cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

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.     

Characterization of acetylcholine receptor isolated from Torpedo californica electroplax through the use of an easily removable detergent, β-d-octylglucopyranoside

Non-ionic detergents used for the solubilization and purification of acetylcholine receptor from Torpedo californica electroplax may remain tightly bound to this protein. The presence of detergent greatly hinders spectrophotometric and hydrodynamic studies of the receptor protein. β-d-Octylglucopyranoside, however, is found to be effective in solubilizing the receptor from electroplax membranes with minimal interference in the characterization of the protein. The acetylcholine receptor purified from either octylglucopyranoside or Triton X-100-solubilized extracts exhibits identical amino acid compositions, α-Bungarotoxin and (+)-tubocurarine binding parameters, and subunit distributions in SDS-polyacrylamide gels. The use of octylglucopyranoside allows for the assignment of a molar absorptivity for the purified receptor at 280 nm of approx. 530 000 $\text{M}{}^{\mathrm{?1}}\text{·}\text{cm}{}^{\mathrm{?1}}$. Additionally, successful reconstitution of octylglucopyranoside-extracted acetylcholine receptor into functional membrane vesicles has recently been achieved (Gonzales-Ros, J.M., Paraschos, A. and Martinez-Carrion, M. (1980) Proc.Natl. Acad. Sci. U.S.A. 77, 1796–1799).Removal of octylglucopyranoside by dialysis does not alter the specific toxin and antagonist binding ability of the receptor or its solubility at low protein concentrations. Sedimentation profiles of the purified acetylcholine receptor in sucrose density gradients reveal several components. Sedimentation coefficients obtained for the slowest sedimenting species agree with previously reported molecular weight values. Additionally, the different sedimenting forms exhibit distinctive behavior in isoelectric focusing gels. Our results suggest that both the concentration and type of detergent greatly influence the physicochemical behavior of the receptor protein.     

Some molecular properties of an isolated acetylcholine receptor ion-translocation protein

An improved procedure for isolation and purification of acetylcholine receptor from Torpedo californica electroplax membranes is described. The purified material contains the neurotransmitter recognition site and a second binding subsite which complexes inorganic cations and bis-quaternary cholinergic analogs. In addition to the transmitter recognition site the isolated macromolecule contains the molecular features necessary for ion-translocation during postsynaptic depolarization, since a chemically excitable membrane can be formed from purified acetylcholine receptor and Torpedo phospholipids.     

Carbamylcholine-induced rapid cation efflux from reconstituted membrane vesicles containing purified acetylcholine receptor.

Membrane preparations containing essentially only the four polypeptides considered to constitute the acetylcholine receptor are purified from $\text{Torpedo}\text{californica}$ electroplax. Treatment of these membranes with 2% ($\text{w}\text{v}$ aqueous sodium cholate followed by removal of all insoluble matter results in a solubilized purified receptor preparation that can be reassociated with phospholipids during dialysis to remove the detergent. Such reconstituted receptor is shown to retain the capability of translocating ²²Na⁺ across the membrane in response to carbamylcholine binding in a highly reproducible manner. The dose response for this effect is similar to that observed for the original electroplax membrane preparation and the carbamylcholine induced signal is completely blocked by α-bungarotoxin.     

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.     

cAMP, not Ca/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Characterization of polypeptide neurotoxins from the venom of Bungarus caeruleus

The venom of the krait Bungarus caeruleus has been fractionated into several components. Two of the basic components were highly toxic to mice and had significant levels of phospholipase A activity. These components appear to be similar in their action to the presynaptic neurotoxin β-bungarotoxin. Two other components were toxic to mice and also reduced the rate of α-bungarotoxin binding to the purified acetylcholine receptor: These components appear to be postsynaptic neurotoxins similar to α-bungarotoxin. Two acidic components displayed A-type phospholipase activity and perturbed the carbamylcholine binding properties of acetylcholine receptor-rich membrane preparations.     

