Urological Surgery Research Institute, Southwest Hospital, Third Military Medical University, Chongqing, China.
Cell Calcium. 2010 May;47(5):449-57. doi: 10.1016/j.ceca.2010.03.006. Epub 2010 May 7.
We examined the role and molecular mechanism of cADPR action on Ca(2+) spark properties in mouse bladder smooth muscle. Dialysis of cADPR with patch pipettes increased frequency and amplitude of spontaneous transient out currents (STOCs) to 6.1+/-0.87 currents/min from 1.2+/-0.36 currents/min (control) and to 179.8+/-48.7pA from 36.4+/-22.6pA (control), respectively, in wildtype (WT) cells, and the effects of cADPR on STOCs were significantly blocked by JVT-591, a RYR2 stabilizer. In contrast, no significant changes were observed in FKBP12.6 null cells. Further studies indicated that Ca(2+) spark properties were altered by cADPR in WT but not FKBP12.6 null cells, namely, Ca(2+) spark frequency was increased by about 3.4-fold, peak Ca(2+) (F/F0) increased to 1.72+/-0.57 from 1.56+/-0.13, size increased to 2.86+/-0.26 microM from 1.92+/-0.14 microM, rise time and half-time decay were prolonged 1.6-fold and 2.3-fold, respectively, in WT cells. Furthermore, in the presence of thapsigargin cADPR still altered Ca(2+) spark properties, and cADPR increased F/F0 without affecting Ca(2+) spark decay time in voltage clamping cells. Dissociation studies demonstrated that application of cADPR resulted in significant removal of FKBP12.6 proteins from sarcoplasmic reticulum (SR) microsomes, and that treatment of the RyR2 immunoprecipitation complexes with cADPR or FK506 disrupted the interaction between RyR2 and FKBP12.6. Finally, cADPR altered SR Ca(2+) load in WT myocytes but not in FKBP12.6-null myocytes. Taken together, these results suggest that Ca(2+) release induced by cADPR is mediated by FKBP12.6 proteins in mouse bladder smooth muscle.
我们研究了 cADPR 在小鼠膀胱平滑肌细胞 Ca(2+)火花性质中的作用和分子机制。用膜片钳微电极将 cADPR 注入细胞内，可使野生型（WT）细胞中的自发性瞬态外向电流（STOC）频率从 1.2+/-0.36 次/分钟增加到 6.1+/-0.87 次/分钟，幅度从 36.4+/-22.6pA 增加到 179.8+/-48.7pA；而 cADPR 对 STOC 的作用可被 RYR2 稳定剂 JVT-591 显著阻断。相比之下，在 FKBP12.6 缺失细胞中则没有观察到明显的变化。进一步的研究表明，cADPR 可改变 WT 细胞的 Ca(2+)火花性质，但对 FKBP12.6 缺失细胞则没有影响，即 Ca(2+)火花频率增加约 3.4 倍，峰值 Ca(2+)（F/F0）从 1.56+/-0.13 增加到 1.72+/-0.57，大小从 1.92+/-0.14 microM 增加到 2.86+/-0.26 microM，上升时间和半衰期衰减分别延长 1.6 倍和 2.3 倍。此外，在 thapsigargin 存在的情况下，cADPR 仍然改变 Ca(2+)火花性质，并且 cADPR 增加了 F/F0，而不影响电压钳制细胞中 Ca(2+)火花衰减时间。解离研究表明，应用 cADPR 可导致 FKBP12.6 蛋白从肌浆网（SR）微粒体中显著去除，并且 cADPR 或 FK506 处理 RyR2 免疫沉淀复合物会破坏 RyR2 与 FKBP12.6 之间的相互作用。最后，cADPR 改变了 WT 心肌细胞中的 SR Ca(2+)负荷，但对 FKBP12.6 缺失心肌细胞则没有影响。综上所述，这些结果表明，cADPR 诱导的 Ca(2+)释放是由小鼠膀胱平滑肌细胞中的 FKBP12.6 蛋白介导的。
