Conformational change of the foot protein of sarcoplasmic reticulum as an initial event of calcium release.
Heavy sarcoplasmic reticulum vesicles were labeled with the thiol-reacting fluorescent probe N-(7-dimethylamino-4-methyl-4-coumarinyl)maleimide (DACM), and the DACM-labeled foot protein moiety was purified. The fluorescence intensity of the DACM attached to the foot protein decreased by the addition of low (activating) concentrations of ryanodine, while it increased at higher (inhibitory) concentrations, suggesting that the lower fluorescence represents the active state of the foot protein, while the higher fluorescence, its inactive state. Under conditions that induce Ca2+ release from SR (Ca2+ jump, addition of Ca2+ release inducing reagents such as caffeine and polylysine), the fluorescence intensity of the protein-attached DACM decreased rapidly (e.g. k congruent to 70 s-1 under optimum conditions). The initial rate of Ca2+ release from the DACM-labeled SR showed a close correlation with the amplitude of the fluorescence change of the foot protein-attached DACM under variety of conditions; e.g. in the presence of Ca2+, polylysine, ATP, and ruthenium red, etc. The fluorescence change of the foot protein was much faster than Ca2+ release from SR under a variety of conditions of Ca2+ release. We propose that the binding of release triggering reagents to the foot protein induces a rapid conformational change, which in turn regulates Ca2+ release.
Ohkusa
T
T
Department of Muscle Research, Boston Biomedical Research Institute, MA 02114.
Kang
J J
JJ
Morii
M
M
Ikemoto
N
N
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008301:Maleimides; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D011107:Polylysine / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D005456:Fluorescent Dyes; D008301:Maleimides; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D011107:Polylysine; D002110:Caffeine; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D002118:Calcium