Interacting Partners of Bestrophin Channels

项目摘要/摘要 Bestrophin蛋白是一类钙激活的阴离子通道，广泛分布于细菌和 哺乳动物，在大多数后生动物中有代表，包括在人类中的四个(Best1-4)。作为回应，他们打开了 通过细胞内钙离子浓度的升高来调节氯离子、−等阴离子的被动流动。Best1和Best2都有 眼睛中的关键角色。Best1主要在视网膜的色素上皮(RPE)中表达 在产生与视觉有关的电信号的过程中扮演着重要的角色，这种电信号被称为“光峰”，而人类的突变 Best1基因与至少五种视网膜退行性疾病有关，统称为 脑扁桃病。Best2在睫状体的无色素上皮(NPE)中高表达，是 参与房水的形成和排出，最终决定眼压(IOP)。 在小鼠中敲除Best2可降低眼压，提示靶向Best2的药物潜力 及其调节剂，用于缓解高眼压，高眼压是多种眼病的常见危险因素 包括开角型青光眼。然而，尽管Bestrophins具有生物学和药学意义，但几乎没有 已知它们在生理环境中的细胞调节。更具体地说，在给定的细胞/组织中， 贝斯特罗芬通道调制传导哪个阴离子(S)用于什么下游目的(S)？答案是 这个问题存在于与不同组织/细胞中的Bestrophins相互作用的蛋白质网络中。虽然 大量证据表明，Bestrophins存在组织特异性的相互作用调节因子，目前还不知道有什么蛋白质 与Best2相互作用，而只有少数报告与Best1相互作用。我们的实验室研究生物物理学。 以及使用由低温电子组成的多学科平台调节Bestrophin通道 显微镜(冷冻-EM)、质谱学、电生理记录、CRISPR/Cas9介导的基因组 编辑和干细胞重新编程/分化。未来五年的目标是确定Best1和Best2 分别在RPE和NPE中相互作用的蛋白质复合体，并了解 这些互动。总体而言，拟议的工作将对多个研究领域做出贡献，包括 钙信号、离子转运、膜蛋白结构和眼生理，以及管道的建立 本工作可普遍应用于研究不同的离子通道和其他感兴趣的膜蛋白。

Project Summary/Abstract The bestrophin proteins are a family of Ca2+-activated anion channels widely distributed from bacteria to mammals, with representatives in most metazoans including four in humans (Best1-4). They open in response to increases of intracellular Ca2+ to mediate the passive flow of Cl− and other anions. Best1 and Best2 both have critical roles in the eye. Best1 is predominantly expressed in retinal pigment epithelium (RPE) of the retina playing an essential role in generating a vision-related electrical signal named “light peak”, while mutations in the human BEST1 gene have been genetically linked to at least five retinal degenerative disorders collectively known as bestrophinopathies. Best2 is highly expressed in non-pigmented epithelium (NPE) of the ciliary body, and is involved in aqueous humor formation and drainage, which ultimately determine intra-ocular pressure (IOP). Knockout of Best2 in mice leads to a reduction of IOP, suggesting the pharmaceutical potential of targeting Best2 and its regulators for relieving ocular hypertension, which is a common risk factor for numerous eye diseases including open-angle glaucoma. However, despite bestrophins’ biological and pharmaceutical significance, little is known about their cellular regulation in a physiological context. More specifically, in a given cell/tissue, how is the bestrophin channel modulated to conduct which anion(s) for what downstream purpose(s)? The answer to this question lies within the protein network that interacts with bestrophins in different tissues/cells. Although ample evidence suggest the existence of tissue-specific interacting regulators of bestrophins, no protein is known to interact with Best2, while only a few have been reported to interact with Best1. Our lab studies the biophysics and regulation of bestrophin channels using a multidisciplinary platform consisting of cryogenic electron microscopy (cryo-EM), mass spectrometry, electrophysiological recording, CRISPR/Cas9-mediated genome editing, and stem cell reprogramming/differentiation. The goal of the next five years is to identify Best1 and Best2 interacting protein complexes in RPE and NPE, respectively, and to understand the functional consequences of these interactions. Overall, the proposed work will make contributions to multiple fields of research including calcium signaling, ion transport, membrane protein structure and ocular physiology, and the pipelines established in this work can be generally applied to study different ion channels and other membrane proteins of interest.