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

在可兴奋细胞中，细胞内Ca2+释放和摄取分别通过Ryanodine受体（RyR）和SERCA-Ca2+- atp酶从内质网和进入肌浆网是功能偶联的。这允许对局部Ca2+信号（Ca2+火花）进行严格的时空控制，并通过低静息Ca2+浓度保障细胞活力。尽管RyR Ca2+释放通道和SERCA Ca2+泵在大脑、心脏、胰腺和骨骼肌中在健康和疾病中发挥着重要作用，但它们的分子靶向操作仍然具有挑战性和复杂性。例如，患者突变通过增加Ca2+泄漏通过心脏中的RyR2通道引起危险的心律失常。然而，在实验细胞模型中，Ca2+泄漏通常是由非生理性的、典型的药理学干预引起的，其代价是显著的脱靶效应。重要的是，RyR-和serca特异性工具的缺乏损害和延迟了大量疾病药物化合物的开发，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RyR通道和SERCA泵的光遗传学操作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工具不仅有希望，而且可以为目标特异性功能研究开辟重要机会，这可能有助于药物开发和测试。在第一个资助期，我们开发了光诱导Ca2+释放的从头策略。最成功的策略是将Ca2+导电的channel rhodopsin2变体与RyR2通道融合。该OptoRyR2依赖于内源性Ca2+诱导的Ca2+释放的放大机制，通过胞质Ca2+激活RyR2通道。在第二个资助期，我们将通过三个主要方向显着扩展计划的工作：在Aim 1中，我们将利用CRISPR/Cas9介导的基因编辑将OptoRyR2的机制概念应用于秀丽隐杆线虫和人类干细胞衍生的心肌细胞，以解决完整动物和人类心肌细胞中的基本生物学问题，开发一个新的光遗传学平台，用于ryr2靶向化合物的药物安全性测试。Aim 2通过在RyR2通道的大细胞质壳结构中插入LOV结构域，通过直接光-机械光门控的新机制方法拓宽了实用概念。这通过现有的高分辨率RyR通道CryoEM结构成为可能。Aim 3，闭合循环，扩展光遗传学工具箱，通过两种平行策略来控制Ca2+摄取：光激活与磷蛋白分子相关的cAMP产生和光机械控制SERCA都将调节Ca2+摄取。因此，我们将开发亚细胞Ca2+控制工具箱，并将其应用于体外和体内，以探索Ca2+稳态的基本问题，以及药物安全测试的新概念。

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.