconversion from low‐affinity to high‐affinity states of the receptor is correlated with the physiological process of ‘desensitization’ on the basis of its dependence on agonist concentration and temperature and of the effect of local anesthetics; the features of the intermediate relaxation process and its possible correlation with the ‘activation’ reaction are discussed.

Fast Kinetic Studies on the Allosteric Interactions between Acetylcholine Receptor and Local Anesthetic Binding Sites

Preincubation of receptor-rich membrane fragments from Torpedo marmorata with tertiary amine local anesthetics and several toxins such as histrionicotoxin, crotoxin and cerulotoxin, modifies the amplitude and time course of the relaxation processes monitored upon rapid mixing of the membrane fragments with the fluorescent agonist, Dns-C6-Cho. In particular, the amplitude of the rapid relaxation process, which is proportional to the fraction of acetylcholine receptor sites in a high-affinity state, increases; accordingly, the rate constant of the 'slow' and 'intermediate' relaxation processes also increases up to ten times (except with histrionicotoxin) whereas in a higher range of local anesthetic concentrations the rate constant of the 'rapid' relaxation process decreases. The data are accounted for by a two-state model of the acetylcholine regulator, assuming distinct binding sites for cholinergic agonists and local anesthetics and allosteric interactions between these two classes of sites; local anesthetics stabilize the regulator in a high-affinity state for agonists even in the absence of agonist, and modify the rate constants for th interconversions between the low-affinity and high-affinity states. The model accounts for the 'slow' fluorescence increase monitored upon addition of local anesthetics to a suspension of receptor-rich membranes supplemented with trace amounts of Dns-C6-Cho. The effect of local anesthetics on the apparent rate constant of the 'rapid' relaxation process can be accounted for on the basis of an additional low-affinity binding of local anesthetics to the acetylcholine receptor site. Finally the increase of the apparent rate constant of the 'intermediate' relaxation process can be simply accounted for by assuming the existence of a third state, corresponding to the 'active' state, to which local anesthetics bind and block ionic transport.

Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog.

Procaine amide azide, a derivative of the local anesthetic procaine amide, was prepared and its interactions with acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax were studied. Procaine amide azide was radiolabeled by a method that may be of general use for the preparation of other radioactive tertiary amines. When low concentrations of 3H-labeled procaine amide azide were photolyzed in the presence of receptor-rich membrane preparations, a simple pattern of incorporated radioactivity was seen after electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. Only two major labeled bands were seen, corresponding to apparent Mr of about 43,000 and 90,000. When the length of the gels was increased, the labeled band of lower Mr was resolved into two labeled proteins, one major and one minor, with apparent Mr of 43,000 and 40,000, respectively. The radioactivity incorporated into the protein of Mr 40,000 could be attributed to interaction of [3H]procaine amide azide with cholinergic ligand-binding sites, whereas labeling of the polypeptide of Mr 43,000 appears to represent interaction of the photolabile derivative with another class of sites. The labeling component of 90,000 Mr could be removed by preparation of membrane fragments in iodoacetamide-containing buffer and therefore appeared unrelated to the acetylcholine receptor.

Crotoxin effects on [formula omitted] cholinergic excitable vesicles and the role of its phospholipase a activity

The phospholipolytic neurotoxin from Crotalus durissus terrificus , crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated 22Na efflux from pre-loaded Torpedo californica excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of [ 125I]-α-bungarotoxin to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A 2 enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have in vitro post-synaptic effects, possibly mediated by its endogeneous phospholipase A 2 activity.

Fast kinetic studies on the interaction of cholinergic agonists with the membrane-bound acetylcholine receptor from Torpedo marmorata as revealed by quinacrine fluorescence.

