scholarly journals Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells.

1985 ◽  
Vol 100 (5) ◽  
pp. 1698-1705 ◽  
Author(s):  
H B Peng ◽  
S C Froehner
Keyword(s):  
Acetylcholine Receptor ◽  
Binding Sites ◽  
Electric Organ ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Latex Beads ◽  
Peripheral Membrane Protein ◽  
Double Label ◽  
Torpedo Electric Organ ◽  
Receptor Clusters

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.

scholarly journals Increased expression of the 43-kD protein disrupts acetylcholine receptor clustering in myotubes

1993 ◽  
Vol 122 (1) ◽  
pp. 169-179 ◽  
Author(s):  
CM Yoshihara ◽  
ZW Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Cluster Formation ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Achr Cluster ◽  
Number Of Clusters ◽  
Vertebrate Muscle ◽  
Peripheral Membrane Protein ◽  
Achr Clustering

The 43-kD protein is a peripheral membrane protein that is in approximately 1:1 stoichiometry with the acetylcholine receptor (AChR) in vertebrate muscle cells and colocalizes with it in the postsynaptic membrane. To investigate the role of the 43-kD protein in AChR clustering, we have isolated C2 muscle cell lines in which some cells overexpress the 43-kD protein. We find that myotubes with increased levels of the 43-kD protein have small AChR clusters and that those with the highest levels of expression have a drastically reduced number of clusters. Our results suggest that the 1:1 stoichiometry of AChR and 43-kD protein found in muscle cells is important for AChR cluster formation.

scholarly journals 300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

1987 ◽  
Vol 104 (4) ◽  
pp. 939-946 ◽  
Author(s):  
ML Woodruff ◽  
J Theriot ◽  
SJ Burden
Keyword(s):  
Acetylcholine Receptor ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Synaptic Membrane ◽  
Binding Assays ◽  
Western Blots ◽  
Neuromuscular Synapses ◽  
Peripheral Membrane Proteins ◽  
Torpedo Electric Organ ◽  
Skeletal Muscle Antibodies

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.

scholarly journals Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides.

1982 ◽  
Vol 92 (2) ◽  
pp. 333-342 ◽  
Author(s):  
P A St John ◽  
S C Froehner ◽  
D A Goodenough ◽  
J B Cohen
Keyword(s):  
Binding Sites ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Relative Molecular Mass ◽  
Alkaline Extraction ◽  
Before And After ◽  
Torpedo Electric Organ ◽  
Extracellular Side ◽  
Alpha Bungarotoxin

Experiments were conducted to examine the topographic arrangement of the polypeptides of the acetylcholine receptor (AcChR) and the nonreceptor Mr 43,000 protein in postsynaptic membranes isolated from Torpedo electric organ. When examined by electron microscopy, greater than 85% of vesicles were not permeable to ferritin or lactoperoxidase (LPO). Exposure to saponin was identified as a suitable procedure to permeabilize the vesicles to macromolecules with minimal alteration of vesicle size or ultrastructure. The sidedness of vesicles was examined morphologically and biochemically. Comparison of the distribution of intramembrane particles on freeze-fractured vesicles and the distribution found in situ indicated that greater than 85% of the vesicles were extracellular-side out. Vesicles labeled with alpha-bungarotoxin (alpha-Bgtx) were reacted with antibodies against alpha-BgTx or against purified AcChR of Torpedo. Bound antibodies were detected by the use of ferritin-conjugated goat anti-rabbit antibody and were located on the outside of greater than 99% of labeled vesicles. Similar results were obtained for normal vesicles or vesicles exposed to saponin. Quantification of the amount of [3H]-alpha-BgTx bound to vesicles before and after they were made permeable with saponin indicated that less than 5% of alpha-BgTx binding sites were cryptic in normal vesicles. It was concluded that greater than 95% of postsynaptic membranes were oriented extracellular-side out. LPO-catalyzed radioiodinations were performed on normal and saponin-treated vesicles and on vesicles from which the Mr (relative molecular mass) 43,000 protein had been removed by alkaline extraction. In normal vesicles, polypeptides of the AcChR were iodinated while the Mr 43,000 protein was not. In vesicles made permeable with saponin, the pattern of labeling of AcChR polypeptides was unchanged, but the Mr 43,000 protein was heavily iodinated. The relative iodination of AcChR polypeptides was unchanged in membranes equilibrated with agonist or with alpha-BgTx or after alkaline-extraction. It was concluded that the Mr 43,000 protein is present on the intracellular surface of the postsynaptic membrane and that AcChR polypeptides are exposed on the extracellular surface.

scholarly journals Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein.

