Binding of -bungarotoxin to acetylcholine receptors in mammalian muscle.

Experiments were performed to determine the specificity of Il25I]a-bungarotoxin binding to skeletal muscle. In adult rat diaphragm, [1251]a-bungarotoxin was found to bind almost exclusively to those regions of the muscle that contain endplates and are known to be sensitive to acetylcholine. In contrast, chronically denervated adult muscle and muscle from neonatal rats, both of which are sensitive along their entire lengths, bound substantial amounts of toxin in all regions. Toxin binding to all muscles was inhibited by d-tubocurarine and by carbamylcholine, but not by atropine. The bound Il251ltoxin was solubilized by homogenization of the tissue in 1]% Triton X-100 and was recovered as a single band, distinct from free toxin, after zone sedimentation. Treatment of the solubilized, toxin-bound complex with 2-mercaptoethanol and sodium dodecyl sulfate resulted in the recovery of free toxin. A toxin-bound complex was also obtained when toxin was incubated directly with extracts of muscle endplate regions prepared by homogenization in Triton X-100. No such complex was observed with extracts prepared from muscle lacking endplates. These results are consistent with the interpretation that a-bungarotoxin binds specifically to the acetylcholine receptor of mammalian skeletal muscle. At the mammalian neuromuscular junction, stimulation of the presynaptic nerve causes the release of the transmitter acetylcholine from the nerve terminals. Released acetylcholine diffuses across the gap separating nerve and muscle cells and interacts with specific receptors associated with the postsynaptic muscle membrane to produce an increase in membrane permeability to sodium and potassium ions (1). The relative amounts of this acetylcholine receptor and its distribution along the muscle have been estimated by measurement of the depolarization produced by the iontophoretic application of acetylcholine onto discrete regions of the muscle surface (2). Using this method, Axelsson and Thesleff (3) and Miledi (4, 5) have shown that adult vertebrate muscle fibers are highly sensitive to acetylcholine only in the region of the neuromuscular junction. After denervation, the muscle fibers become sensitive to acetylcholine along their entire length. Muscle fibers of fetal and neonatal rats are also sensitive to acetylcholine in regions outside the neuromuscular junction (6). The recent characterization of several snake toxins has suggested another method for estimating the amount and distribution of acetylcholine receptors. Toxins purified from Abbreviations: SDS, sodium dodecyl sulfate; +EP, containing endplates; -EP, without endplates; EGTA, [Ethylenebis(oxyethylenenitrilo)] tetraacetic * Present address: Department of Biochemistry, University of California Medical Center, San Francisco, Calif. 94122 147 the venoms of Bungarus multicinctus, Lacticauda semifasciata, and Naja naja (cobra) disrupt neuromuscular transmission by blocking the postsynaptic response to acetylcholine (7-9). In addition, a-bungarotoxin blocks the response to acetylcholine of denervated muscle fibers (10). Autoradiographic studies on muscles to which [13I]a-bungarotoxin has been bound have shown that binding occurs preferentially to endplate regions of normal fibers and to the entire length of denervated fibers (11). These experiments suggest that the binding of a-bungarotoxin may allow direct measurement of the acetylcholine-receptor content in muscle preparations. Both cobra toxin and a-bungarotoxin, which block cholinergic transmission in the electric organs of the marine ray and eel, have been used in studies on the isolation and characterization of the acetylcholine receptor from these tissues (12-14). Muscle preparations, which are relatively poor in synaptic components, are less attractive as a source of acetylcholine receptor for biochemical studies, but may offer special advantages for the study of the synthesis of the receptor and its control. In order to develop an assay for the acetylcholine receptor in this system, we have further examined the specificity of a-bungarotoxin binding in mammalian muscle. We report here that [la5I]a-bungarotoxin binds to a component of muscle that has the distribution and pharmacology expected of the acetylcholine receptor. MATERIALS AND METHODS Preparation of [1i5I]a-Bungarotoxin. Crude Bungarus multicinctus venom was fractionated by passage over a Sephadex G-50 column in 0.1 M ammonium acetate (pH 5.0). The protein peak eluted at about 0.6 column volume was applied directly to a carboxymethyl Sephadex column, and the column was developed with a linear gradient of 0.05 M ammonium acetate (pH 5.0) to 1.0 M ammonium acetate (pH 7.0) and concentrated by ultrafiltration with an Amicon Diaflo filter. After 10% polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) buffer, as described below, more than 95% of the purified toxin was found to migrate as a single component, with a molecular weight of about 9 X 10' (15). The purified toxin had a toxicity comparable to that reported by Chang and Lee (7). No loss of toxicity was observed after 3 months of storage at 0°C in 0.05 M sodium phosphate (pH 7.5). The postsynaptic action of the purified toxin was confirmed by demonstrating that it depressed the amplitude of miniature endplate potentials and blocked the depolarization caused by iontophoretically applied acetylcholine. a-Bungarotoxin was labeled with [l251]iodide by the general Proc. Nat. Acad. Sci. USA 69 (1972) procedure of Greenwood, Hunter, and Gloves (16). To 0.4 mg of a-bungarotoxin in 