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

1.用不同浓度(10 nM~(-1)mM)的游离Ca~(2+)和Gt~(2+)研究了氟烷对天然和FKBP耗竭的骨骼肌肌浆网(SkSR)组分[~(3 H)]ryanodine结合的影响。总的来说，FKBP耗竭的SkSR的[Ca^&lt；2+&gt；]_&lt；Free&gt；，[^~3H]ryanodine结合量显著高于天然SR(2-2.5倍)。氟烷的加入略微(高达20%)增强了[~(3 H)]Ryanodine与天然SkSR的结合。在FKBP耗竭的SkSR中也观察到了这一现象，但在接近生理的[Ca^&lt；2+&gt；]_&gt；的100 nM处，Ryanodine结合开放RyR的70%的Ryanodine结合增强了[^~3H]ryanodine的结合，因此我们的结果表明，从RYR1中去除FKBP12显著增加了在生理[Ca^&lt；2+&gt；]_&lt；氟烷对天然和FKBP缺失的SKS…钙离子负荷和释放的影响在SR悬浮液中加入ATP和Ca~(2+)&gt；可启动兔天然SkSR和FKBP耗竭SkSR的更多R-ATP能量负荷。与天然SR相比，FKBP耗尽的SkSR的加载速度要慢一些。当[Ca^&lt；2+；gt；]降至基线后加入氟烷时，可观察到SR囊泡中的Ca^&lt；2+&gt；释放。在原生SkSR中，[Ca^&lt；2+]降至基线。相反，在FKBP耗竭的SkSR中，[Ca^&lt；2+&gt；]的下降很小，并达到了显著高于基线水平的平台期。当在平台期加入将RyR锁定在关闭状态的药物钌红时，[Ca^&lt；2+]重新开始下降，并达到基线水平。这些结果提示，FKBP12的缺失使RYR1对氟烷更加敏感，延长了药物对通道的激活。犬以外物种的RYR2与FKBP12的相互作用-据报道，不仅FKBP12.6，而且FKBP12也与狗以外的各种物种的心脏SR(CSR)结合，因此这些动物中的RYR2有望与FKBP12结合。我们通过免疫共沉淀和(His)_6FKBP12的Pull-down实验，研究了FKBP12与CSR的结合是否与RYR2紧密相关。FKBP12和FKBP12.6与大鼠、小鼠和兔的FKBP CSR结合。然而，即使在CSR与高水平(10μM)的FKBP12孵育后，FKBP12与从FKBP12结合的CSR中产生的RYR2免疫共沉淀也没有关联。反之，RYR2既不与FKBP12共沉淀，也不与(His)_6FKBP12共纯化。我们的结果表明FKBP12与CSR结合，但不与RYR2紧密结合。FKBP12可能不是与RYR2结合，而是与CSR膜的其他组分结合，或者FKBP12与RYR2的相互作用具有较快的开关速度。较少

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