Disulfide linkage of biotin identifies a 106-kDa Ca2+ release channel in sarcoplasmic reticulum.
Reactive disulfide reagents (RDSs) with a biotin moiety have been synthesized and found to cause Ca2+ release from sarcoplasmic reticulum (SR) vesicles. The RDSs oxidize SH sites on SR proteins via a thiol-disulfide exchange, with the formation of mixed disulfide bonds between SR proteins and biotin. Biotinylated RDSs identified a 106-kDa protein which was purified by biotin-avidin chromatography. Disulfide reducing agents, like dithiothreitol, reverse the effect of RDSs and thus promoted active re-uptake of Ca2+ and dissociated biotin from the labeled protein indicating that biotin was covalently linked to the 106-kDa protein via a disulfide bond. Several lines of evidence indicate that this protein is not Ca2+, Mg2+-ATPase and is not a proteolytic fragment or a subunit of the 400-kDa Ca2+-ryanodine receptor complex (RRC). Monoclonal antibodies against the ATPase did not cross-react with the 106-kDa protein, and polyclonal antibodies against the 106-kDa did not cross-react with either the ATPase or the 400-kDa RRC. RDSs did not label the 400-kDa RRC with biotin. Linear sucrose gradients used to purify the RRC show that the 106-kDa protein migrated throughout 5-20% linear sucrose gradients, including the high sucrose density protein fractions containing 400-kDa RRC. Protease inhibitors diisopropylfluorophosphate used to prevent proteolysis of 400-kDa proteins did not alter the migration of 106-kDa in sucrose gradients nor the patterns of biotin labeling of the 106-kDa protein. Incorporation of highly purified 106-kDa protein (free of RRC) in planar bilayers revealed cationic channels with large Na+ (gNa+ = 375 +/- 15 pS) and Ca2+ (gCa2+ = 107.7 +/- 12 pS) conductances which were activated by micromolar [Ca2+]free or millimolar [ATP] and blocked by micromolar ruthenium red or millimolar [Mg2+]. Thus, the SR contains a sulfhydryl-activated 106-kDa Ca2+ channel with apparently similar characteristics to the 400-kDa "feet" proteins.
Zaidi
N F
NF
Department of Physiology, School of Medicine, University of Pittsburgh, Pennsylvania 15261.
Lagenaur
C F
CF
Hilkert
R J
RJ
Xiong
H
H
Abramson
J J
JJ
Salama
G
G
D000818:Animals; D001710:Biotin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D004220:Disulfides / Q000138:chemical synthesis / Q000494:pharmacology*; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D013329:Structure-Activity Relationship
D015220:Calcium Channels; D004220:Disulfides; D007473:Ion Channels; D008565:Membrane Proteins; D001710:Biotin; D002118:Calcium