Functional stability of Torpedo acetylcholine receptor. Effects of protease treatment

The effect of tryptic degradation on structural and functional properties of the membrane-bound acetylcholine receptor from Torpedo californica has been investigated. Under conditions of proteolysis which resulted in extensive degradation of receptor subunits, the membrane preparations retained their full capability of mediating agonist-induced cation flux as measured in rapid kinetic experiments. Low concentrations on trypsin also cleaved receptor dimers to monomers, and this effect was paralleled by degradation of the Mr 65 000 subunits which are known to contain sulfhydryl group(s) involved in receptor dimerization through an interchain disulfide bond(s). This conversion to monomers occurred at lower trypsin concentrations when the enzyme was added to the outside of the vesicles compared with the effects observed when the enzyme was present inside the vesicles. Similarly Mr 43 000 protein consistently found in preparations of the membrane-bound acetylcholine receptor, which can readily be removed without apparent effect on receptor function, displayed greater susceptibility to proteolysis when trypsin was added to the exterior medium rather than inside the vesicles. The results emphasize the full functionality of the monomeric form of the acetylcholine receptor comprised of four polypeptides.     

Functional equivalence of monomeric and dimeric forms of purified acetylcholine receptors from Torpedo californica in reconstituted lipid vesicles

Acetylcholine receptors from Torpedo californica electric organ were solubilized and purified under conditions which prevent inactivation of the agonist-regulated cation channels. The dimer form of the receptors was preserved during purification. Treatment with reducing agents converted dimers into monomers. Receptor monomers and dimers were separately reconstituted into soybean lipid vesicles by the cholate dialysis technique. Reconstituted monomers and dimers were functionally equivalent with respect to their carbamylcholine-induced dose-dependent uptake of 22Na+, the total flux of 22Na+ per receptor during the permeability response, and the occurrence of desensitization. Evidence against non-covalent association of monomers to produce dimeric functional units was obtained using glutaraldehyde as a crosslinking agent. These results show that both the acetylcholine-binding sites and the agonist-regulated cation-specific channel are contained within the alpha 2 beta gamma delta subunit structure of the acetylcholine receptor monomer.     

Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action?

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring 86Rb+ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: integrated net ion efflux (in 10 s) from untreated vesicles, integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with alpha-bungarotoxin, and initial rates of efflux (5-100 ms) from vesicles that were partially blocked with alpha-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10(8)-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum 86Rb+ efflux by 50% (KB values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, KB values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times KB did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. We conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.     

Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes.     

Dimeric arrangement and structure of the membrane-bound acetylcholine receptor studied by electron microscopy.

The acetylcholine receptor protein (AChR) from the electric organ of Torpedo marmorata is studied in its membrane-bound form by electron microscopy and single-particle image averaging. About half the molecule protrudes from the membrane surface by approximately 5 nm. The low-resolution 3-D structure of this hydrated portion, including its handedness, can be deduced from averaged axial and lateral projections and from freeze-etched membrane surfaces. In native membrane fragments, a dimeric form of the AChR is observed and the relative orientation of the AChR monomers within the dimer is established. The dimers disappear upon disulfide reduction of the membrane preparations, whereas the average axial projections of the AChR monomer remain unaffected. Since the existence of disulfide bonds linking AChR monomers between their respective delta-subunits is well documented, the approximate position of the delta-subunit within the low-resolution structure of the AChR molecule can be deduced from the structure of the dimers.     

Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit.     

The sidedness of the COOH terminus of the acetylcholine receptor delta subunit

The nicotinic acetylcholine receptor from Torpedo sp. occurs as a dimer, disulfide-cross-linked between delta subunits. We determined the sidedness of the COOH terminus of the acetylcholine receptor delta subunit by locating the delta-delta disulfide relative to the membrane and by identifying the Cys residue forming the disulfide. We used receptor-rich native membrane vesicles isolated from Torpedo californica electric tissue and characterized as to orientation and intactness. These vesicles had not been extracted and retained v ("43-kDa protein") as a marker of the cytoplasmic surface. Using the reduction of v as an assay of permeability, we showed that two reductants, 2-mercaptoethanesulfonate and reduced glutathione, were relatively impermeant. Both of these reductants reduced the delta-delta disulfide in sealed right-side-out vesicles equally in the presence and absence of saponin, and 2-mercaptoethanesulfonate reduced this disulfide equally in the presence and absence of Triton X-100. By contrast, surfactants enhanced the reduction of dimer in inside-out and sequestered vesicles. We conclude that the disulfide is extracellular. To identify the Cys residue forming the disulfide, we labeled the sulfhydryls both in receptor dimer and in monomer generated by mild reduction of dimer. By high performance liquid chromatography and NH2-terminal sequencing of cyanogen bromide fragments of labeled delta-delta dimer and delta monomer, we found that the penultimate residue, delta-Cys-500, uniquely formed an intersubunit disulfide and that this disulfide was uniquely reduced when receptor dimer was reduced to monomer. Therefore, the delta COOH terminus is extracellular.     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.     

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.     

Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel.

The reversible acetylcholine esterase inhibitor (-)-physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR-rich membrane vesicles from Torpedo marmorata electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maelicke, Neuron, in press). This suggests that eserine exerts its channel-activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholine. Thus the conformational state of the receptor corresponding to desensitization is confined to the transmitter binding region, leaving the channel fully activatable-albeit only from other than the transmitter binding site(s).     

Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor. A model is proposed which accounts for all the experimental data obtained thus far on the transmembrane orientation of the subunit polypeptide chains.     

