ABSTRACT['15I]Monoiodo-and [la5I]diiodo-a-bungarotoxin were synthesized and shown to bind specifically to the acetylcholine receptor of cultured embryonic chickand rat-muscle cells. The pharmacologic properties of the receptor of cultured embryonic chick muscle resembled those of the nicotinic acetylcholine receptor of adult vertebrate muscle. Autoradiography of muscle cells labeled with toxin showed that acetyleholine receptors were distributed over the entire cell surface. In addition, discrete areas with a high receptor concentration were found.a-Bungarotoxin, a protein of known amino-acid-sequence (1) obtained from the venom of the Formosan banded krait, Bungarus multicinctus, and similar proteins from cobra and other elapid snake venoms bind with high specificity to acetylcholine receptors of striated muscle (2-8), electroplax (9-11), and brain (12). In this way, neurotoxins such as abungarotoxin inhibit the response of certain cells to acetylcholine.Acetylcholine receptors are distributed over the entire surface of embryonic and neonatal striated muscle cells of vertebrates (7, 13); however, in adult innervated muscle, the acetylcholine receptors are restricted to the area of the synapse (14, 15). Since the binding of a-bungarotoxin to the acetylcholine receptor of muscle is not readily irreversible in the systems examined, labeled a-bungarotoxin can be used to assay acetylcholine-receptor concentration as well as sites of synaptic connections (4-12).The purpose of this communication is to show that [125"]I a-bungarotoxin binds to the acetylcholine receptor of cultured embryonic chick-and rat-muscle cells, and to describe the properties of this receptor. Fractionation of Monoiodo-and Diiodo-a-bungarotoxin. The solution of iodinated toxin was diluted 2-fold with a solution containing 2 mg of albumin per ml of H20 and adsorbed onto a 1.0-ml column of CM-Sephadex (C-50) equilibrated with solution B (3.3 mM sodium phosphate buffer, pH 7.4; 2 mg of albumin per ml). The column was washed with solution B and the column effluent (peak I, Fig. 1), containing 3-10% of the applied radioactivity, was discarded. Only 20-30% of this material was precipitated by 10% Cl3CCOOH. The iodinated toxin was eluted (peaks II and III, Fig. 1) with a linear gradient consisting of 40 ml of solution B and 40 ml of solution B containing 80 mM NaCl. More than 90% of the radioactivity of peaks II and III was precipitated by 10% CI3CCOOH. METHODS

No abstract