Acetylcholine Receptors of Muscle Grown In Vitro

Abstract

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

Ultrastructure of acetylcholine receptor clusters on cultured muscle fibers.

“…The distribution of aBT-binding sites visualized by immunoperoxidase staining confirmed and extended the results of autoradiography with 1251-labeled aBT (3,9,10). Mature myotubes had a light brown stain over most of their surface, causing them to stand out in contrast to the substrate and fibroblasts (Figs.…”

“…4 b). Also, in cross sections, the submembrane regions were (3,9,10). Since these techniques could not resolve the ultrastructural distribution of the receptors, two distinct models could be used to interpret those results: (a) there were more receptors per unit area membrane in the clusters; and (b) the concentration per unit area was the same both within and outside of clusters, but folding of the plasma membrane within the clusters made the concentration appear higher.…”

“…The preparation and maintenance of the cultures has been previously described in detail (9,10). in brief, 10 l l-day old chick embryo thigh muscle was dissociated with collagenase.…”

“…ACh receptors on myotubes grown in culture from dissociated chick embryo skeletal muscle cells initially have an even distribution. However, as the myotubes mature, discrete regions with high receptor concentration (clusters) are formed (3,9,10). Analysis of these clusters may yield information on the mechanisms which serve to restrict the distribution of neurotransmitter receptors to (and within) the neuromuscular junction and other (5) synaptic connections.…”

Multiple forms of acetylcholinesterase in clonal muscle cells

“…[12sI] Diiodo-a-bungarotoxin ([12SI]o~-BT) was prepared according to the method [9]. The cell homogenates in solution A were assayed for the toxin binding by a Millipore filtration assay [10] : homogenates were incubated with 8 nM [12sI]a-BT at 37°C for 60 rain, diluted with detergent-free saline solution more than 300-fold, filtered and washed through Millipore EGWP filters, and counted in a Packard Gamma Scintillation Spectrometer 5375.…”