| Project/Area Number |
07458168
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
KASAI Michiki GRADUATE SCHOOL ENGINEER.SCI., Osaka University, PROFESSOR, 基礎工学研究科, 教授 (40022595)
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| Co-Investigator(Kenkyū-buntansha) |
UYEDA Atsuko GRADUATE SCHOOL ENGINEER.SCI., Osaka University, ASSISTANT, 基礎工学研究科, 助手 (90252634)
IDE Toru GRADUATE SCHOOL ENGINEER.SCI., Osaka University, ASSISTANT, 基礎工学研究科, 助手 (60231148)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1996: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1995: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | Sarcoplasmic reticulum / Ryanodine receptor / Excitation-contraction coupling / Calcium channel / Calsequestrin / Triad / Depolarization / Fura-2 |
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| Research Abstract |
In order to clarify the molecular mechanism of excitation-contraction of skeletal muscle, intrinsic factors regulating excitation-contraction coupling was surveyed by using isolated sarcoplasmic reticulum (SR) vesicles. It is known that Ca^<2+> release channel (ryanodine receptor) opens by micromolar Ca^<2+> from outside the vesicles. We found that the channel became inexcitable when intravesicular Ca^<2+> concentration was lowered accompanying release of calsequestrin (CSQ). From the result, we concluded that CSQ regulates the ryanodine receptor channel. By using affinity column conjugated with CSQ,we found CSQ binds to 96-kDa triadin, 30-kDa DIDS binding protein, and 25-kDa junctin. By affinity column with 30-kDa protein, 30-kDa protein was found to bind to CSQ and junctin. As the result, we concluded that 3 proteins make ternary complex and regulate Ca^<2+> release from SR.
Next, triads, ternary complex of transverse tubular membrane (TTM) and SR,were purified and Kinetic studies of ca^<2+> release from SR was carried out. depolarization of TTM was caused by ionic replacement, and Ca^<2+> release was assayd by using Fura-2 fluorescence. Ca^<2+> release curve could be divided into two phases. In contrast to other previous studies, in the fast phase the amount of released Ca^<2+> increased with an increase in the magnitude of depolarization but the release rate did not ; on the other hand, in the slow phase the Ca^<2+> release rate increased but the amount of Ca^<2+> did not. These dual kinetics of Ca^<2+> release were explained by the CSQ regulation model for the fast release phase.
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