Role of FKBP in the interaction between ryanodine raeptor/Ca^2 release channel and volatile anesthetics

1.氟烷对[^3H]ryanodine与天然和FKBP耗竭的骨骼肌SR（SkSR）组分结合的影响在不同浓度（10 nM-1 mM）的游离Ca^2+下，对兔天然和FKBP耗竭的SkSR进行了[^3H]ryanodine结合研究。在所有的[Ca^&lt;2+&gt;]_<free>中，[^3H]ryanodine在FKBP耗竭的SkSR中的结合显著高于天然SR（2-2.5倍）。在整个[Ca^&lt;2+]_检测中，氟烷的加入略微（高达20%）增强了[^3H]ryanodine与天然SkSR的结合<free>。这也在FKBP耗尽的SkSR中观察到，除了在<free>接近生理[Ca ^&lt;2 +&gt;]_的[Ca ^&lt;2 +&gt;]_为100 nM<free>时，[^3H]ryanodine结合被70%的Ryanodine与开放的RYR结合增强，因此我们的结果表明，从RYR 1中去除FKBP 12显著增加了在生理[Ca^&lt;2+&gt;]_下氟烷刺激时开放的通道数量<free>。氟烷对天然和去FKBP SkS钙离子负载和释放的影响 ...更多信息 将ATP和Ca^&lt;2 +&gt;加入SR悬浮液中，可启动兔天然和FKBP耗竭的SkSR的R-ATP激活Ca^&lt;2 +&gt;负荷。与天然SR相比，FKBP耗尽的SkSR的Ca^2+负载速率在某种程度上较慢。当[Ca^2+]降至基线后加入氟烷时，观察到SR囊泡中的Ca^2+释放。在天然SkSR中，[Ca^&lt;2+&gt;]下降至基线。相反，在FKBP耗尽的SkSR中，[Ca^2+]的下降很少，并达到显著高于基线水平的平台。当在平台期加入钌红（一种将RYR锁定在关闭状态的药物）时，[Ca^2+]重新开始下降并达到基线水平。这些结果表明，FKBP 12的缺失使RYR 1对氟烷更敏感，药物对通道的激活时间延长.犬以外物种的RYR 2与FKBP 12的相互作用-据报道，不仅FKBP 12.6，而且FKBP 12与犬以外的各种物种（包括大鼠、小鼠和兔）的心脏SR（CSR）结合，因此预期这些动物中的RYR 2与FKBP 12结合。我们通过免疫共沉淀和（His）_6FKBP 12的pull down试验检测了FKBP 12与CSR的结合是否与RYR 2紧密相关。FKBP 12和FKBP 12.6与大鼠、小鼠和兔的FKBP CSR结合。然而，即使在CSR与高浓度（10 μM）FKBP 12孵育后，FKBP 12也不与从FKBP 12结合的CSR免疫沉淀的RYR 2相关。反之亦然，RYR 2既不与FKBP 12共沉淀，也不与（His）_6FKBP 12共纯化。我们的结果表明FKBP 12与CSR结合，但与RYR 2不紧密结合。FKBP 12可能不与RYR 2结合，而是与CSR膜的其他成分结合，或者FKBP 12与RYR 2的相互作用具有快速的开关速率。少

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 … More 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