Structural analysis of the bestrophin anion channel Best2

Bestrophins是一个五聚体钙激活的氯离子通道家族，它对 通过允许单价阴离子流过细胞膜，细胞内的钙离子(Ca~(2+))。 Bestrophins在存在纳摩尔到微摩尔浓度的Ca~(2+)和 表现出时间和浓度依赖的电流失活。背后的结构基础 响应增加的钙离子浓度的激活和随后的失活还没有 在分子水平上得到了充分的阐明。Bestrophins在各种各样的组织中表达，并且 通常在特定的细胞类型中。Best1在视网膜色素上皮(RPE)中表达。 眼部细胞和BEST1基因突变导致一类视网膜退行性疾病 叫做青春症。另一方面，BEST2在基底膜上表达 眼睛的无色素睫状上皮(NPE)细胞，在那里它已经与世代有关 这表明它可能是治疗青光眼的药理靶点。 Best1-4的序列分析表明，Best2在胞质孔隙收缩方面存在差异，a 先前与通道选通有关的区域。这个项目的目标是1)生成一个结构 解释哺乳动物Best2通道中钙依赖的激活和失活的模型， 包括电生理分析和合理设计突变体的结构分析 以检验该模型，以及2)研究胞质收缩内的结构差异 Best2，这是它与其他Bestrophin的区别。这些目标的实现将有助于 Bestrophins在全身的作用，并将指导基于结构的药物设计 专门针对Best2，而不是其他可能有治疗青光眼潜力的bestrophins。

Bestrophins are a family of pentameric calcium-activated chloride channels that respond to intracellular calcium (Ca2+) by allowing the flow of monovalent anions across the membrane. Bestrophins are activated in the presence of nanomolar to micromolar concentrations of Ca2+ and exhibit a time- and concentration -dependent inactivation of current. The structural basis behind activation and subsequent inactivation in response to increasing Ca2+concentrations has not been fully elucidated at the molecular level. Bestrophins are expressed in a wide variety of tissues, and often within specialized cell types. Best1 is expressed in the retinal pigmented epithelial (RPE) cells of the eye and mutations in the BEST1 gene lead to a class of retinal degenerative diseases called bestrophinopathies. BEST2, on the other hand, is expressed in the basolateral membrane of the nonpigmented ciliary epithelial (NPE) cells of the eye, where it has been linked to generation of intraocular pressure, suggesting it may represent a pharmacological target to treat glaucoma. Sequence analysis of Best1-4 reveals Best2 has a difference in the cytosolic pore constriction, a region previously implicated in channel gating. The goal of this project is to 1) generate a structural model to explain Ca2-dependent activation and inactivation in a mammalian Best2 channel, including electrophysiological analysis and structural analysis of rationionally designed mutants to test the model, and 2) investigate the structural differences within the cytosolic constriction of Best2 that distinguish it from other bestrophins. Completion of these aims will inform on the function of bestrophins throughout the body, and will guide structure-based drug design to specifically target Best2 and not other bestrophins, which may have potential to treat glaucoma.