Structural and functional correlation of the trypsin-digested Ca2+ release channel of skeletal muscle sarcoplasmic reticulum.
The effect of trypsin digestion on the (i) fragmentation pattern, (ii) activity, (iii) [3H]ryanodine binding, and (iv) sedimentation behavior of the skeletal sarcoplasmic reticulum (SR) ryanodine receptor-Ca2+ release channel complex has been examined. Mild tryptic digestion of heavy, junctional-derived SR vesicles resulted in the rapid disappearance of the high molecular weight (Mr approximately 400,000) Ca2+ release channel protein on sodium dodecyl sulfate gels and appearance of bands of lower Mr upon immunoblot analysis, without an appreciable effect on [3H]ryanodine binding or the apparent S value (30 S) of the 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized channel complex. Further degradation to bands of Mr greater than 70,000 on immunoblots correlated with a reduction of channel size from 30 S to 10-15 S and loss of high affinity [3H]ryanodine binding to the trypsinized receptor, while low affinity [3H]ryanodine binding and [3H]ryanodine bound prior to digestion were retained. Parallel 45Ca2+ efflux measurements also indicated retention of the Ca2+, Mg2+, and ATP regulatory sites, although Ca2+-induced 45Ca2+ release rates were changed. In planar lipid bilayer-single channel measurements, addition of trypsin to the cytoplasmic side of the high conductance (100 pS in 50 mM Ca2+), Ca2+-activated SR Ca2+ channel initially increased the fraction of channel open time and was followed by a complete and irreversible loss of channel activity. Trypsin did not change the unitary conductance, and was without effect on single channel activity when added to the lumenal side of the channel.
Meissner
G
G
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7260.
Rousseau
E
E
Lai
F A
FA
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism*
D015220:Calcium Channels; D012433:Ryanodine; D000249:Adenosine Monophosphate; D014357:Trypsin; D008274:Magnesium