Treatments that extract the 43K protein from acetylcholine receptor clusters modify the conformation of cytoplasmic domains of all subunits of the receptor.
The nicotinic acetylcholine receptor (AChR) of Torpedo electric organ and vertebrate skeletal muscle is closely associated with a Mr 43,000 protein (43K). In this study, we have examined the effects on the AChR of treatments which remove the 43K protein. We used semiquantitative fluorescence techniques to measure the binding of antibodies to clustered AChR in cultured rat myotubes. We found that labeling by antibodies to the cytoplasmic portions of each of the four receptor polypeptides increased significantly upon extraction of the 43K protein. Labeling by an antibody to an extracellular epitope of the alpha subunits was not affected by removal of the 43K protein, suggesting that changes were restricted to the cytoplasmic domains of the AChR. Increases in labeling by antibodies were more limited following protease treatment, which removes most cytoskeletal structures but leaves the 43K protein bound to the membrane. Competition between an antibody to the beta subunit and an antibody to the gamma and delta subunits suggests that the cytoplasmic portion of the AChR still retains a degree of native structure in the absence of the 43K protein. Our results suggest that, although some of these changes may be due to simply exposing additional epitopes on the AChR, the cytoplasmic portions of all the subunits of the AChR undergo significant conformational changes upon extraction of the 43K protein.
Krikorian
J G
JG
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201.
Bloch
R J
RJ
D000818:Animals; D000918:Antibody Specificity; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry*; D014101:Torpedo
D008565:Membrane Proteins; D009124:Muscle Proteins; D011978:Receptors, Nicotinic