Reconstitution of a purified acetylcholine receptor

Dialysis of the purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organ results in a vesicular membrane system in which the receptor is embedded in the bilayer and oriented so that most of the neurotoxin-binding sites appear to be on the outer surface. The constituted vesicles are chemically excitable by acetylcholine and carbamylcholine, as measured by ²²Na⁺ efflux. The excitability is specifically blocked by the antagonist α-bungarotoxin. These results demonstrate that the purified reconstituted receptor system not only can specifically bind neurotransmitter but also can trigger ion translocation. It therefore has the properties necessary to effect postsynaptic depolarization in vivo.     

The characterization of actin associated with postsynaptic membranes from Torpedocalifornica

SDS-polyacrylamide gel electrophoresis of acetylcholine receptor from $\text{Torpedo}\text{californica}$ electroplax membrane fragments shows, in addition to the four receptor subunits of 40,000, 50,000, 60,000 and 65,000 daltons, other components of apparent molecular weights 43,000, 47,000 and 90,000 daltons. In this study deoxyribonuclease I inhibitory activity has been used to identify actin in $\text{Torpedo}\text{californica}$ receptor-enriched membranes and affinity chromatography on a deoxyribonuclease I agarose column has been used to purify this protein from the membrane preparations. In addition the membrane protein components have been analyzed by electrophoresis on a series of SDS-polyacrylamide gels of varying acrylamide concentrations. Evidence is presented that actin is a component of most preparations of receptor-enriched membrane fragments, having an apparent molecular weight of 47,000 daltons, and is distinct from the 43,000 dalton protein.     

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.     

Antigenic differences between a proteolipid and a proteodetergent from Torpedo electroplax having similar cholinergic binding properties

The nicotinic acetylcholine receptor from Torpedo marmorata was extracted and purified from the electroplax membranes by using both aqueous detergent (proteodetergent) or chloroform-methanol (proteolipid). When studied with a highly sensitive radioimmunoassay, it was found that both proteins do not cross-react immunologically against an antireceptor antiserum prepared with the proteodetergent. Treatment with organic solvents of the electroplax membranes, as well as of the proteodetergent receptor purified by affinity chromatography, impaired the radioimmunoassay. This suggests that the antigenicity has been affected by the change in solvent polarity, even though both proteins have similar binding properties for cholinergic ligands. These findings do not allow a simple immunological comparison between the cholinergic proteodetergent and the proteolipid as previously stated in the literature.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

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.     

Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes

Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti-Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha-neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state.     

Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization,purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000–6000 pmol per mg protein of α-[¹²⁵]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius $\text{125 ± 10}$ Å and on sucrose gradient centrifugation one major peak was observed of 20–22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.     

Conversion of high affinity acetylcholine receptor from Torpedo californica electroplax to an altered form.

Most Triton extracts of fresh electroplax membranes showed a single kind of acetylcholine binding, of high affinity, with K_d = 17 nM. Fresh membrane preparations without Triton also showed only high-affinity binding (K_d = 14 nM), but membrane preparations from electroplax stored for 5 months in liquid nitrogen gave equal amounts of low-affinity binding material (K_d = 0.2 μM). Heating Triton extracts at 40 °C for 40 min or treating with 10^?4mp-chloromercuribenzoate or 5,5′-dithiobis(2-nitrobenzoic acid) converted the preparation to more than 90% low-affinity binding. It is suggested that the high-affinity is the native form and the low-affinity is an oxidation product.     

Comparison of the subunits of Torpedo californica acetylcholine receptor by peptide mapping.

The acetylcholine receptor from Torpedo californica electroplax was purified approximately 100-fold by affinity chromatography on alpha-neurotoxin-Sepharose 6B. Four putative subunits (alpha, beta, gamma, delta) of apparent molecular weights of 43,000, 52,000, 58,000, and 63,000 were found when the purified material was analyzed by sodium dodecyl sulfate (NaDodSO4) gel electrophoresis. In some preparations, however, the amount of the gamma polypeptide was small. The presence of N-ethylmaleimide throughout the purification procedure greatly enhanced the amount of the gamma chain. To investigate the possibility that the putative subunits may be structurally related, they were isolated by preparative NaDodSO4 gel electrophoresis and subjected to peptide mapping analyses. The patterns of fragments generated by Staphylococcus aureus V8 protease, papain, or chymotrypsin were different for each of the polypeptides. Thus, it is unlikely that they are derivatives of each other.