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出现了用光控制电活动的光化学方法。我们在这个项目中的目标是开发一种广泛适用的方法，通过生成工具来控制细胞功能的其他方面，以赋予广泛的细胞表面受体和离子通道光敏性。为了迎接这一挑战，我们将使用模块化的方法，利用一个单独的“通用光开关”作为关键的光敏元件。光开关的核心含有可异构化的小的偶氮苯部分，它对380 nm和500 nm的光分别有缩短和延长的响应。我们将使用这种光开关来间接调节受体和通道的活性，方法是通过一个“适配肽”和一个配位域来间接调节受体和通道的活性，“适配肽”包含一个“捕捉结构域”，它识别光开关的短构型，而不是长构型，配体结构域包含靶细胞表面受体或离子通道的多肽激活剂或抑制物。捕获结构域在所有适配肽中保持不变，允许由单个通用光开关控制，但配基结构域的性质是为调节特定受体而定制的。将使用几种策略将适配肽的光依赖捕获转化为受体激活或抑制。这些措施包括用二聚体光开关将适配肽二聚化以激活生长因子受体，以及通过抗体拴系的光开关将适配肽(包括配体)输送到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。我们将在两个例子受体上测试通用光开关方法的有效性：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。建立一种对这些受体进行光敏调节的方法，将使人们能够以前所未有的精度研究它们在完整组织中的发育和神经功能中的作用，但更重要的是，它将展示一种强大的受体调节新技术的出现，该技术可以应用于任何已知存在多肽配体的细胞表面蛋白。

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.