The intactness and orientation of acetylcholine receptor-rich membrane from Torpedo californica electric tissue

By a mild and highly reproducible fractionation of Torpedo californica electric tissue, we prepared membrane which was 30 times enriched in nicotinic acetylcholine receptor (AChR). This preparation was neither alkali-stripped nor reconstituted and consequently contained nu (43-kDa protein), which is associated with the cytoplasmic aspect of the receptor. We tested this membrane for the presence of sealed vesicles and determined the orientation of these vesicles by combining three methods. Two of these methods were based on the accessibilities, in the presence and absence of detergent, of the extracellular acetylcholine binding site to alpha-bungarotoxin and of the intracellular nu to trypsin. These two methods are specific for AChR-containing membrane. The third method was morphometry of electron micrographs, by which we estimated the proportion of sequestered membrane. These methods taken together indicated that approximately 45% of the AChR-containing membrane was in the form of leaky vesicles or sheets, 33% was sealed right-side-out vesicles, 11% was sealed inside-out vesicles, and 11% was sequestered within multilamellar or multivesicular vesicles. The complexity of this membrane needs to be taken into account in sidedness studies of the AChR.     

Electron microscopic localization of choline acetyl transferase activity in the electric organ of Torpedo marmorata

The light microscopic method for demonstration of choline acetyltransferase (CAT) activity based on the formation of a lead mercaptide of free SH-acetyl Coenzyme A was adapted for electron microscopy. In samples of electric organ of Torpedo marmorata CAT activity was found to be restricted to synaptic vesicles and cysternae. The precipitate formed was mostly fine grained and distributed more or less evenly throughout the vesicles. Generally, the reaction product seemed not to adhere to the inner side of the vesicle membrane. CAT activity was found only in the presynaptic region of the synapse, neither the synaptic cleft nor the postsynaptic region reacted positively. CAT activity was found also within synaptic vesicles in nerve endings prepared from electric organ. Samples of Torpedo brain reacted positively too. Complete suppression of CAT activity with inhibitors, judged on the basis of lead mercaptide deposited, was rather difficult to achieve. From a group of 10 presumed enzyme inhibitors, only 2 compounds reacted satisfactorily, namely trans-1,2-dihydro-2-imino-4-(1-naphthylvinyl)-1-pyridine-ethanol hydrobromide and 5,5-dithio-bis-(2-nitrobenzoic acid) (3,3'-6). On the whole, the results obtained show the viability of the method used and furthermore it offers also some new insight into the turnover of acetylcholine, since it may be deduced from the results that under certain circumstances acetylcholine may be synthesized in synaptic vesicles.     

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)     

Topological dispositions of lysine alpha 380 and lysine gamma 486 in the acetylcholine receptor from Torpedo californica

The locations have been determined, with respect to the plasma membrane, of lysine alpha 380 and lysine gamma 486 in the alpha subunit and the gamma subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the alpha subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the gamma subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to a portion of the vesicles prior to labeling to render them permeable to pyridoxal phosphate. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine alpha 380 and lysine gamma 486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The peptides bound and released by the immunoadsorbents were positively identified and quantified by high-pressure liquid chromatography. Modification of lysine alpha 380 in the native acetylcholine receptor in sealed vesicles increased 5-fold in the presence of saponin, while modification of lysine gamma 486 was unaffected by the presence of saponin. The conclusions that follow from these results are that lysine alpha 380 is on the inside surface of a vesicle and lysine gamma 486 is on the outside surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of subunits of acetylcholine receptor that interact with a cholesterol photoaffinity probe

All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with [3H]cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.     

A second pathway of activation of the Torpedo acetylcholine receptor channel.

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine (D-eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles and of equilibrium binding. We find that (-) physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as D-tubocurarine, alpha-bungarotoxin or antibody WF6. The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor. Antibodies FK1 and benzoquinonium antagonize channel activation (and binding) of eserine, suggesting that the eserine binding site(s) is separate from, but adjacent to, the acetylcholine binding site at the receptor. In addition to the channel activating site(s) with an affinity of binding in the 50 microM range, there exists a further class of low-affinity (Kd approximately mM) sites from which eserine acts as a direct blocker of the acetylcholine-activated channel. Our results suggest the existence of a second pathway of activation of the nAChR channel.     

Interaction of the 43K protein with components of Torpedo postsynaptic membranes

Interactions of the major Mr 43 000 peripheral membrane protein (43K protein) with components of Torpedo postsynaptic membranes have been examined. Treatment of membranes with copper o-phenanthroline promotes the polymerization of 43K protein to dimers and higher oligomers. These high molecular weight forms of 43K protein can be converted to monomers by reduction with dithiothreitol and do not contain any of the other major proteins found in these membranes, including the subunits of the acetylcholine receptor, as shown by immunoblotting with monoclonal antibodies. To study directly its interactions with the membrane, the 43K protein was radioiodinated and purified by immunoaffinity chromatography. Purified 43K protein binds tightly to pure liposomes of various compositions in a manner that is not inhibited by KCl concentrations up to 0.75 M. The binding can be reversed by adjusting the pH of the reaction to 11, the same treatment that removes 43K protein from postsynaptic membranes. Unlabeled 43K protein solubilized from Torpedo membranes with cholate can be reconstituted with exogenously added lipids in the absence of the receptor. The results suggest that 43K protein molecules are amphipathic and that they may interact with each other and with the lipid bilayer. These interactions cannot explain the coextensive distribution of 43K proteins with acetylcholine receptors in situ. However, they could account for the association of the 43K protein with the postsynaptic membrane and may contribute to the maintenance of the structure of the cytoplasmic specialization of which this protein is a major component.     

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.     

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.