Rapid cation flux from Torpedo californica membrane vesicles: comparison of acetylcholine receptor enriched and selectively extracted preparations.

Rapid efflux of ²²Na from within closed vesicles derived from $\text{Torpedo}\text{californica}$ electroplax membranes has been studied as an $\text{in}\text{vitro}$ assay of acetylcholine receptor functionality. The most highly purified membrane preparations contained major polypeptides of M.W. 43 and 90 × 10³ daltons in addition to the four peptides characteristic of the acetylcholine receptor (40, 50, 60, 65 × 10³ daltons). Removal of these extra peptides by base extraction did not significantly alter the characteristics of carbamylcholine induced ²²Na efflux: the agonist dose response curve was similar, preequilibration with agonist caused desensitization, the irreversible antagonist α-Bungarotoxin blocked the efflux and the reversible blockade by the neurotoxin perhydrohistrionicotoxin was also retained. The dose response curve for perhydrohistrionicotoxin corresponded closely to its known binding characteristics for base extracted membranes.     

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica

Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali- stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor.     

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.     

Labeling of functionally sensitive sulfhydryl-containing domains of acetylcholine receptor from Torpedo californica membranes

N-(1-Pyrene)maleimide, a fluorescent, lipophilic, alkylating agent, was used as a probe for the nicotinic acetylcholine receptor (AChR). Preincubation with N-(1-pyrene)maleimide under nonreducing conditions inhibits agonist-induced cation permeability of AChR-enriched membranes. This inhibition is dependent on the concentration of N-(1-pyrene)maleimide used. This correlation was also exhibited by resonance energy transfer of tryptophan fluorescence to N-(1-pyrene)maleimide and by the labeling stoichiometries. However, agonist-induced desensitization, as based on the time-dependent inhibition of alpha-bungarotoxin binding upon preincubation with the agonist carbamylcholine, was unaffected by N-(1-pyrene)maleimide. Alkylation of the AChR by N-(1-pyrene)maleimide is pH-dependent with an apparent pKa of 7.5 and is unaffected by preincubation with carbamylcholine, alpha-bungarotoxin, tubocurarine, or decamethonium. Preincubation with a 25-fold molar excess of N-ethylmaleimide partially protects against N-(1-pyrene)maleimide, yet simultaneous incubation with an equimolar concentration does not protect. In contrast, simultaneous incubation with equimolar concentrations of phenylmaleimide or naphthylmaleimide inhibited N-(1-pyrene)maleimide alkylation by 52 and 67%, respectively. Each AChR subunit is labeled by N-(1-pyrene)maleimide. Prior alkylation with N-ethylmaleimide does not alter the labeling profile but lowers the amount of labeling of all subunits. Reductive methylation of membranes under conditions which dimethylate all or most protein amino groups does not inhibit alkylation by N-(1-pyrene)maleimide. The above results, as well as amino acid analysis of N-(1-pyrene)maleimide-alkylated receptor, indicate that a homologous class of cysteines, which reside in each subunit within the AChR domain embedded in the membrane, are involved in the reaction with N-(1-pyrene)maleimide.     

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.     

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 alpha-[125I]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius 125 +/- 10 A 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 beta-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

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.     

Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes

By use of a quench-flow technique to measure tracer ion flux rates in a physiologically significant time domain, the kinetics of activation and inactivation of purified reconstituted acetylcholine receptor (AChR) were investigated. After solubilization in sodium cholate, purification by affinity chromatography, and reconstitution into soybean lipids, the AChR from Torpedo californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4 degrees C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 = 7 ms) first-order filling of vesicle internal volume that presented a 10(4)-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 X 10(-4) M Carb. In the presence of Carb, the purified AChR also underwent a two-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (milliseconds to minutes) and then measuring the 86Rb+ influx rate. The two inactivation processes were each characterized by a distinct maximum rate (5.3 and 0.10 s-1) and by a different dependence on Carb concentration. The slow phase of inactivation gave a half-maximal rate at 2.5 X 10(-4) M Carb, and the fast inactivation was half-maximal at 1.3 X 10(-3) M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. The results are discussed in terms of models that describe the relationship between ligand binding and the processes of channel activation and desensitization.     

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.     

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.     

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica

We investigated the differential repair of DNA lesions induced by bifunctional mitomycin C, monofunctional decarbamoyl mitomycin C and ultraviolet irradiation in normal human, Xeroderma pigmentosum and Fanconi's anemia cells using assays for the survival of clone-forming ability, alkaline sucrose sedimentation and hydroxyapatite chromatography of DNA. Four FA cell lines exhibited about 5 to 15 times higher sensitivity to MC killing, despite normal resistance to u.v. and DMC, than did normal human cells. The XP cells, however, were highly sensitive to u.v. and DMC killings due to their deficiency in excision repair, but the cells unexpectedly had an almost normal capacity for surviving MC and repairing the MC interstrand cross-links.In experiments to determine the sedimentation velocity of the DNA in alkaline sucrose gradients, normal and XP cells showed evidence for single-strand cutting following MC treatment. The sedimentation velocity of the DNA covalently cross-linked by MC in an FA strain was 2.5 times faster than that of the untreated control, and remained unaltered during post-incubation due to the lack of half-excision⁴ of cross-links. However, FA cells, but not XP cells, had the normal ability to incise DNA with the DMC monoadducts. Hydroxyapatite chromatography revealed the reversibly bihelical property of MC cross-linked DNA after denaturation. Normal and XP cells lost such reversibility during post-MC incubation as the result of cross-link removal with first-order kinetics (half-life = 2 h). The three FA lines studied exhibited two- to eightfold reduced rates of cross-link removal than normal and XP cells, indicating a difference in the repair deficiency of the FA strain. Thus we have been led to conclude that FA cells may have different levels of deficiency in half-excision repair of interstrand cross-links induced by MC, despite having normal mechanisms for repair of u.v.-induced pyrimidine dimers and DMC monoadducts, and vice versa in XP cells.     

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.     

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.     

Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranes

The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing.     

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.     

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.     

Electrogenic behavior of synaptic vesicles from Torpedo californica.

Electrical potential changes in pure synaptic vesicles from Torpedo californica were monitored with the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. Vesicles resuspended in variable external sodium ion in the presence of gramicidin established sodium ion membrane diffusion potentials. Vesicles resuspended in choline or acetylcholine chloride became hyperpolarized upon addition of gramicidin. Hyperpolarization was subsequently partially reversed spontaneously by choline or acetylcholine influx, which was confirmed by gel filtration, to yield a new, less negative, stable membrane potential. Thus, acetylcholine and choline are taken up electrogenically by synaptic vesicles.