1991 ◽  
Vol 115 (2) ◽  
pp. 435-446 ◽  
Author(s):  
R J Bloch ◽  
W G Resneck ◽  
A O'Neill ◽  
J Strong ◽  
D W Pumplin
Keyword(s):  
Acetylcholine Receptors ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Membrane Domains ◽  
Intact Cells ◽  
Fluorescence Measurements ◽  
Peripheral Membrane Proteins ◽  
Vertebrate Muscle ◽  
Torpedo Electric Organ ◽  
Receptor Clusters

A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.

Formation and dispersal of acetylcholine receptor clusters in muscle cells

1986 ◽  
Vol 9 ◽  
pp. 125-129 ◽  
Author(s):  
H. Benjamin Peng ◽  
Mu-ming Poo
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Receptor Clusters

scholarly journals Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters.

1984 ◽  
Vol 98 (2) ◽  
pp. 550-557 ◽  
Author(s):  
H B Peng
Keyword(s):  
Acetylcholine Receptor ◽  
Divalent Cations ◽  
Simple Procedure ◽  
Latex Beads ◽  
Immunofluorescence Studies ◽  
Cellular Machinery ◽  
The Stability ◽  
Receptor Clusters ◽  
Myotomal Muscle ◽  
Achr Clustering

The formation of acetylcholine receptor (AChR) clusters can be experimentally induced in cultured Xenopus myotomal muscle cells by positive polypeptide-coated latex beads (Peng, H.B., P.-C. Cheng, and P.W. Luther, 1981, Nature [Lond.], 292:831-834). This provides a simple procedure for studying the cellular process of AChR clustering. In this study, the involvement of calcium and calmodulin in this process was examined. A deprivation in extracellular calcium by calcium-free medium or by the addition of calcium antagonists such as divalent cations Co2+ and Ni2+ (1-5 mM) or organic compounds verapamil and D-600 (0.1-0.5 mM) suppressed the formation of AChR clusters induced by the latex beads in a largely reversible manner. Antagonists against calmodulin, including trifluoperazine (1-5 microM) and the naphthalene sulfonamide W-7 (20 microM), also suppressed AChR clustering. However, the effect of W-7 was much weaker than that of trifluoperazine (TFP). Although the formation of AChR clusters is inhibited by these drugs, the stability of the existent clusters is relatively insensitive to them. These data suggest that the clustering of AChR involves a Ca2+ and calmodulin-activated process. Immunofluorescence studies using an antibody against calmodulin indicate that calmodulin is diffusely distributed in the cytoplasm in addition to its localization at the I-bands. Thus I propose that a local rise in intracellular calcium caused by a locally applied stimulus, exemplified here by the polypeptide-coated latex beads, may trigger the formation of AChR clusters. Furthermore, the cellular machinery for this process may involve calmodulin and is diffusely distributed in the muscle cell.

scholarly journals Development of Acetylcholine Receptor Clusters on Cultured Muscle Cells

1973 ◽  
Vol 70 (1) ◽  
pp. 270-274 ◽  
Author(s):  
A. J. Sytkowski ◽  
Z. Vogel ◽  
M. W. Nirenberg
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Receptor Clusters

scholarly journals Freeze-fracture and electrophysiological studies of newly developed acetylcholine receptors in Xenopus embryonic muscle cells.

1984 ◽  
Vol 98 (6) ◽  
pp. 2160-2173 ◽  
Author(s):  
P C Bridgman ◽  
S Nakajima ◽  
A S Greenberg ◽  
Y Nakajima
Keyword(s):  
Size Distribution ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Freeze Fracture ◽  
Muscle Cells ◽  
Embryonic Muscle ◽  
Freeze Fracture Electron Microscopy ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin ◽  
Solitary Form

The development of acetylcholine receptors on Xenopus embryonic muscle cells both in culture and in situ was studied using electrophysiology and freeze-fracture electron microscopy. Acetylcholine sensitivity first appeared at developmental stage 20 and gradually increased up to about stage 31. Freeze-fracture of muscle cells that were nonsensitive to acetylcholine revealed diffusely distributed small P-face intramembraneous particles. When cells acquired sensitivity to acetylcholine, a different group of diffusely distributed large P-face particles began to appear. This group of particles was analyzed by subtracting the size distribution found on nonsensitive cells from that found on sensitive cells. We call this group of particles difference particles. The sizes of difference particles were large (peak diameter 11 nm). The density of difference particles gradually increased with development. The density of small particles (less than 9 nm) did not change with development. At later stages (32-36) aggregates of large particles appeared, which probably represent acetylcholine receptor clusters. The size distribution of difference particles was close to that of the aggregated particles, suggesting that at least part of difference particles represent diffusely distributed acetylcholine receptors. Difference particles exist mostly in solitary form (occasionally double), indicating that an acetylcholine receptor can be functional in solitary form. This result also shows that diffuse acetylcholine receptors that have previously been observed with 125I-alpha-bungarotoxin autoradiography do indeed exist in solitary forms not as microaggregates.

Sign in / Sign up

Export Citation Format

Share Document