Interacting Partners of Bestrophin Channels

项目总结/摘要 雌激素蛋白是广泛分布于细菌和哺乳动物细胞中的钙离子激活的阴离子通道家族。 哺乳动物，在大多数后生动物中具有代表性，包括人类中的四种（Best1 - 4）。他们打开回应 增加细胞内Ca2+介导Cl −和其他阴离子的被动流动。Best1和Best2都有 眼睛中的关键角色。Best1主要表达于视网膜色素上皮细胞（RPE）， 在产生与视觉相关的电信号“光峰”中起重要作用，而人类中的突变 BEST 1基因与至少五种视网膜退行性疾病有遗传联系，这些疾病统称为 雌激素病Best2在睫状体的非色素上皮（NPE）中高度表达， 参与最终决定眼内压（IOP）的房水形成和引流。 小鼠中Best2的敲除导致IOP降低，表明靶向Best2的药物潜力 及其调节剂，用于缓解高眼压，高眼压是许多眼部疾病的常见风险因素 包括开角型青光眼。然而，尽管bestrophins的生物学和药学意义， 在生理学背景下对它们的细胞调节是已知的。更具体地，在给定的细胞/组织中， 调节斑萎蛋白通道以传导哪种阴离子用于什么下游目的？的答案 这个问题存在于与不同组织/细胞中的雌激素相互作用的蛋白质网络中。虽然 大量证据表明存在组织特异性的雌激素相互作用调节因子，但目前还没有已知的蛋白质 与Best2相互作用，而据报道只有少数与Best1相互作用。我们的实验室研究生物物理学 和调控的bestrophin通道使用一个多学科的平台组成的低温电子 显微镜（cryo-EM），质谱，电生理记录，CRISPR/Cas9介导的基因组 编辑和干细胞重编程/分化。未来五年的目标是确定最佳1和最佳2 分别在RPE和NPE中相互作用的蛋白质复合物，并了解 这些互动。总体而言，拟议的工作将为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学，以及建立的管道 在这项工作中，可以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.