Structure-function Analysis of Bestrophins

 描述(申请人提供)：许多生理事件，如心跳和肌肉收缩，都是由某些离子的细胞浓度调节的，这些离子通过细胞膜上称为离子通道的指定结构流入和流出细胞。离子通道/通量的失调在临床上与许多疾病有关。人Bestrophin-1(HBest1)是一种眼部高表达的氯离子传导通道。到目前为止，已经发现120多种不同的hBest1突变与多种导致视力丧失的眼病有关。尽管许多hBest1致病突变体被证明是有缺陷的氯通道，但这种缺陷如何在病理上导致眼部疾病仍然是个谜。此外，目前还不清楚hBest1的氯离子通道功能是如何调节/激活的。因此，深入了解hBest1的结构和功能具有重要的生物学意义和生物医学意义。本研究的目的是从结构和功能两个方面分别从结晶学和电生理学方面阐明hBest1的作用机制。重要的是，最近获得的细菌Bestrophin同源物(KpBest，来自肺炎克雷伯菌)的结构模型为本提案中的研究目标提供了新的线索和结构基础。具体地说，在目标1中，根据KpBest结构模型的建议，将在KpBest和hBest1中检验假定的Bestrophin上的两个离子渗透门；在目标2中，将通过共结晶ATP和KpBest来检验ATP直接相互作用并激活bestrophins的假设，并进一步用KpBest和hBest1进行功能研究；在目标3中，将使用KpBest结构作为搜索模型来获得真核生物贝斯特罗芬(S)和/或hBest1的晶体结构。在K99阶段(目标1，以及目标2和3的部分内容)，我将得到蛋白质结晶学领域的领军人物韦恩·亨德里克森博士的指导。这项工作将对致病的hBest1突变如何影响通道的结构和功能从而导致眼部疾病提供新的线索，并为未来基于结构的特异性通道激活剂/抑制剂的设计提供有价值的见解。

 DESCRIPTION (provided by applicant): Many physiological events, such as heart beat and muscle contraction, are orchestrated by the cellular concentrations of certain ions, which flow in and out of the cell through designated structures on the cell membrane called ion channels. Dysregulation of ion channel/flux is clinically linked to many diseases. The human bestrophin-1 (hBest1) is a chloride (Cl-) conducting channel highly expressed in eyes. So far, over 120 distinct mutations of hBest1 have been identified to associate with multiple kinds of eye diseases that cause vision loss. Although many hBest1 disease-causing mutants have been documented as defective Cl- channels, how this defect pathologically results in eye disorders remains elusive. Moreover, it is largely unclear how the Cl- channel function of hBest1 is regulated/activated. Therefore, it is of both biological and biomedical significance to thoroughly understand the structure and function of hBest1. The goal of this proposal is to elucidate the working mechanisms of hBest1 by crystallography and electrophysiology from the structural and functional aspects, respectively. Importantly, a recently obtained structure model of a bacterial bestrophin homolog (KpBest, from Klebsiella pneumoniae) provides novel clues and a structural basis for the research aims in this proposal. To be specific, in Aim 1, the two putative ion permeation gates on bestrophins, as suggested by the KpBest structure model, will be examined in both KpBest and hBest1; in Aim 2, the hypothesis that ATP directly interacts and activates bestrophins will be tested by co-crystallizing ATP and KpBest, and further functionally examined with both KpBest and hBest1; in Aim 3, the crystal structure of eukaryotic bestrophin(s) and/or hBest1 will be obtained using the KpBest structure as a search model. During the K99 phase (Aim 1, and parts of Aims 2 and 3), I will be mentored by Dr. Wayne Hendrickson, a leader in the field of protein crystallography. This work will shed new light on how disease-causing hBest1 mutations influence the structure and function of the channel to contribute to eye diseases, and provide valuable insight for structure-based specific channel activator/inhibitor design in the future.