Retinal degeneration and chloride channels

描述(申请人提供)：视网膜疾病在美国估计每28人中就有1人受到影响，并对个人和经济造成巨大影响。这个应用程序将研究一种快速增长的遗传性视网膜变性，称为脑桥蛋白病变。Bestrophin病是由人类Bestrophin-1基因(HBest1)突变引起的。HBest1基因突变导致一系列显性和隐性遗传性视网膜变性，呈现出一系列不同的临床病理。众所周知，hBest1编码一个钙激活的氯离子通道(CACC)，该通道表达在视网膜色素上皮(RPE)的基侧膜上，而眼球疾病的特征是由RPE CACC产生的眼电光峰减少。这些观察结果导致了脑病的“CACC假说”。尽管相当多的数据支持这样的观点，即bestrophinopsis是由Best1的氯离子运输缺陷引起的，但对敲除和敲门突变小鼠模型的研究提供了强烈矛盾的结论。本项目的目标是深入研究脑病的CACC假说。我们将详细探索光峰的分子机制，以建立光峰背后的离子机制，从启动光峰的钙信号到产生光峰的阴离子通道。我们的假设是CRAC通道Orai-1负责钙内流，激活钙激活的阴离子通道TMEM16A/Ano1来产生光峰。我们假设Best1突变通过两种机制减少光峰：与Ano1的相互作用和调节Ano1的活性，以及通过改变钙信号。这些研究将结合使用膜片钳记录和从野生型和转基因小鼠以及猴子分离的RPE的最新钙成像来完成。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。虽然氯离子通道显然是正常RPE功能所必需的，但它们在调节和在视网膜中的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，而且越来越明显的是，Best1功能障碍在视网膜疾病中发挥的作用比以前认识的要大得多。Best1基因突变除了是导致视网膜病变的主要因素外，还可能导致疾病易感性，或者Best1蛋白可能是其他原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.