Svensson Bengt, Nitu Florentin R, Rebbeck Robyn T, McGurran Lindsey M, Oda Tetsuro, Thomas David D, Bers Donald M, Cornea Razvan L
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis.
Department of Pharmacology, University of California, Davis.
bioRxiv. 2023 Jul 8:2023.07.07.548138. doi: 10.1101/2023.07.07.548138.
Ca leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide, (DPc10) detects leaky RyR2 in cardiomyocytes. Conversely, calmodulin (CaM) inhibits RyR2 leak. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. Here we used FRET to understand the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca , DPc10 decreased both FRET (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca , DPc10 decreased FRET without affecting CaM/RyR2 binding affinity. This correlates with analysis of fluorescence-lifetime-detected FRET indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca and lengthens D-FKBP/A-CaM distances independent of [Ca ]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, this effect being larger in micromolar vs. nanomolar Ca . Moreover, A-DPc10/RyR2 binding is cooperative in CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.
心肌细胞肌浆网（SR）通过过度活跃的静息心肌兰尼碱受体通道（RyR2）发生钙泄漏会促发心律失常。一种外源性肽（DPc10）可检测心肌细胞中泄漏的RyR2。相反，钙调蛋白（CaM）可抑制RyR2泄漏。这些观察结果促使人们设计一种用于靶向RyR2的药物发现的荧光共振能量转移（FRET）生物传感器。在此，我们利用FRET来了解在SR囊泡中结合RyR2时驱动DPc10-CaM相互依赖性的分子机制。我们使用供体-FKBP12.6（D-FKBP）来解析受体-CaM（A-CaM）与RyR2的结合。在低纳摩尔钙浓度下，DPc10降低了FRET（在饱和[A-CaM]情况下）以及CaM/RyR2的结合亲和力。在微摩尔钙浓度下，DPc10降低了FRET，但不影响CaM/RyR2的结合亲和力。这与荧光寿命检测的FRET分析结果相关，表明DPc10在纳摩尔（而非微摩尔）钙浓度下降低了RyR2钙调蛋白结合位点的占有率，并延长了D-FKBP/A-CaM的距离，且与[Ca]无关。为了观察DPc10/RyR2的结合，我们使用了受体-DPc10（A-DPc10）。CaM会削弱A-DPc10/RyR2的结合，这种效应在微摩尔钙浓度下比在纳摩尔钙浓度下更大。此外，A-DPc10/RyR2的结合以CaM和FKBP依赖的方式协同进行，这表明两种内源性调节剂都促进了RyR2亚基之间协调的结构变化以进行通道调节。借助冷冻电镜结构分析，这些见解为针对RyR2的治疗发现的DPc10-CaM范式的进一步发展提供了信息。
