兰尼碱受体新型穿膜肽配体calcin家族的结构-功能关系及其对儿茶酚氨多形性室性心动过速的拮抗效应

Calcin is a set of globular cell-penetrating peptide ligands of ryanodine receptors (RyRs) with high affinity and exquisite selectivity. The defining characteristic of calcins is their capacity to stabilize RyR openings in a long-lasting subconducting state. They show a wide range of activities from 0.3 nM to 375 nM, therefore displaying a great potential to treat RyR-related diseases. Previously, we have provided a comprehensive analysis of the structure–function relationships of the nine known calcins, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs. Using the RyR2-R4496C CPVT mouse model, we further confirmed the inhibition of Imperacalcin (the 1st calcin) on spontaneous Ca2+ releases by partial depletion of Ca2+ from sarcoplasmic reticulum in molecular and cellular levels. In intact mice, our investigations showed that Imperacalcin significantly antagonized the moderate symptoms of CPVT like premature ventricular contraction and discontinuous bidirectional ventricular tachycardia, but not the serious symptoms continuous bidirectional/polymorphic ventricular tachycardia. In this study, our first aim is to dissect the pivotal amino acids, functional important amino acids, non-pivotal amino acids, as well as the motif distribution that determine RyR pharmacology and thus to analyze the structure-function relationships, and the interaction of calcin-RyRs using a serious of manually synthesized calcin mutants. Then, we will design and optimize the calcins from primiary sequence and spatial structure, which will be further selected to optimize their capacity to antagonize the bidirectional/polymorphic tachyarrhythmias using the RyR2-R4496C mutant mice from the molecular, cellular and intact mice levels, therefore, to develop a novel paradigm for the treatment of calcium-dependent arrhythmias like CPVT, results may also be applied to other cardiomyopathies where controlled unloading of SR Ca2+ may be desirable.

calcin是一组特异性激活兰尼碱受体（RyRs）的高亲和力蝎毒素穿膜多肽配体，活性0.3~375 nM，具有很好的应用于RyRs相关疾病的潜力。我们在前期研究8种天然calcin结构功能特性的基础上，从分子、细胞与整体动物三层面证实了代表性Imperacalcin具有主动释放并部分耗竭肌浆网Ca2+，抑制心肌细胞自发性Ca2+释放，拮抗以RyR2-R4496C突变小鼠为模型的CPVT室性期前收缩、非连续性双向室速等轻、中度症状的效应，但对连续性双向/多形性室速等严重症状的作用则相对有限。因此，本课题我们拟人工获取系列突变calcin，通过活性检测与生物信息学分析明确关键氨基酸、影响功能的重要氨基酸、非关键氨基酸以及模体片段信息等阐述其结构功能关系的同时对calcin进行人工优化，验证并提升其对CPVT的拮抗效应，期望筛选出活性更强、更具治疗CPVT等Ca2+紊乱性心律失常潜力的先导多肽。

心血管疾病是我国居民疾病死亡的首要原因，而Ca2+紊乱性心律失常是最常见的难治性心血管疾病之一。蝎毒素Calcin多肽具有通过作用于心肌细胞RyRs部分释放肌浆网Ca2+达到拮抗Ca2+紊乱性心律失常的潜力。围绕Calcin项目组开展了系列工作：（1）先后采集国内外20个不同地区的蝎子样品开展转录组测序工作，筛选到12条全新的天然Calcin序列，并进行了结构功能预测分析。（2）同步构建了重组表达和化学合成的Calcin人工获取方法，并最终选择化学合成的方法开展了Calcin的线性肽、折叠肽以及突变肽的合成与鉴定工作，并检测了Calcin的生物活性。（3）根据Calcin的特征性模体序列，先后合成了8条Calcin部分肽段，探究了Calcin部分肽段的穿膜特性，发现其中有2条多肽FITC-OpiCa1 17-33和FITC-OpiCa1 23-33与经典的TAT穿膜肽相当甚至更佳的穿膜效果，有望成为基于蝎毒素Calcin的新型穿膜多肽而进行广泛的应用研究。（4）项目组开展了PLGA-Tetracaine-Calcin复合纳米颗粒的系列合成与表征工作，该工作为进一步提升Calcin对RyRs相关的Ca2+紊乱性心律失常的拮抗效力提供了新的方向。在项目执行期间先后发表SCI论文11篇，其中第1或通讯（共同）作者10篇；发表中文综述1篇；授权国家发明专利1项，实用新型专利1项。培养博士研究生1名，硕士研究生3名。