Synthesis of acetylcholine receptor during differentiation of cultured embryonic muscle cells.

Abstract

Acetylcholine receptor, a component of the specialized muscle membrane, appears during the differentiation of embryonic myogenic cells in tissue culture. Demonstration of incorporation of the radioactive precursor L-[a5Simethionine into purified receptor polypeptides is presented as evidence for its synthesis de novo. The identity of the purified radioactive species is established by cosedimentation of the [35Sireceptor with [3H]a-toxin binding activity on sucrose gradients and by crossreaction with antiserum to purified acetylcholine receptor of Electrophorus electricus. Myogenic cells in tissue culture undergo morphological, functional, and biochemical alterations analogous to those observed in differentiating muscle tissue in situ (1). Mononucleate replicating cells derived from fetal skeletal muscle, when maintained in tissue culture, withdraw from the cell cycle, align, and subsequently fuse to form multinucleate fibers exhibiting cross-striations and spontaneous or induced contractions. The myogenic process is accompanied by accumulation of muscle specific isoenzymes, increases in the rates of synthesis of contractile proteins, and development of a specialized plasma membrane manifest by its chemical and electrical excitability. As in the case of contractile elements and specific enzymes, the specialized components of the membrane are elaborated during the period of cell fusion. One of the components of this membrane system is the acetylcholine (ACh) receptor. Until now, study of the accumulation and turnover of the receptor in cultured muscle cells has been limited to indirect methods using radioactively labeled a-neurotoxins as tags in vvo (2-5).The ACh receptor from fish electric tissue has been the subject of extensive biochemical, pharmacological, and immunological investigation (6) which has resulted in a technology applicable to the direct study of the metabolic activity of this protein in cultured cells. In Membrane Fraction from SS-Labeled Cells. Cells were removed from the culture dishes and suspended in PBS (line 1, Table 2). Whole cells, nuclei, and membrane fragments were collected by low-speed centrifugation at 1000 X g for 5 min. The pellet was homogenized in 8.5% sucrose 0.05 M Tris-HCl (pH 7.6) and the unbroken nuclei removed by lowspeed centrifugation. The nuclear pellet was treated in the same manner twice more, and the pooled supernatants containing membrane fragments (line 2, Table 2) were adjusted to 0.5 M KCl to aid in the removal of contractile proteins.Membrane fragments were collected by centrifugation at 100,000 X g for 1 hr. The pellet was extracted with 0.1 M Tris-HCl, 0.1 M NaCl, 1.0% Triton X-100, pH 8.0 for 1 hr at 4°. The extract was subsequently cleared by centrifugation at 100,000 X g for 1 hr, followed by filtration of the supernatant through a plug of glass wool (line 3, Table 2). Toxin Affinity Columns. Erabutoxin B was coupled to cyanogen bromide activated Sepharose 4B by the method of Ratner (8). Toxin was coupled to activated washed beads at a concentrati...

Evidence for degradation of the acetylcholine (nicotinic) receptor in skeletal muscle during the development of the chick embryo

“…Values of 16-18 h for intrinsic half-lives have been found with either denervated adult muscle [28] or developing non-innervated myotubes in vitro after labeling of the acetylcholine receptor molecule with &bungarotoxin [26,29,30] or incorporation of labelled amino acids [26,30].…”

Allosteric Receptors: From Electric Organ to Cognition

“…In particular, α-bungarotoxin allowed early on (in an electron microscopy study performed by my student Jean-Pierre Bourgeois in collaboration with Antoinette Ryter) the evaluation of the number of receptor molecules per unit of postsynaptic membrane surface, showing that their density is extremely high (approximately 15,000 μm 2 ) and persists, in the electric organ, several weeks after denervation (200,201). The snake α-toxin became also an exceptional tool in the hands of John Merlie, an American postdoctoral fellow who initiated a fruitful collaboration with François Gros' laboratory (202,203) and in the hands of Heinrich Betz (my third German postdoctoral fellow), who investigated the expression of the muscle receptor genes and their epigenetic repression by electrical activity during muscle development (204,205).…”

Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers.

“…In primary cultures of chick myotubes, both a low density uniform distribution and high density "clusters" have been revealed by optical autoradiography with '25I-labeled neurotoxins that specifically bind to AChR (2)(3)(4). The time course of synthesis and turnover of AChR in cultured myotubes has been studied with radioactive snake toxins (9,10) and with radioactive amino acid precursors (11).…”