Lumenal sites and C terminus accessibility of the skeletal muscle calcium release channel (ryanodine receptor).
The membrane topology of the skeletal muscle ryanodine receptor (RyR1) was investigated using site-directed antibodies directed against amino acid sequences 2804-2930, 4581-4640, 4860-4886, and 4941-5037. Ab(2804-2930) bound with identical affinity to either closed or permeabilized sarcoplasmic reticulum vesicles, confirming the cytoplasmic location of this segment. Ab(4581-4640) did not bind to closed vesicles but bound well to permeabilized vesicles, supporting a lumenal location for this segment. Ab(4860-4886) did not bind to closed vesicles but exhibited weak binding to the permeabilized vesicles, suggesting that a portion of the epitope may be exposed on the lumenal surface. The C-terminal antibody (Ab(4941-5037)) bound weakly to closed vesicles, and binding was not significantly enhanced by permeabilizing vesicles with low concentrations of non-denaturing detergent. However, the C-terminal antibodies bound efficiently to vesicles which were transiently incubated at alkaline pH or subjected to trypsinolysis, conditions where few of the vesicles were permeabilized. These results support a model for the membrane topology of the ryanodine receptor as proposed by Takeshima et al. (Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., and Numa, S. (1989) Nature 339, 439-445). The results also suggest that the native conformation of the C terminus is inaccessible to antibodies.
Grunwald
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Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill 27599-7260, USA.
Meissner
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D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D001666:Binding Sites, Antibody; D015220:Calcium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D009124:Muscle Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D018482:Muscle, Skeletal / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D014357:Trypsin
D000906:Antibodies; D015220:Calcium Channels; D009124:Muscle Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D014357:Trypsin