Interrelationship of the antigenic and toxin-binding sites of the acetylcholine receptor of chick cultured muscle cells

1980 ◽  
Vol 8 (6) ◽  
pp. 741-741 ◽  
Author(s):  
MARTIN BIRD ◽  
ROGER HARRISON ◽  
GEORGE G. LUNT
Keyword(s):  
Acetylcholine Receptor ◽  
Binding Sites ◽  
Muscle Cells ◽  
Toxin Binding

Acetylcholine Receptors of Cultured Muscle Cells Demonstrated with Ferritin-α-Bungarotoxin Conjugates

1974 ◽  
Vol 16 (2) ◽  
pp. 473-479
Author(s):  
B. T. HOURANI ◽  
B. F. TORAIN ◽  
M. P. HENKART ◽  
R. L. CARTER ◽  
V. T. MARCHESI ◽  
...  
Keyword(s):  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Muscle Cells ◽  
Muscle Fibres ◽  
Toxin Binding ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Small Clusters

α-Bungarotoxin-ferritin conjugates were used to visualize by freeze-fracture and thin-section electron microscopy toxin-binding sites, presumably acetylcholine (ACh) receptors, in membranes of muscle cells grown in tissue culture. Toxin conjugated to ferritin by a glutaraldehyde reaction and purified by column chromatography in a buffer of high ionic strength remains active in blocking the effect of iontophoretically applied ACh. The potency of the conjugates was decreased 5-10 times compared to native α-bungarotoxin. Toxin-ferritin conjugates were identified in small clusters which were not uniformly distributed over the surface membrane. Binding was inhibited by preincubation in D-tubocurare or unconjugated toxin. The relation of the clusters to the non-uniform distribution of ACh sensitivity and α-bungarotoxin binding on cultured muscle fibres is discussed.

scholarly journals BiP forms stable complexes with unassembled subunits of the acetylcholine receptor in transfected COS cells and in C2 muscle cells

1992 ◽  
Vol 117 (4) ◽  
pp. 841-847 ◽  
Author(s):  
JR Forsayeth ◽  
Y Gu ◽  
ZW Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Binding Activity ◽  
Alpha Subunit ◽  
Stable Complex ◽  
Cos Cells ◽  
Toxin Binding ◽  
Immunoglobulin Binding Protein ◽  
Alpha Bungarotoxin ◽  
Immunoglobulin Binding

We have investigated the role of the immunoglobulin-binding protein (BiP) in the folding and assembly of subunits of the acetylcholine receptor (AChR) in COS cells and in C2 muscle cells. Immunoprecipitation in COS cells showed that alpha, beta, and delta subunits are associated with BiP. In the case of the alpha subunit, which first folds to acquire toxin-binding activity and is then assembled with the other subunits to form the AChR, BiP was associated only with a form that is unassembled and does not bind alpha-bungarotoxin. Similar results were found in C2 cells. Although the alpha and beta subunits of the AChR are minor membrane proteins in C2 cells, they were prominent among the proteins immunoprecipitated by antibodies to BiP, suggesting that BiP could play a role in their maturation or folding. In pulse-chase experiments in C2 cells, however, labeled alpha subunit formed a stable complex with BiP that was first detected after most of the alpha subunit had acquired toxin-binding activity and whose amount continued to increase for several hours. These kinetics are not compatible with a role for the BiP complex in the folding or assembly pathway of the AChR, and suggest that BiP is associated with a misfolded form of the subunit that is slowly degraded.

scholarly journals Genetic variants of C2 muscle cells that are defective in synthesis of the alpha-subunit of the acetylcholine receptor.

1987 ◽  
Vol 105 (3) ◽  
pp. 1329-1336 ◽  
Author(s):  
R Black ◽  
D Goldman ◽  
S Hochschwender ◽  
J Lindstrom ◽  
Z W Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Genetic Variants ◽  
Muscle Cells ◽  
Binding Activity ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Wild Type ◽  
S1 Nuclease ◽  
Toxin Binding ◽  
Alpha Bungarotoxin

We have analyzed two genetic variants of C2 muscle cells that have reduced levels of binding activity for alpha-bungarotoxin and have found that both synthesize only low levels of the alpha-subunit of the acetylcholine receptor. In both variants the uptake of 22Na in response to carbachol is diminished in proportion to the reduction in toxin-binding activity. In addition, the kinetic and sedimentation properties of the residual toxin-binding activity in both is indistinguishable from that seen in wild-type cells. Immunoblotting experiments on extracts of the variants using subunit-specific antibodies to alpha- and beta-subunits of the acetylcholine receptor demonstrated that the beta-subunit was present, but failed to detect alpha-subunit. In both variants, the amount of alpha-subunit accumulated after a 5-min period of labeling with [35S]methionine was reduced by over 90%, leading to the conclusion that the alpha-subunit is synthesized at greatly reduced rates. Northern blot and S1 nuclease analysis showed no differences between the alpha-subunit mRNA in wild-type and variant cells.

scholarly journals Identification of regions involved in the binding of alpha-bungarotoxin to the human alpha7 neuronal nicotinic acetylcholine receptor using synthetic peptides

2003 ◽  
Vol 372 (2) ◽  
pp. 543-554 ◽  
Author(s):  
Martha MARINOU ◽  
Socrates J. TZARTOS
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Binding Sites ◽  
Synthetic Peptides ◽  
Extracellular Domain ◽  
Muscle Type ◽  
Nicotinic Acetylcholine ◽  
Toxin Binding ◽  
Α1 Subunit

The neuronal α7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin α-bungarotoxin (α-Bgt). Fine mapping of the α-Bgt-binding site on the human α7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human α7 subunit (residues 1–206). Screening of these peptides for 125I-α-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186–197, which exhibited the best 125I-α-Bgt binding, and one at residues 159–165, with weak toxin-binding capacity; these correspond, respectively, to loops C and IV of the agonist-binding site. Toxin binding to the α7(186–197) peptide was almost completely inhibited by unlabelled α-Bgt or d-tubocurarine. Alanine substitutions within the sequence 186–198 revealed a predominant contribution of aromatic and negatively charged residues to the binding site. This sequence is homologous to the α-Bgt binding site of the α1 subunit (residues 188–200 in Torpedo AChR). In competition experiments, the soluble peptides α7(186–197) and Torpedo α1(184–200) inhibited the binding of 125I-α-Bgt to the immobilized α7(186–197) peptide, to native Torpedo AChR, and to the extracellular domain of the human α1 subunit. These results suggest that the toxin-binding sites of the neuronal α7 and muscle-type AChRs bind to identical or overlapping sites on the α-Bgt molecule. In support of this, when synthetic α-Bgt peptides were tested for binding to the recombinant extracellular domains of the human α7 and α1 subunits, and to native Torpedo and α7 AChR, the results indicated that α-Bgt interacts with both neuronal and muscle-type AChRs through its central loop II and C-terminal tail.

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.

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