Calcins as Membrane-permeable Ligands of Ryanodine Receptors

描述(申请人提供)：Ryanodine受体(RyR)是肌浆网钙释放通道，在可兴奋和不可兴奋细胞的钙信号转导中发挥关键作用。RyRs之所以得名，是因为它们的特性在很大程度上要归功于ryanodine，一种以高度亲和力和特异性与RyRs结合的植物生物碱。Ryanodine一直是RyRs的一种无价配体，但其功能作用复杂，阻碍了其在细胞研究中的应用。为了寻找新的配体来克服ryanodine的一些功能和结构上的缺陷，我们在选定的蝎子的毒液中发现了一组多肽毒素，称为Calcins，对RyRs具有高亲和力和良好的选择性。钙蛋白的决定性特征是它们在长期亚导状态下稳定RyR开口的能力。这种作用几乎与ryanodine相似，但与ryanodine不同的是，calcins与RyRs(快速结合率)结合迅速，自由地从其结合部位解离(可逆效应)，表现出剂量和序列可变的效应，并且可以进行衍生化，而不会造成受体亲和力的重大损失。Calcins还以惊人的速度和不同程度的效力调节完整心肌细胞内的钙离子，从而作为第一个高动态范围的细胞穿透肽(CPP)RyR特异性钙激动剂进入该领域。这项研究计划将首先确定这组新的多肽毒素的特征，然后开发这组新的多肽毒素，以揭示分子、细胞和整个心脏水平上RyR功能的基本机制。我们的多学科计划，有明确的交付成果和里程碑，可能包含两个具体目标。在第一个目标中，我们将首先鉴定和修饰参与RyR识别和细胞穿透的钙蛋白的结构域，以产生一组功能多样化的CPP，能够以广泛的动态范围调节RyR功能并将货物运送到心肌细胞内部。在第二个目标中，我们将在完整的心肌细胞和兰登多夫灌流的工作心脏中对天然和重组的RyR使用钙蛋白来创造急性或持续的RyR过度活动期，并揭示RyR门控、钙触发的心律失常和机电交替的机制。这些研究将使用心肌细胞作为钙蛋白表征的细胞模型，但我们的最终目标是为科学界产生一组功能不同的CPP，能够调节RyR功能并将货物运送到广泛的细胞内部。

DESCRIPTION (provided by applicant): Ryanodine receptors (RyR) are sarcoplasmic reticulum Ca2+ release channels that play a critical role in Ca2+ signaling of excitable and non-excitable cells. RyRs owe their name to the fact that they were characterized in great part thanks to ryanodine, a plant alkaloid that binds to RyRs with high affinity and specificity. Ryanodine has been an invaluable ligand of RyRs, but its functional effects are complex and hamper its use in cellular studies. In search of novel ligands that could overcome some of the functional and structural disadvantages of ryanodine, we found in the venom of selected scorpions a set of peptide toxins, termed calcins, displaying high affinity and exquisite selectivity against RyRs. The defining characteristic of calcins is their capacity to stabilize RyR openings in a long-lasting subconducting state. This effect is nearly analogous to that of ryanodine, but unlike ryanodine, calcins bind rapidly to RyRs (fast association rate), freely dissociate from their binding site (reversible effect), display a dose- and sequence-variable effect, and are amenable for derivatization without undergoing major loss in receptor affinity. Calcins also modulate intracellular Ca2+ in intact cardiomyocytes with remarkable speed and with several degrees of potency, thus entering the field as the first cell-penetrating peptides (CPP) RyR-specific Ca2+ mobilizer of high dynamic range. This research program will characterize first and then exploit this novel group of peptide toxins to unravel fundamental mechanisms of RyR function at the molecular, cellular and whole heart level. Our multidisciplinary program, with well defined deliverables and milestones, may be enveloped in two specific aims. In the first aim, we will first identify and modify the structural domains of calcins involved in RyR recognition and cell penetration to generate a group of functionally diverse CPPs capable of modulating RyR function with wide dynamic range and of delivering cargo to the interior of cardiomyocytes. In the second aim, we will use calcins on native and recombinant RyR, in intact cardiomyocytes, and Langendorff-perfused working hearts to create acute or sustained periods of RyR hyperactivity and reveal mechanisms of RyR gating, Ca2+-triggered arrhythmias and electromechanical alternans. These studies will use ventricular cardiomyocytes as the cell model for characterization of calcins, but our ultimate goal is to generate for the scientific community a group of functionally diverse CPPs capable of modulating RyR function and of carrying cargo to the interior of a wide range of cells.