Surface topography analysis of the ryanodine receptor/junctional channel complex based on proteolysis sensitivity mapping.
The ryanodine receptor/junctional channel complex (JCC) has been isolated and identified morphologically as the foot structure spanning the gap between sarcoplasmic reticulum and the transverse tubule. Functionally, the JCC contains the calcium release channel which mediates calcium release in excitation-contraction coupling in skeletal muscle. The three-dimensional structure of the JCC, obtained by image analyses of electron micrographs, reveals two distinct faces and an efflux pathway indicated by a central pore with a diameter of approximately 20 A and four radial canals leading to the myoplasm. The JCC consists of four identical protomers, with Mr = 565,233 each. In the present study, proteolytic digestion of native JCC was carried out to obtain insight into its surface topography. Peptides generated by cleavages with endoproteinases Lys-C and Glu-C were separated by reverse-phase high pressure liquid chromatography and sequenced. Twenty-four cleavage sites have been identified and are equated to surface exposed peptides. Surface probability analysis, combined with protease sensitivity, has been used to provide independent information identifying sequences with high likelihood of appearing on the surface. This analysis also predicts sequences with low surface probability which may be buried within the hydrophobic core of the structure. Furthermore, regions of high surface probability, not cleaved by proteases, are potential candidates for surface-surface association and/or the hydrophilic internal calcium efflux pathway.
Marks
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Department of Cardiology, Children's Hospital, Boston, Massachusetts.
Fleischer
S
S
Tempst
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels / Q000648:ultrastructure*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011336:Probability; D011487:Protein Conformation; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000648:ultrastructure*; D019837:Ryanodine Receptor Calcium Release Channel
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel