Residues 377-389 from the δ subunit ofTorpedo californica acetylcholine receptor are located in the cytoplasmic surface

1994 ◽  
Vol 13 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Bernardo Perez-Ramirez ◽  
Ana Iriarte ◽  
Marino Martinez-Carrion
Keyword(s):  
Acetylcholine Receptor ◽  
Cytoplasmic Surface

scholarly journals Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting.

1982 ◽  
Vol 92 (2) ◽  
pp. 514-522 ◽  
Author(s):  
R Sealock
Keyword(s):  
Acetylcholine Receptor ◽  
Tannic Acid ◽  
Cytoplasmic Membrane ◽  
Membrane Surfaces ◽  
Peripheral Membrane Proteins ◽  
Cytoplasmic Surface ◽  
Thin Section Electron Microscopy ◽  
Surface Image ◽  
Section Electron ◽  
Hydrophobic Portion

In this study, acetylcholine receptor-rich postsynaptic membranes from electric tissues of the electric rays Narcine brasiliensis and Torpedo californica are negatively contrasted for thin-section electron microscopy through the use of tannic acid. Both outer (extracellular) and inner (cytoplasmic) membrane surfaces are negatively contrasted, and can be studied together in transverse sections. The hydrophobic portion of the membrane appears as a thin (approximately 2 nm), strongly contrasted band. This band is the only image given by membrane regions which are devoid of acetylcholine receptor. In regions of high receptor density, however, both surfaces of the membrane are seen to bear or be associated with material which extends approximately 6.5 nm beyond the center of the bilayer. The material on the outer surface can be identified with the well-known extracellular portion of the receptor molecule. A major portion of the inner surface image is eliminated by extraction of the membranes at pH 11 to remove peripheral membrane proteins, principally the 43,000 Mr (43K) protein. The images thus suggest a cytoplasmic localization of the 43K protein, with its distribution being coextensive with that of the receptor. They also suggest that the 43K protein extends farther from the cytoplasmic surface than does the receptor.

scholarly journals Immunoelectron microscopy of acetylcholine receptors and 43 KD protein after rapid freezing, freeze-substitution, and low-temperature embedding in lowicryl K11M.

1991 ◽  
Vol 39 (5) ◽  
pp. 625-634 ◽  
Author(s):  
G W Phillips ◽  
P C Bridgman
Keyword(s):  
Monoclonal Antibodies ◽  
Low Temperature ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Uv Light ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Cytoplasmic Surface ◽  
Cytoplasmic Proteins ◽  
Anti Acetylcholine

To label intracellular determinants of the acetylcholine receptor and associated cytoplasmic proteins while preserving optimal ultrastructure, we developed a post-embedment labeling technique that uses rapid-frozen specimens and freeze-substitution without chemical fixatives. This procedure has been made possible through the use of a low-temperature resin (Lowicryl K11M) that can be polymerized with UV light at -60 degrees C. Rapid-frozen muscle cells were used to evaluate the preservation of structure, and Torpedo electroplaque cells and purified postsynaptic membranes were used to quantitatively evaluate the labeling specificity, efficiency, and resolution of the technique. The labeling efficiency of seven different monoclonal antibodies (MAb) to the acetylcholine receptor varied from 3-13%; there was a correlation between the degree of efficiency and the number of epitopes with which the antibodies reacted. The resolution of the technique was not sufficient to determine whether the anti-acetylcholine receptor MAb were bound to the cytoplasmic or the extracellular surface, but was sufficient to correctly determine the location of the receptor-associated 43 KD protein on the cytoplasmic surface.

scholarly journals Visualization of the cytoplasmic surface of Torpedo postsynaptic membranes by freeze-etch and immunoelectron microscopy.

1987 ◽  
Vol 105 (4) ◽  
pp. 1829-1846 ◽  
Author(s):  
P C Bridgman ◽  
C Carr ◽  
S E Pedersen ◽  
J B Cohen
Keyword(s):  
Monoclonal Antibodies ◽  
Acetylcholine Receptor ◽  
Immunoelectron Microscopy ◽  
Dense Array ◽  
Dense Particle ◽  
Membrane Surfaces ◽  
Cytoplasmic Surface ◽  
Direct Association ◽  
Particle Aggregates ◽  
Smooth Membrane

The synapse-specific Mr 43,000 protein (43K protein) and the acetylcholine receptor were visualized by freeze-etch immunoelectron microscopy in preparations of purified Torpedo postsynaptic membranes. Vesicles were immobilized on glass and then sheared open by sonication to expose the cytoplasmic surface. Membranes were labeled with monoclonal antibodies to the 43K protein or the acetylcholine receptor. The cytoplasmic surface was devoid of filamentous structure, and the 43K protein and the cytoplasmic projection of the acetylcholine receptor were associated with prominent surface particles. Acetylcholine receptor and 43K protein, in membrane surfaces in direct contact with glass coated with polyornithine, segregated into dense particle aggregates separated by smooth membrane patches, whereas those in contact with glass coated with Alcian Blue underwent little or no detectable rearrangement. After treatment of vesicles at alkaline pH to remove the 43K protein, the cytoplasmic surfaces were still covered by a dense array of particles that were more uniform in shape and appeared slightly shorter than those seen on unextracted membranes, but similar in height to the extracellular projection. Monoclonal antibodies to the acetylcholine receptor labeled these particles, while antibodies to 43K protein did not. We conclude that the 43K protein is in direct association with the receptor and that complexes of the receptor and 43K protein can undergo surface-induced lateral redistribution. In addition, the cytoplasmic projection of the acetylcholine receptor is sufficiently large to be readily detected by freeze-etch electron microscopy and is similar in height to the extracellular projection.

scholarly journals Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies.

1984 ◽  
Vol 98 (6) ◽  
pp. 2239-2244 ◽  
Author(s):  
R Sealock ◽  
B E Wray ◽  
S C Froehner
Keyword(s):  
Monoclonal Antibodies ◽  
Acetylcholine Receptor ◽  
Protein A ◽  
Spatial Relationship ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Postsynaptic Membrane ◽  
Specific Protein ◽  
Cytoplasmic Surface ◽  
Membrane Bound

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.

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