Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Effects on the Ca2+ release channel.
The Ca2+ release channel and ryanodine receptor are activities copurifying with the 400,000-450,000 Da high molecular weight protein of cardiac and skeletal junctional sarcoplasmic reticulum. Calpain II, an endogenous cytosolic protease, was used to selectively degrade the high molecular weight protein in cardiac and skeletal muscle sarcoplasmic reticulum vesicles, and its effects on the activity of the Ca2+ release channel and [3H]ryanodine binding sites were analyzed. Degradation of the high molecular weight protein was associated with appearance of 315,000 and 150,000 Da proteolytic fragments and with a change in the ultrastructure of the "feet," extravesicular projections that protrude from the junctional sarcoplasmic reticulum membrane. The maximal number of [3H]ryanodine binding sites and the affinities of the sites for ryanodine were not remarkably affected by calpain II. Ca2+ release channels recorded from nondegraded cardiac and skeletal membrane vesicle preparations had slope conductances of 85 and 110 pS, respectively, measured with 1 microM cis-Ca2+ and 50 mM trans-Ba2+. Proteolysis did not alter the unitary channel conductances but did increase the percentage of channel open times from 36% to more than 90%. After proteolysis, channel opening remained dependent on micromolar cis-Ca2+, and high concentrations of ryanodine (300 microM) still blocked the channel. Our results suggest that proteolysis of the Ca2+ release channel with calpain II selectively impairs its inactivation, leaving its unitary conductance and the requirement for micromolar Ca2+ intact.
Rardon
D P
DP
Krannert Institute of Cardiology, Indiana University, School of Medicine, Indianapolis 46202.
Cefali
D C
DC
Mitchell
R D
RD
Seiler
S M
SM
Hathaway
D R
DR
Jones
L R
LR
D000818:Animals; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D002154:Calpain / Q000494:pharmacology*; D004285:Dogs; D006321:Heart / Q000187:drug effects*; D006652:Histological Techniques; D008854:Microscopy, Electron; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism; D009132:Muscles / Q000187:drug effects* / Q000648:ultrastructure; D009206:Myocardium / Q000648:ultrastructure; D010447:Peptide Hydrolases / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism
D015220:Calcium Channels; D009124:Muscle Proteins; D012433:Ryanodine; D010447:Peptide Hydrolases; D002154:Calpain