Rational Design from Cryo-EM Structures of High-Affinity Ryanodine Receptor Ligands Based on Natural Peptides

总结 Ryanodine受体（RyRs）是肌浆网（SR）的Ca ~（2+）释放通道，在肌浆网的钙通道中起重要作用。 在心脏和骨骼肌细胞的兴奋-收缩偶联中起重要作用。心脏RyR基因突变 （RYR 2）与儿茶酚胺能多形性室性心动过速（CPVT）有关，CPVT是一种致心律失常性心动过速。 以肾上腺素能介导的室性心动过速和猝死为特征的综合征。 CPVT显然是一种由RyR 2引起的细胞内Ca 2+处理不当引起的致瘤性疾病 功能障碍因此，从CPVT综合征中获得的见解有助于我们对其他疾病的机械理解。 RyR 2连接的Ca 2+处理不当起关键作用的心肌病。RyR 2功能障碍导致 通过诱导SR Ca 2+“泄漏”和/或自发Ca 2+释放（SCR），心脏突然舒张， Ca 2+浓度达到临界值，触发了脑电活动。因此，控制RyR 2过度活性以减轻SR Ca 2 + “泄漏”和避免SCR是主要心肌病治疗方案的主要目标。一群球状的 称为钙蛋白的肽以高亲和力和特异性靶向RyR。Imperacalcin（IpCa）， 钙蛋白家族通过进入RyR的宽前庭并精确地结合到一个位点， 在通道的胞质“帽”深处，邻近跨膜区，充当“推动” 横向的跨膜螺旋线的通道孔，使它打开，并产生一个长期的 亚电导态在CPVT的动物模型中，IpCa穿透心室肌细胞的外膜 并诱导SR Ca 2+的部分耗尽，从而防止SCR并有效地缓解肾上腺素介导的 Ca 2+超载触发Ca 2+依赖性心律失常。因此，IpCa天然地表现为RyR的激动剂， 在SCR是心律失常主要触发因素的心肌病中的抗心律失常作用，并通过合理设计 IpCa类似物能够关闭它与RyR一起创建的天然“凹槽”，它具有成为 第一个高亲和力RyR阻断剂，可以防止SR Ca 2+“泄漏”。使用钙蛋白-RyR复合物的冷冻-EM结构 和一系列的功能分析，目标1将确定钙蛋白的结构域，使他们能够结合到 RyR具有精致的亲和力和特异性，并产生能够阻断RyR离子传导的钙蛋白类似物。 目的2将使用在交感神经刺激期间容易呈现SCR的CPVT小鼠系和新的兔CPVT 该模型呈现了组成性SR Ca 2+渗漏和病理性心脏重构，以确定是否是天然的和 突变钙调蛋白能够预防RyR功能障碍触发的心律失常。我们的多学科计划，与 明确定义的可交付成果和里程碑，由结构（货车Petegem）和功能方面的两位PI专家领导 （Valdivia）RyRs的生物学，将产生一种治疗Ca 2+依赖性心律失常的新范式， 现有的药物治疗严重不足。

SUMMARY Ryanodine receptors (RyRs), are the Ca2+ release channels of sarcoplasmic reticulum (SR) that play an essential role in excitaton-contraction coupling of cardiac and skeletal muscle cells. Mutations in the cardiac RyR gene (RYR2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia and sudden death. CPVT is clearly an arrhythmogenic disorder stemming from intracellular Ca2+ mishandling caused by RyR2 dysfunction. As such, insights gained from CPVT syndromes benefit our mechanistic understanding of other cardiomyopathies where RyR2-linked Ca2+ mishandling plays a pivotal role. RyR2 dysfunction brings about deleterious effects in the heart by inducing SR Ca2+ “leak” and/or spontaneous Ca2+ release (SCR), a sudden, diastolic Ca2+ bolous of critical mass that triggers arrhythmic activity. Thus, reigning in RyR2 hyperactivity to mitigate SR Ca2+ “leak” and avoid SCR is a primary goal of therapeutic regimes for major cardiomyopathies. A group of globular peptides termed calcins target RyRs with high affinity and specificity. Imperacalcin (IpCa), the founding member of the calcin family, engages RyRs by entering through their wide vestibule and binding with exquisite precision to a site deep in the cytosolic “cap” of the channel, adjacent to the transmembrane region, acting as a wedge that “pushes” laterally the transmembrane helices that line the channel pore, causing it to open and giving rise to a long-lived subconductance state. In animal models of CPVT, IpCa penetrates the external membrane of ventricular myocytes and induces a partial depletion of SR Ca2+, thus preventing SCR and in effect relieving the adrenergically-mediated Ca2+ overload that triggers Ca2+-dependent arrhythmias. Thus, IpCa behaves naturally as an agonist of RyRs, with anti-arrhytmic effect in cardiomyopathies where SCR is the primary trigger of arrhythmias, and by rational design of IpCa analogs capable of closing the natural “grooves” it creates with RyRs, it has the tangible potential to become the first high-affinity RyR blocker that may prevent SR Ca2+ “leak”. Using cryo-EM structures of calcin-RyR complexes and a series of functional assays, aim 1 will determine the structural domains of calcins that allow them to bind to RyRs with exquisite affinity and specificity, and to generate calcin analogs capable of blocking RyR ion conduction. Aim 2 will use CPVT mouse lines that readily present SCR during sympathetic stimulation and a novel rabbit CPVT model that presents constitutive SR Ca2+ leak and pathological cardiac remodeling to determine whether native and mutant calcins are capable of preventing RyR dysfunction-triggered arrhythmias. Our multi-disciplnary program, with well-defined deliverables and milestones and led by two PIs experts in structural (Van Petegem) and functional (Valdivia) biology of RyRs, will produce a novel paradigm for the treatment of Ca2+-dependent arrhythmias, an area severely underserved by existent pharmacotherapy.