| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
1. Effects of halothane on [^3H]ryanodine binding to native and FKBP-depleted skeletal muscle SR (SkSR) fraction [^3H]ryanodine binding study was performed against rabbit native and FKBP-depleted SkSR at various concentrations (10 nM-1 mM) of free Ca^<2+>. At all [Ca^<2+>]_<free>, [^3H]ryanodine binding were significantly (2-2.5 fold) higher in FKBP depleted SkSR compared to native SR. Addition of halothane slightly (up to 20 %) enhanced the [^3H]ryanodine binding to native SkSR throughout the [Ca^<2+>]_<free> examined. This was also observed in FKBP-depleted SkSR except at the [Ca^<2+>]_<free> of 100 nM, which is close to physiological [Ca^<2+>]_<free>, the [^3H]ryanodine binding was enhanced by 70 % Ryanodine binds to open RYR, thus our results suggest that removal of FKBP12 from RYR1 significantly increases the number of channels open upon stimulation by halothane at physiological [Ca^<2+>]_<free>.
2. Effects of halothane on Ca^<2+> loading and release of native and FKBP-depleted SkS
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R-ATP-energized Ca^<2+> loading of rabbit native and FKBP-depleted SkSR was initiated by adding ATP and Ca^<2+> into SR suspension. Ca^<2+> loading rate of FKBP-depleted SkSR was somehow slower compared to native SR. When halothane was added after the [Ca^<2+>] run down to baseline, Ca^<2+> release from the SR vesicles was observed. In native SkSR, [Ca^<2+>] ran down to baseline. In FKBP-depleted SkSR, in contrast, run down of [Ca^<2+>] was little and reached a plateau which was significantly higher than the baseline level. When ruthenium red, a drug that locks the RYR at the closed state, was added at the plateau phase, [Ca^<2+>] restarted running down and reached the baseline level. These results suggested that depletion of FKBP12 makes RYR1 more sensitive to halothane and the channel activation by the drug is prolonged.
3. Interaction of RYR2 of species other than dog with FKBP12-It has been reported that not only FKBP12.6 but also FKBP12 binds to cardiac SR (CSR) from various species other than dog including rat, mouse and rabbit, thus RYR2 in those animals are expected to bind FKBP12. We examined by co-immunoprecipitation and pull down assay of (His)_6FKBP12, whether FKBP12 bound to the CSR is tightly associated with RYR2. FKBP12 as well as FKBP12.6 bound to FKBP CSR of rat, mouse and rabbit. However, FKBP12 was not associated with RYR2 immunoprecipitated from FKBP12-bound CSR even after CSR was incubated with high (10 μM) of FKBP12. Vice versa, RYR2 was neither co-immunoprecipitated with FKBP12 nor co-purified with (His)_6FKBP12 by Ni-NTA. Our results suggested FKBP12 binds to CSR but does not tightly associate with RYR2. FKBP12 might bind not to RYR2 but other component of CSR membrane, or the interaction of FKBP12 with RYR2 is with rapid on-off rate. Less
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