Rapid changes of fluorescence intensity take place after fast mixing of quinacrine‐labelled receptorrich membrane fragments from Torpedo marmorata with cholinergic effectors and are recorded in the time range of 1 – 1000 ms, accessible to the stopped‐flow technique. Agonists such as acetylcholine, phenyltrimethylammonium, carbamylcholine, suberyldicholine or choline cause the change, but not antagonists such as d‐tubocurarine, flaxedil or Naja nigricollisα‐toxin. In addition, preincubation of the membrane fragments with N. nigricollisα‐toxin blocks the response to agonists. Ceruleotoxin, a toxin from Bungarus ceruleus venom which blocks the postsynaptic response to acetylcholine at a site distinct from the α‐toxin site, causes a decrease of the apparent amplitude of the fast signal. In the presence of 0.05 mM tetram, an acetylcholinesterase inhibitor, the kinetics of the fast response to acetylcholine do not change; in the second or minute range and in the case of acetylcholine, however, a decrease of fluorescence intensity occurs in the absence of acetylcholinesterase inhibitor showing that the fast fluorescence signal is reversible.Under the present experimental conditions the observed fast kinetics do not vary with the concentration of receptor sites or quinacrine but do vary with the concentration of agonist. The data can be analyzed in terms of the phenomenological reaction scheme , when k2»k3, if the fluorescence signal monitors the isomerization from A to A*. The following values of the constants are found: for acetylcholine: k4= (1.5 ± 0.5) s−1, k3= (60 ± 40) s−1, k2≥ 5 × 102 s−1 and k1≥ 107l·mol−1 s−1, K1=k2/k1= (7 ± 3) × 10−5 M, K=K1·k4/k3= (2 ± 1) × 10−6M; for carbamylcholine: k4= (0.2 ± 0.1) s−1, k3= (10 ± 5) s−1, k2× 102 s−1 and k1≥ 3 × 105 s−1, K1= (3 ± 1) × 10−4 M and K= (4 ± 3) × 10−6 M. The values of the apparent dissociation constants determined from the variation of the amplitudes of the fast effect for acetylcholine and carbamylcholine are respectively (1.0 ± 0.5) × 10−6 M and (5 ± 2) × 10−6 M. Some variability in the absolute values of the constants is noticed from one preparation to another and, for a given preparation, changes with age. Sets of kinetic constants have also been determined in the case of choline and suberyldicholine. At 20 °C, the Q10 is about 2.5 in the presence of 0.03 mM carbamylcholine and 1.3 in the presence of 0.01 M carbamylcholine; it is 1.3 with 0.05 M acetylcholine.The data are interpreted in terms of a three‐state model and their physiological significance is discussed.

Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ.

A method is described for the large‐scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol α‐toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor‐rich membrane fragments is composed of only one class of membrane fragments covered with 8‐nm rosettes identified as acetylcholine receptor molecules.This preparation is used as a starting material for the detergent solubilization and the large‐scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X‐100 extract of receptor‐rich membranes done in the presence of 2‐mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy.Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecylsulfate reveals three bands of apparent molecular weights 40000 ± 1000, 50000 ± 2000 and 66000 ± 2000, the two heaviest ones being present in significantly lower amounts than the 40000‐Mr one. The comparison of several samples of purified protein with different specific activities reveals a striking variability of the ratios of the 40000‐Mr band to the 50000 and 66000‐Mr ones.

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ : II. The permeability response of the receptor-rich membrane fragments to cholinergic agonists in vitro

Purified receptor-rich membrane fragments (spec. act. 1000 ± 400 nmol 3H-labelled α-toxin sites/g protein) from Torpedo marmorata make closed vesicles which retain 22Na + in free and membrane-bound compartments. Acetylcholine (after blocking acetylcholinesterase with 0.01 m m-Tetram † † Abbreviations used: Tetram, O,O′-diethyl S-(β-diethylamino)ethyl phosphorothiolate; SKF-525A, diethylamino ester of diphenylpropylacetic acid; methyldilvasene, 2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide; EGTA, ethyleneglycol bis(β-aminoethylether)- N,N′-tetraacetic acid. ), carbamylcholine, phenyltrimethylammonium, suberyldicholine and methyldilvasene enhance the release of the free 22Na + from these microsacs and the effect of carbamylcholine is blocked by Naja nigricollis α-toxin, d-tubocurarine, flaxedil and hexamethonium. Decamethonium, an agonist on T. marmorata electroplaque, acts as a competitive antagonist on this in vitro system. Two local anaesthetics, dimethisoquin and SKF-525A block the response in vitro. Apparent dissociation constants for cholinergic ligands are determined from the dose-response curves. The values of the constants are, for most of them, significantly larger than those measured in vivo with the electroplaque by following steady-state depolarization in the presence of agonist added in the bath (on the other hand, they coincide rather well with the equilibrium dissociation constants given by direct binding studies with the cholate-solubilized receptor). The real dissociation constants measured with the membrane-bound receptor are close to the apparent ones for all the antagonists tested. However, the agonists bind at concentrations two orders of magnitude smaller than those necessary to trigger the response.