0.15 ml of 0.05 M sodium phosphate (pH 7.5) was added 0.10 ml of 2 M potassium phosphate buffer (pH 7.5) followed by 10 mCi of carrier-free Nal1BI in 0.10 ml of 0.1 M NaOH. Iodination was begun by addition of 0.01 ml of a freshly prepared solution of 25 mg/ml of Chloramine T in 0.05 M sodium phosphate (pH 7.5) to the chilled reaction solution and mixing. The reaction was terminated 2-3 sec later by the addition of 0.01 ml of a freshly prepared solution of 50 mg/ml of sodium metabisulfite in 0.05 M sodium phosphate (pH 7.5). [l25I]a-Bungarotoxin was separated from Nalal5 by passage through a Sephadex G-25 column in 0.05 M sodium phosphate (pH 7.5). SDS-polyacrylamide gel electrophoresis (10% gels) of the labeled toxin demonstrated that 85-90% of the 125I label migrated as a single component, with a molecular weight of about 9 X 103. Recovery of protein was 60-90%, as measured by absorbance at 280 nm. With the assumption that this material had an average molecular weight of 9 X 108, specific activities were calculated to be 1.5-1.7 X 105 cpm per pmol (three preparations). This is equivalent to 0.05 mol of 1251 label per mol of toxin. The relative biological activity of [l25I]a-bungarotoxin preparations was determined by dilution of aliquots with various amounts of untreated a-bungarotoxin, followed by measurement of the maximum amount of labeled toxin that could bind to muscle in the standard binding assay (see below). The [125I ]a-bungarotoxin (two different preparations) was 60% as active as untreated a-bungarotoxin. Since labeled molecules comprise only 5% of the [1251 ]a-bungarotoxin preparation, dilution experiments of this kind serve to define the limits of activity of only the unlabeled population of molecules in the preparation. Binding [125I]a-Bungarotoxin to Muscle. Rat diaphragms were removed with ribs attached and incubated with [l121]abungarotoxin at room temperature in Kreb's solution, at pH 7.3, containing 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM KH2PO4, and 12 mM NaHCO3. The rate at which toxin was bound to diaphragm muscle depended on the age and concentration of the [125I]a-bungarotoxin preparation. The maximum extent of binding was independent of these parameters. With freshly prepared toxin, 2 hr of incubation with 1.4 ,g/ml was adequate to achieve maximum binding. In all experiments reported here, [125I ] ebungarotoxin was used within 10 days of preparation; with the exception of the experiments described in Table 2, incubation conditions were always chosen to yield maximum binding of toxin to adult diaphragm muscle. After incubation with labeled toxin, the muscle was washed overnight in several changes of a solution containing 0.15 M NaCl-0.4 mM EGTA-0.02 M Tris HCl (pH 7.4) (standard buffer). The muscle was then divided under a dissecting microscope into regions containing endplates (+EP) and regions lacking endplates (-EP). The +EP regions consisted of a central strip of the muscle containing over 95% of the endplates (17). The -EP regions consisted of peripheral strips lacking nerve branches. The dissection was done so that +EP and -EP portions were of about equal weight. Care was taken to trim away any fibers that had been damaged before incubation. The strips were then weighed and homogenized, at a concentration of 100 mg/ml in standard buffer containing 1% Triton X-100, with a Kontes were counted in a Packard scintillation counter, with a scintillation fluid containing one part Triton X-100 to two parts of a solution of 0.2% 2,5-diphenyloxazole (PPO) and 0.01% p-bis 2-(5-phenyloxazolyl)-benzene (POPOP) in toluene. SDS-Polyacrylamide Gel Electrophoresis. SDS gels were prepared as described by Davies and Stark (18). Protein samples were incubated with a 10-fold excess by weight of SDS and 2-mercaptoethanol at 1000C for 5 min, followed by incubation at 370C for 10 hr. Electrophoresis was at 8 mA/ tube, with 0.1% mercaptoacetic acid in the upper reservoir buffer. Pyronin Y was used as the tracking dye. Gels were stained by the method of Weber and Osborn (19) and destained in 42.5% methanol, which contained 7.5% acetic acid. Destained gels were scanned with a Gilford model 240 spectrophotometer and model 2410 linear transport attachment. The distribution of radioactivity in the gels was determined by the method of Young and Fulhorst (20). Zone Sedimentation. 0.20-ml samples were layered onto 4.8-ml gradients in polyallomer tubes containing 5-20% sucrose in standard buffer and 1% Triton X-100. Zone centrifugation was performed in a Spinco model L-2 preparative ultracentrifuge using an SW 50.2 rotor at 49,000 rpm for 8 hr at 20C. Protein was determined by the method of Lowry et al. (21), with bovine serum albumin as a standard. Since samples containing Triton X-100 gave a precipitate upon addition of the phenol reagent, all such samples, including appropriate controls with albumin, were filtered through a Millipore filter before measurement of the absorption at 750 nm. Materials. White rats were obtained from Charles River Breeding Co. Adults weighed 170-220 g. Rats were denervated by transection of the left phrenic nerve in the thorax (22). Neonatal diaphragms were taken from rats on the day of birth. Crude Bungarus multicinctus venom was purchased from Sigma Chemical Co. Carrier-free Na 125I was purchased from New England Nuclear Corp. Chloramine T was obtained from Eastman Kodak Co., atropine sulfate from Calbiochem., d-tubocurarine from Mann Research Lab., and carbamylcholine chloride from K. and K. Laboratories, Inc. TABLE 1. Binding of [251I]cx-bungarotoxin to rat diaphragm Femtomoles of toxin per mg of tissue