Going full circle - optogenetic control of Ca2+ release from and reuptake into the endoplasmic reticulum

完整循环——光遗传学控制Ca2从内质网释放和再摄取

• 批准号: 315402240
• 负责人: Professor Dr. Alexander Gottschalk
• 金额: --
• 依托单位: Buchmann Institute for Molecular Life Sciences
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/315402240?language=en
• 关键词: Going; full; circle; optogenetic; control

In excitable cells, intracellular Ca2+ release and uptake from and into the endo/sarcoplasmic reticulum, via the Ryanodine Receptor (RyR) and SERCA-Ca2+-ATPase, respectively, are functionally coupled. This allows for tight spatiotemporal control of local Ca2+ signals (Ca2+ sparks) and safeguards cell viability through low resting Ca2+ concentrations. Despite the eminent role of RyR Ca2+ release channels and SERCA Ca2+-pumps in the brain, heart, pancreas and skeletal muscle, both in health and disease, their molecularly targeted manipulation remains challenging and complex. For example, patient mutations cause dangerous arrhythmias via RyR2 channels in the heart through increased Ca2+ leak. However, in experimental cellular models Ca2+ leak is commonly induced by non-physiological, typically pharmacological interventions at the cost of significant off-target effects. Importantly, the lack of RyR- and SERCA-specific tools compromises and delays the development of drug compounds for a large number of diseases, including arrhythmias, cancer, cognitive dysfunction, diabetes, epilepsy, heart failure, and muscle fatigue. While optogenetic manipulation of RyR channels and SERCA pumps addresses a challenging area, our collaborative efforts to develop the first OptoRyR tool are not only promising, but can open major opportunities for target-specific functional studies, which may facilitate drug development and testing.In the first funding period we have developed de novo strategies for light-induced Ca2+ release. The most successful strategy used a Ca2+-conducting Channelrhodopsin2 variant fused to the RyR2 channel. This OptoRyR2 relies on an endogenous amplification mechanism of Ca2+-induced Ca2+-release through cytosolic Ca2+-activation of RyR2 channels. For the second funding period we will significantly extend the planned work through three major directions: In Aim 1 we will apply the mechanistic concept of OptoRyR2 to C. elegans and human stem cell-derived cardiomyocytes using CRISPR/Cas9 mediated gene editing to address fundamental biological questions in intact animals and human cardiomyocytes to develop a novel optogenetic platform for drug-safety testing of RyR2-targeted chemical compounds. Aim 2 broadens the utility concept through a new mechanistic approach of direct opto-mechanical light-gating of RyR2 through insertion of LOV domains in the channel’s large cytosolic shell structure. This became possible through existing high-resolution CryoEM structures of RyR channels. Aim 3, and closing the circle, extends the optogenetic toolbox to control Ca2+ uptake through two parallel strategies: light-activation of cAMP production molecularly linked to phospholamban and opto-mechanical control of SERCA will both modulate Ca2+ uptake. Thus, we will develop a toolbox for subcellular Ca2+ control and apply this in vitro and in vivo to enable exploring basic questions of Ca2+ homeostasis, as well as a new concept of drug-safety testing.

在可兴奋细胞中，细胞内钙的释放和摄取分别通过Ryanodine受体(RyR)和SERCA-Ca~(2+)-ATPase功能偶联。这允许对局部钙信号(钙火花)进行严格的时空控制，并通过低静息钙浓度保护细胞存活。尽管在健康和疾病中，RyR钙释放通道和SERCA钙泵在大脑、心脏、胰腺和骨骼肌中都发挥着重要的作用，但它们的分子靶向操作仍然具有挑战性和复杂性。例如，患者突变通过心脏中的RyR2通道增加钙离子泄漏而导致危险的心律失常。然而，在实验细胞模型中，钙离子泄漏通常是由非生理性的，通常是药物干预引起的，代价是显著的非靶点效应。重要的是，缺乏RyR和SERCA特定的工具会损害和推迟治疗许多疾病的药物化合物的开发，包括心律失常、癌症、认知功能障碍、糖尿病、癫痫、心力衰竭和肌肉疲劳。虽然光基因操作RyR通道和SERCA泵解决了一个具有挑战性的领域，但我们合作开发的第一个OptoRyR工具不仅前景光明，而且可以为靶向特定功能研究打开重大机遇，这可能会促进药物开发和测试。在第一个资助期，我们开发了光诱导钙释放的从头策略。最成功的策略是使用与RyR2通道融合的钙离子传导通道视紫红质2变异体。这种OptoRyR2依赖于一种内源性的放大机制，即通过激活RyR2通道内的钙离子来诱导钙离子释放。在第二个资助期，我们将通过三个主要方向大幅扩展计划的工作：在目标1，我们将使用CRISPR/Cas9介导的基因编辑将OptoRyR2的机械概念应用于线虫和人类干细胞来源的心肌细胞，以解决完整动物和人类心肌细胞的基本生物学问题，为RyR2靶向化合物的药物安全性测试开发一个新的光遗传平台。目的2通过在通道的大胞质壳结构中插入LOV结构域来直接对RyR2进行光机械光门的新的机械方法，拓宽了实用概念。通过现有的RyR通道的高分辨率低温EM结构，这成为可能。目的3，并结束循环，扩展光遗传工具箱，通过两个平行的策略来控制钙摄取：光激活与磷蛋白分子相连的cAMP产生和光机械控制SERCA都将调节钙摄取。因此，我们将开发一个用于亚细胞内钙调控的工具箱，并将其应用于体外和体内，以探索钙稳态的基本问题，以及药物安全性测试的新概念。