Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum.
Ca2+ release from heavy sarcoplasmic reticulum (SR) vesicles was induced by 2 mM caffeine, and the amount (A) and the rate constant (k) of Ca2+ release were investigated as a function of the extent of Ca2+ loading. Under both passive and active loading conditions, the A value increased monotonically in parallel to Ca2+ loading. On the other hand, k sharply increased at partial Ca2+ loading, and upon further loading, it decreased to a lower level. Since most of the intravesicular calcium appears to be bound to calsequestrin both under passive and under active loading conditions, these results suggest that the kinetic properties of induced Ca2+ release show significant variation depending upon how much calcium has been bound to calsequestrin at the time of the induction of Ca2+ release. An SR membrane segment consisting of the junctional face membrane (jfm) and attached calsequestrin (jfm-calsequestrin complex) was prepared. The covalently reacting thiol-specific conformational probe N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM) was incorporated into several proteins of the jfm, but not into calsequestrin. The fluorescence intensity of DACM increased with Ca2+. Upon dissociation of calsequestrin from the jfm by salt treatment, the DACM fluorescence change was abolished, while upon reassociation of calsequestrin by dilution of the salt it was partially restored. These results suggest that the events occurring in the jfm proteins are mediated via the attached calsequestrin rather than by a direct effect of Ca2+ on the jfm proteins. We propose that the [Ca2+]-dependent conformational changes of calsequestrin affect the jfm proteins and in turn regulate the Ca2+ channel functions.
Ikemoto
N
N
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Ronjat
M
M
Mészáros
L G
LG
Koshita
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002155:Calsequestrin / Q000378:metabolism*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D008301:Maleimides; D009124:Muscle Proteins / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D002155:Calsequestrin; D005456:Fluorescent Dyes; D008301:Maleimides; D009124:Muscle Proteins; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D002118:Calcium