A universal photoswitch system for optical control of neuronal receptors

描述（由申请人提供）： 近年来生物学中最令人兴奋的技术发展之一是用光来控制电活动的光化学方法的出现。我们在这个项目中的目标是开发一种广泛适用的方法，通过生成用于在广泛的细胞表面受体和离子通道上赋予光敏性的工具来控制细胞功能的其他方面。为了应对这一挑战，我们将使用模块化的方法，利用一个单一的“通用光电开关”作为关键的光传感组件。光开关在其核心含有小的可异构化的偶氮苯部分，其分别响应于380和500 nm的光而缩短和延长。我们将使用这种光开关通过“衔接肽”间接调节受体和通道活性，所述衔接肽含有“捕获结构域”和配体结构域，所述“捕获结构域”识别光开关的短构型，但不识别长构型，所述配体结构域含有靶细胞表面受体或离子通道的肽激活剂或抑制剂。捕获结构域在所有衔接肽中保持恒定，允许通过单个通用光开关进行控制，但配体结构域的性质被定制以调节特定受体。将使用几种策略将衔接肽的光依赖性捕获转化为受体激活或抑制。这些包括用二聚光开关使衔接肽二聚化以激活生长因子受体，并通过抗体系留的光开关将衔接肽（包括配体）递送至G蛋白偶联受体。可以开发其他策略来激活不同类型的受体和离子通道。我们将测试通用光开关方法对两个示例受体的有效性：脑源性生长因子（BDNF）的TrkB受体，受体酪氨酸激酶的神经营养因子家族的成员，以及神经肽Y的受体，这是一种GPCR型受体。产生这些受体的光敏调节的方法将允许检查它们在发育和完整组织中的神经功能中的作用，具有前所未有的精度，但更重要的是，它将证明出现一种强大的新技术，用于受体调节，可以应用于任何细胞表面蛋白，其中已知的肽配体存在。

DESCRIPTION (provided by applicant): One of the most exciting technical developments in biology in recent years is the emergence of photochemical methods for controlling electrical activity with light. Our goal in this project is to develop a broadly applicable method for controlling other aspects of cellular function by generating tools for conferring light sensitivity on a broad range of cell surface receptors and ion channels. To meet this challenge we will use a modular approach, utilizing a single "Universal Photoswitch" as the key light-sensing component. The photoswitch contains at its core the small isomerizable azobenzene moiety, which shortens and lengthens in response to 380 and 500 nm light, respectively. We will use this photoswitch to indirectly regulate receptor and channel activity, through an "adapter peptide", which contains a "capture domain", which recognizes the short, but not the long configuration of the photoswitch, and a ligand domain, which contains a peptide activator or inhibitor of the targeted cell surface receptor or ion channel. The capture domain is kept constant among all adapter peptides, allowing control by a single Universal Photoswitch, but the nature of ligand domain is tailored to regulate a specific receptor. Several strategies will be used to translate light-dependent capture of the adapter peptide into receptor activation or inhibition. These include dimerizing the adapter peptide with a dimeric photoswitch to activate growth factor receptors, and delivering the adapter peptide, including the ligand, to a G-protein coupled receptor via an antibody-tethered photoswitch. Other strategies may be developed to activate different types of receptors and ion channels. We will test the effectiveness of the Universal Photoswitch approach on two example receptors: the TrkB receptor for brain-derived growth factor (BDNF), a member of the neurotrophin family of receptor tyrosine kinases, and the receptor for neuropeptide Y, which is a GPCR-type receptor. Generating a method for light-sensitive regulation of these receptors will allow examination of their roles in development and neural function in intact tissue with unprecedented precision, but more importantly, it will demonstrate the emergence of a powerful new technique for receptor regulation that can be applied to any cell surface protein for which a known peptide ligand exists.