Retinal degeneration and chloride channels

描述（由申请人提供）：视网膜疾病影响估计1在美国的28人，并有一个巨大的个人和经济影响。这项申请将调查一个快速增长的类别遗传性视网膜变性称为雌激素。Bestrophinopathies是由人类bestrophin-1基因（hBest 1）突变引起的。hBest 1突变产生一系列显性遗传和隐性遗传的视网膜变性，表现出多种临床病理。已经确定hBest 1编码在视网膜色素上皮（RPE）的基底外侧膜中表达的Ca激活的氯离子通道（CaCC），并且bestrophinopathies的特征在于由RPE CaCC产生的眼电图光峰的减少。这些观察结果导致了雌激素病的“CaCC假说”。虽然大量的数据支持的想法，bestrophinopathies是由Best 1的氯离子转运的缺陷，敲除和敲入突变小鼠模型的研究提供了强烈的矛盾的结论。该项目的目标是深入研究雌激素病的CaCC假说。我们将详细探讨光峰的分子机制，以建立光峰背后的离子机制，从启动它的Ca信号到产生它的阴离子通道。我们的假设是，CRAC通道奥赖-1负责Ca内流，激活Ca激活的阴离子通道Tmem 16 a/Ano 1产生光峰。我们假设Best 1突变通过两种机制降低光峰：与Ano 1活性相互作用并调节Ano 1活性，以及通过改变Ca信号传导。这些研究将使用膜片钳记录和来自野生型和转基因小鼠以及来自猴的分离的RPE的最先进的Ca成像的组合来完成。这些研究具有创新性，因为它们研究了人们知之甚少的RPE细胞的基本特性。虽然氯离子通道对于正常的RPE功能是不可或缺的，但它们在视网膜中的调节和生理作用方面仍然没有得到充分的研究。这些研究意义重大，因为了解RPE功能的离子机制对于了解视网膜生理学至关重要，并且因为Best 1功能障碍在视网膜病变中的作用比以前认识到的要大得多。除了是导致bestrophinopathies的主要参与者之外，Best 1突变还可能导致疾病易感性，或者Best 1蛋白可能是其他病因不明的视网膜病变的下游靶点。

DESCRIPTION (provided by applicant): Retinal diseases affect an estimated 1 in 28 people in the US, and have a huge personal and economic impact. This application will investigate a rapidly-growing category of inherited retinal degenerations termed bestrophinopathies. Bestrophinopathies are caused by mutations in the human bestrophin-1 gene (hBest1). hBest1 mutations produce a panoply of both dominantly- and recessively-inherited retinal degenerations presenting a diverse range of clinical pathologies. It is well-established that hBest1 encodes a Ca-activated chloride channel (CaCC) that is expressed in the basolateral membrane of the retinal pigment epithelium (RPE) and that bestrophinopathies are characterized by a reduction in the electro-oculogram light peak that is generated by an RPE CaCC. These observations have led to the "CaCC hypothesis" for bestrophinopathies. Although considerable data supports the idea that bestrophinopathies are caused by defects in chloride transport by Best1, studies on knockout and knockin-mutant mouse models have provided strongly conflicting conclusions. The goal of this project is to examine in depth the CaCC hypothesis of bestrophinopathies. We will explore in detail the molecular mechanisms of the light peak to establish the ionic mechanisms underlying the light peak, from the Ca signals that initiate it to the anion channels that generate it. Our hypothesis is that the CRAC channel Orai-1 is responsible for the Ca influx that activates the Ca-activated anion channel Tmem16a/Ano1 to generate the light peak. We hypothesize that Best1 mutations reduce the light peak by two mechanisms: interacting with and regulating Ano1 activity and by altering Ca signaling. These studies will be accomplished using a combination of patch clamp recording and state-of-the-art Ca imaging of isolated RPE from wild type and transgenic mice and from monkey. These studies are innovative because they investigate fundamental properties of RPE cells that are poorly understood. Although it is clear that chloride channels are indispensible for normal RPE function, they remain inadequately investigated, both with regard to their regulation and their physiological roles in the retina. These studies are significant because understanding the ionic mechanisms of RPE function is essential to understanding retinal physiology and because it is becoming apparent that Best1 dysfunction plays a much larger role in retinopathies than previously recognized. In addition to being a prime player in causing bestrophinopathies, Best1 mutations may also contribute disease susceptibility or Best1 protein may be a downstream target in other retinopathies of unknown etiology.