Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum.
Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.
Morii
M
M
Danko
S
S
Kim
D H
DH
Ikemoto
N
N
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008301:Maleimides / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D012430:Ruthenium Red / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D013748:Tetracaine / Q000494:pharmacology
D005456:Fluorescent Dyes; D007473:Ion Channels; D008301:Maleimides; D009124:Muscle Proteins; D013748:Tetracaine; D012430:Ruthenium Red; D002110:Caffeine; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D000255:Adenosine Triphosphate; D002118:Calcium