IMMUNOPHILIN FKBP MODULATES CA2+ RELEASE CHANNEL CLOSURE AND ADAPTATION IN HEART

钙离子诱导的细胞内钙释放(CICR)是一种普遍存在的 通过正离子放大钙离子刺激的钙离子信号传递过程 反馈。终止钙离子释放的信号的性质 从心肌肌浆网中分离出来仍难以捉摸。本研究 旨在检测免疫亲和素FK506结合蛋白 与兰尼定受体密切相关的12.6 KDa多肽(FKBP) (RyR)/Ca++释放通道在CICR的终止中起作用 心。使用共聚焦显微镜和钙离子指示剂Fluo-3 将RyRs的原位门控视觉化为“Ca++火花”。我们观察到 FK506或雷帕霉素对FKBP的抑制作用使短暂的钙火花转变为 在大鼠心室肌细胞中，长时间的“钙+”发光，增加了 RYRS的平均开放时间从14.6ms到93ms。两个级别的CA++火花 幅度，类似于RyRs全电导开口和次导开口， 也观察到了。在电压钳制细胞中，钙离子火花由 在存在的情况下，去偏振脉冲的持续时间增加了6.8倍 FK506。Ca~(++)火花发生频率不受 FK506。此外，FK506还增强和延长了 刺激[Ca~(++)]i瞬变和收缩，随后诱导 潜在致心律失常的[Ca++]i的自发振荡。 这些现场观测通过单通道记录得到扩展 或大鼠心脏RyRs掺入平面脂质双层。FK506延长 RyR开口的寿命，并诱导了 在[Ca~(++)]为0.1M和10M时，双层膜的亚电导态。 此外，在双层中，FK506几乎取消了RyR~适应， [Ca~(++)]阶跃升高引起的RyR活性自发衰减 (0.1~10微米)，由钙离子笼状化合物光解产生 NP-EGTA。总之，我们得出结论，通过促进关闭，FKBP 并使RyR适应持续的Ca++刺激，可能会负担得起 一种内在机制，终止RyR开口，从而施加 心肌细胞CICR的负反馈调节。

Ca++-induced Ca++ release (CICR) from intracellular stores is a ubiquitous Ca++ signalling process that amplifies a Ca++ stimulus via positive feedback. The nature of the signal that terminates the release of Ca++ from the cardiac sarcoplasmic reticulum has remained elusive. This study was intended to examine whether the immunophilin FK506-binding protein (FKBP), a 12.6 KDa peptide that is tightly associated to ryanodine receptor (RyR)/Ca++ release channel, plays a role in the termination of CICR in heart. Confocal microscopy and the Ca++ indicator fluo-3 were used to visualize the in situ gating of RyRs as "Ca++ sparks". We observed that inhibition of FKBP by FK506 or rapamycin turned brief Ca++ sparks into long-lasting "Ca++ glows" in rat ventricular myocytes, increasing the mean open time of RyRs from 14.6 to 93 ms. Ca++ sparks with two-level amplitudes, resembling RyRs full and sub-conductance openings, were also observed. In voltage-clamped cells, Ca++ sparks evoked by depolarization pulses had a 6.8-fold increase in duration in the presence of FK506. The frequency of Ca++ spark occurrence was unaffected by FK506. In addition, FK506 potentiated and prolonged the electrically stimulated [Ca++]i transient and contraction, and subsequently induced spontaneous oscillations of [Ca++]i that are potentially arrythmogenic. These observations in situ were extended by single-channel recordings or rat cardiac RyRs incorporated in planar lipid bilayers. FK506 prolonged the lifetime of RyR openings and induced the occurrence of sub-conductance states in bilayers at both 0.1 and 10 muMM [Ca++]. Furthermore, in bilayers, FK506 virtually abolished RyR ~adaptation~, the spontaneous decay of RyR activity elicited by step increases of [Ca++] (from 0.1 to 10 muM) produced by photolysis of Ca++-caged compound NP-EGTA. Altogether, we conclude that FKBP, by facilitating the closure of RyR and enabling RyR to adapt to sustained Ca++ stimuli, may afford an intrinsic mechanism to terminate RyR openings and thus to exert a negative feedback regulation of CICR in heart cells.