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.     

Ligand-induced conformation changes in Torpedo californica membrane-bound acetylcholine receptor

A time-dependent increase in ligand affinity has been studied in cholinergic ligand binding to Torpedocalifornica acetylcholine receptor by inhibition of the kinetics of of [125I]-alpha-bungarotoxin-receptor complex formation. The conversion of the acetylcholine receptor from low to high affinity form was induced by both agonists and antagonists of acetylcholine and was reversible upon removal of the ligand. The slow ligand induced affinity change in vitro resembled electrophysiological desensitization observed at the neuromuscular junction and described by a two-state model (Katz, B., & Thesleff, S. (1957) J. Physiol. 138, 63). A quantitative treatment of the rate and equilibrium constants determined for binding of the agonist carbamoylcholine to membrane bound acetylcholine receptor indicated that the two-state model is not compatible with the in vitro results.     

Interaction of local anesthetics with Torpedo californica membrane-bound acetylcholine receptor

The effects of local anesthetics on the rate of the agonist-induced increase in ligand affinity of membrane-bound acetylcholine receptor from Torpedo californica were examined. The rate of the transition in receptor affinity was determined by following the time-dependent increase in inhibition of iodinated alpha-bungarotoxin binding caused by 1 microM carbamylcholine. At concentrations below those that directly inhibited the binding of iodinated alpha-bungarotoxin, dibucaine increased the rate of the transition to a high-affinity state and tetracaine decreased this rate. The measured rate constants were 0.026 +/- 0.008 s-1 in the presence and 0.010 +/- 0.002 s-1 in the absence of dibucaine while tetracaine decreased the rate to 0.006 +/- 0.002 s-1 as compared to a control value of 0.012 +/- 0.003 s-1. A parallel was observed between the effectiveness of a compound in increasing or decreasing the rate of the agonist-induced transition in affinity and the change in its apparent inhibition constant in the presence of carbamylcholine (increase or decrease) measured by the displacement of tritiated perhydrohistrionicotoxin. This parallel could be explained by assuming (a) that local anesthetics bound directly to the specific histrionicotoxin binding site or (b) that they bound to a different site and the observed effects were caused by conformational changes.     

Immunospecific identification and three-dimensional structure of a membrane-bound acetylcholine receptor from Torpedo californica.

Antibodies, raised against affinity column-purified acetylcholine receptor from Torpedo californica, were used as a basis for immunospecific identification of the receptor in membrane fragments. Rabbit and goat anti-receptor antibodies were coupled directly or indirectly via goat anti-rabbit antibody to colloidal gold spheres or to ferritin. The labeled membranes were visualized by negative stain electron microscopy, and show that the receptor corresponds to the 85 Å diameter rosette seen in membranes derived from electroplaques.Electron micrographs of immunospecifically labeled receptor, in the plane perpendicular to the membrane surface, confirm and extend our previous conclusions based on X-ray diffraction analysis, that the molecule extends above the extracellular membrane surface by approximately 55 Å, and little on the cytoplasmic side. Calculated molecular volumes based on X-ray diffraction and electron microscopy indicate that the membrane receptor has a molecular weight in the range of 250,000 to 310,000, a range consistent with current estimates of detergent-solubilized monomer molecular weight.     

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.     

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.     

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.     

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.     

Electrostatic determinants of the ion channel control of the nicotinic acetylcholine receptor of Torpedo californica

The patch clamp K⁺-conductance G of the nicotinic acetylcholine receptor (AcChoR) dimer (M_r≈ 590 000) of Torpedo californica, reconstituted in lipid vesicles, which decreases with increasing Ca²⁺-concentration in the range 0.1≤[Ca²⁺]/mM≤2, can be quantitatively rationalized by Ca²⁺-binding to negatively charged sites, causing charge reversal reducing the normal K⁺-accumulation in the channel vestibules. Cleavage of the sialic acid residues (up to 20±2 per dimer) reduces the K⁺-accumulation factor α = G₀/G_∞ from α = 3±0.8 of the normal AcChoR to α = 2±0.7 for the desialyated AcChoR. Desialysation also decreases the Ca²⁺-sensitivity of the conductance from G₀ = 96.6±6 pS at [Ca²⁺]→0 of the normal AcChoR to G₀ = 84.2±6 pS. Endogenous hyperphosphorylation (to up to 28±4 phosphates per dimer) enhances the vestibular K⁺-accumulation to α = 3.6±0.7, without affecting the Ca²⁺-dissociation equilibrium constant K_Ca = 0.34± 0.05 mM at 295 K (22 °C). Most interestingly, even in the absence of AcCho, the hyperphosphorylated AcChoR dimer exhibits spontaneously long-lasting open channel events (τ = 200±50 ms). At [AcCho] = 2 μM there are two open states (τ ₁ = 20±10 ms, τ ₂ = 140±60 ms) whereas the normal AcChoR dimer has only one open state (τ = 6±4 ms). – Physiologically important is that (i) the sialic acid and phosphate residues render the AcChoR conductance sensitive to control by divalent ions and (ii) the channel behavior of the hyperphosphorylated AcChoR without AcCho appears to indicate pathophysiologically high phosphorylation activity of the cell leading, among others, to myasthenic syndromes. Received: 10 November 1997 / Revised version: 12 January 1998 / Accepted: 7 March 1998     

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.     

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

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

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.     

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.     

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.     

Cholinergic ligand-induced affinity changes in Torpedo californica acetylcholine receptor

Measurement of small-angle X-ray scattering from a sample of hyaluronic acid of high molecular weight in 0.05 m HNO₃ gave persistence length plots which agreed in form with theory and led to apparent persistence lengths of from 4 to 6 nm. Similar measurements in 0.2 m NaCl gave plots which deviated somewhat in form from theoretical expectation, but which could be interpreted to give a persistence length of 4 nm in this solvent. Data for intrinsic viscosity [η] as a function of molecular weight were in reasonable agreement with the Yamakawa-Fujii treatment of [η] for the worm-like chain model for a persistence length of about 4 nm in both 0.5 m NaCl and 0.1 m HCl, perhaps slightly higher in the latter. The values of persistence length estimated from [η] depend somewhat on the choice of chain parameters and the method of correction of experimental data to unperturbed solvent conditions. Experimental data for the sedimentation coefficient, while less definitive, were consistent within experimental uncertainty with the same parameters of the worm-like chain model. These calculated results are in substantial agreement with the values derived from small-angle X-ray scattering. A fraction of hyaluronic acid of low molecular weight in 0.05 m HNO₃ gave an estimated molecular weight of 2.7 × 10⁴ and a radius of gyration of 8 nm, in reasonable agreement with expected values based on the worm-like chain model for a persistence length of about 4 nm.     

Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.