Calcins as Membrane-permeable Ligands of Ryanodine Receptors

描述（由申请人提供）：Ryanodine受体（RyR）是肌浆网Ca2+释放通道，在可兴奋和不可兴奋细胞的Ca2+信号传导中发挥关键作用。RyR的名字源于这样一个事实，即它们的特征很大程度上要归功于ryanodine，ryanodine是一种以高亲和力和特异性结合RyR的植物生物碱。Ryanodine一直是RyRs的宝贵配体，但其功能作用复杂，阻碍了其在细胞研究中的应用。在寻找新的配体，可以克服一些功能和结构上的缺点ryanodine，我们发现在选定的蝎子的毒液一组肽毒素，称为钙蛋白，显示高亲和力和精致的选择性对RyRs。钙蛋白的定义特征是它们在长期亚导电状态下稳定RyR开口的能力。这种作用与兰尼定的作用几乎类似，但与兰尼定不同的是，钙蛋白与RyR快速结合（快速结合速率），从其结合位点自由解离（可逆作用），显示剂量和序列可变效应，并且易于衍生化而不会发生受体亲和力的重大损失。钙蛋白还以显著的速度和几种程度的效力调节完整心肌细胞中的细胞内Ca 2+，从而作为第一个细胞穿透肽（CPP）RyR特异性高动态范围的Ca 2+动员剂进入该领域。这项研究计划将首先表征，然后利用这组新的肽毒素，在分子，细胞和整个心脏水平上揭示RyR功能的基本机制。我们的多学科计划，具有明确的可交付成果和里程碑，可能包含两个具体目标。在第一个目标中，我们将首先确定和修改参与RyR识别和细胞渗透的钙蛋白的结构域，以产生一组功能多样的CPP，其能够以宽的动态范围调节RyR功能并将货物递送到心肌细胞内部。在第二个目标中，我们将使用天然和重组RyR，在完整的心肌细胞，和Langendorff灌注工作的心脏，以创建急性或持续的RyR活动过度，并揭示RyR门控，Ca2+触发的心律失常和电机械交替的机制。这些研究将使用心室心肌细胞作为表征钙蛋白的细胞模型，但我们的最终目标是为科学界产生一组功能多样的CPP，这些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.