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.