Optical control of G protein-coupled receptor (GPCR) signaling is a highly valuable approach for comprehensive understanding of GPCR-based physiologies and controlling them precisely. However, optogenetics for GPCR signaling is still developing and requires effective and versatile tools with performance evaluation from their molecular properties. Here, we systematically investigated performance of two bistable opsins that activate Gi/Go-type G protein (mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP)) in optical control in vivo using Caenorhabditis elegans . Transgenic worms expressing MosOpn3, which binds 13- cis retinal to form photopigments, in nociceptor neurons showed light-induced avoidance responses in the presence of all- trans retinal, a retinal isomer ubiquitously present in every tissue, like microbial rhodopsins and unlike canonical vertebrate opsins. Remarkably, transgenic worms expressing MosOpn3 were ~7,000 times more sensitive to light than transgenic worms expressing ChR2 in this light-induced behavior, demonstrating the advantage of MosOpn3 as a light switch. LamPP is a UV-sensitive bistable opsin having complete photoregenerative ability by green light. Accordingly, transgenic worms expressing LamPP in cholinergic motor neurons stopped moving upon violet light illumination and restored coordinate movement upon green light illumination, demonstrating color-dependent control of behavior using LamPP. Furthermore, we applied molecular engineering to produce MosOpn3-based tools enabling light-dependent upregulation of cAMP or Ca 2+ levels and LamPP-based tool enabling clamping cAMP levels color dependently and context independently, extending their usability. These findings define the capacity of two bistable opsins with similar retinal requirement as ChR2, providing numerous strategies for optical control of various GPCR-based physiologies as well as GPCR signaling itself.

中文翻译：

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双稳态动物视蛋白 MosOpn3 和 LamPP 以分子特性依赖的方式对 GPCR 信号的高性能光学控制

G 蛋白偶联受体 (GPCR) 信号的光学控制是一种非常有价值的方法，可以全面了解基于 GPCR 的生理学并精确控制它们。然而，用于 GPCR 信号传导的光遗传学仍在发展中，需要有效且通用的工具从其分子特性进行性能评估。在这里，我们系统地研究了激活 Gi/Go 型 G 蛋白的两种双稳态视蛋白（蚊子 Opn3 (MosOpn3) 和七鳃鳗副视蛋白 (LamPP)）在体内光学控制中的性能秀丽隐杆线虫. 表达 MosOpn3 的转基因蠕虫，它结合 13-顺式视网膜形成感光色素，在伤害感受器神经元中显示出在所有存在的情况下光诱导的回避反应反式视网膜，一种普遍存在于每个组织中的视网膜异构体，如微生物视紫红质，不同于典型的脊椎动物视蛋白。值得注意的是，在这种光诱导行为中，表达 MosOpn3 的转基因蠕虫对光的敏感度是表达 ChR2 的转基因蠕虫的约 7,000 倍，证明了 MosOpn3 作为光开关的优势。LamPP 是一种对紫外线敏感的双稳态视蛋白，具有完全的绿光光再生能力。因此，在胆碱能运动神经元中表达 LamPP 的转基因蠕虫在紫光照射下停止移动，并在绿光照射下恢复坐标运动，证明了使用 LamPP 对行为的颜色依赖控制。此外，我们应用分子工程来生产基于 MosOpn3 的工具，从而实现 cAMP 或 Ca 的光依赖性上调2+级别和基于 LamPP 的工具能够根据颜色和上下文独立地夹紧 cAMP 级别，从而扩展它们的可用性。这些发现定义了两种与 ChR2 具有相似视网膜需求的双稳态视蛋白的能力，为各种基于 GPCR 的生理学以及 GPCR 信号本身的光学控制提供了许多策略。