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 Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture.

1982 ◽  
Vol 92 (1) ◽  
pp. 231-236 ◽  
Author(s):  
J Prives ◽  
A B Fulton ◽  
S Penman ◽  
M P Daniels ◽  
C N Christian
Keyword(s):  
Cell Surface ◽  
Internal Structure ◽  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Cells In Culture ◽  
Detergent Extraction ◽  
Embryonic Muscle ◽  
Alpha Bungarotoxin ◽  
Triton X

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent alpha-bungarotoxin and extracted with Triton X-100, using conditions that preserve internal structure. A significant population of the AcCho receptors is retained on the skeletal framework remaining after detergent extraction. The skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction.

scholarly journals Localization and synthesis of the acetylcholine-binding site in the α-chain of the Torpedo californica acetylcholine receptor

1984 ◽  
Vol 224 (3) ◽  
pp. 995-1000 ◽  
Author(s):  
D J McCormick ◽  
M Z Atassi
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Synthetic Peptide ◽  
Solid Phase ◽  
Essential Elements ◽  
Binding Activity ◽  
Binding Assays ◽  
Free Peptide ◽  
Loop Region ◽  
Alpha Bungarotoxin

The sequence of the alpha-chain of the acetylcholine receptor of T. californica has been determined by recent cloning studies. The integrity of the disulphide bond between Cys-128 and cys-142 has been shown to be important for the maintenance of the binding activity of the receptor, thus implicating the regions around the disulphide bridge in binding with acetylcholine. In the present work, a synthetic peptide containing this loop region (residues 125-147) was synthesized. Solid-phase radiometric binding assays demonstrated a high binding of 125I-labelled alpha-bungarotoxin to the synthetic peptide. It was further shown that the free peptide bound well to [3H]acetylcholine. Additional experiments demonstrated that pretreatment of peptide 125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulphide structure was essential for binding. Unlabelled acetylcholine also inhibited the binding of labelled acetylcholine to the synthetic peptide. The region 125-147, therefore, contains essential elements of the acetylcholine binding site of the Torpedo receptor.

scholarly journals Transcripts for the acetylcholine receptor and acetylcholine esterase show distribution differences in cultured chick muscle cells.

1992 ◽  
Vol 118 (5) ◽  
pp. 1201-1212 ◽  
Author(s):  
K W Tsim ◽  
I Greenberg ◽  
M Rimer ◽  
W R Randall ◽  
M M Salpeter
Keyword(s):  
In Situ Hybridization ◽  
Acetylcholine Receptor ◽  
Gaussian Distribution ◽  
Muscle Cells ◽  
Skewed Distribution ◽  
Alpha Subunit ◽  
Subunit Mrna ◽  
Nuclear Label ◽  
The Mean

In situ hybridization of chick cultured muscle cells using exonic DNA probes for both AChR alpha-sub-unit and the catalytic subunit of AChE, revealed major differences in the distribution of label both over nuclei and in their surrounding cytoplasm, although some overlap in these distributions exists. For the AChR alpha-subunit there is a highly skewed distribution of labeled nuclei, with 35% of the nuclei being relatively inactive (less than 0.25 times the mean label) and approximately 10% being very heavily labeled (greater than 2.5 times the mean label). In contrast the nuclei labeled with the exonic probe for the AChE transcripts had a more Gaussian distribution, yet with some slight skewness in the direction of a few heavily labeled nuclei. There was also a difference in the cytoplasmic distribution of the label. The AChR alpha-subunit mRNA was mainly within 4 microns of labeled nuclei while the AChE mRNA was more widely distributed throughout the cytoplasm, possibly within a 10 microns rim around labeled nuclei. An intronic probe for the AChE gave the identical distribution of nuclear label to that of the exonic probe (but without any cytoplasmic label). In addition, calibration of the technique indicated that per myotube the AChE transcript is about sixfold more abundant than the AChR alpha-subunit transcript.

scholarly journals Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

1991 ◽  
Vol 114 (4) ◽  
pp. 799-807 ◽  
Author(s):  
Y Gu ◽  
J R Forsayeth ◽  
S Verrall ◽  
X M Yu ◽  
Z W Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Surface Binding ◽  
Mouse Muscle ◽  
Cos Cells ◽  
Gamma Subunits ◽  
Alpha Beta ◽  
High Concentrations ◽  
Alpha Bungarotoxin ◽  
Transfection Mixture ◽  
In Cells

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.

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.

scholarly journals An acetylcholine receptor precursor alpha subunit that binds alpha-bungarotoxin but not d-tubocurare.

1986 ◽  
Vol 83 (2) ◽  
pp. 498-502 ◽  
Author(s):  
B. E. Carlin ◽  
J. C. Lawrence ◽  
J. M. Lindstrom ◽  
J. P. Merlie
Keyword(s):  
Acetylcholine Receptor ◽  
Alpha Subunit ◽  
Alpha Bungarotoxin
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