Molecular Identity and Physiological Function of Novel Chloride Channels

项目摘要/摘要 氯化物是动物细胞中含量最丰富的游离阴离子。氯离子通道发挥着广泛的功能 包括细胞体积调节、液体分泌、兴奋性调节和细胞内酸化 细胞器。许多与氯化物有关的遗传性疾病令人印象深刻地说明了它们的生理作用。 调节失调，如囊性纤维化、肌强直和癫痫。然而，尽管最近取得了进展，氯化物 长期以来，在离子通道的贵族家族中，通道一直是贫穷的表亲。几十年来，这一领域一直 以钠、钾和钙通道为主。事实上，仍然有许多电生理上的 特性良好的氯通道，没有分子同一性。这一差距使得我们不可能解释他们的 精确的功能以及它们的功能障碍如何导致疾病。在之前的R35 Mira ESI资助期，我们 进行了无偏RNAi筛选，鉴定了一种没有序列相似性的新的膜蛋白Pac 到其他离子通道，如长期受到追捧的酸或质子活化氯(PAC)通道。通过调解 氯离子内流和随后的细胞肿胀、PAC电流与酸诱导的细胞损伤有关。我们 建立PAC基因敲除小鼠并证明PAC在体外酸诱导的细胞死亡中起关键作用 和活体缺血性脑损伤。因此，PAC是治疗中风和其他酸中毒相关的潜在药物靶点。 疾病。通过结合突变、膜片钳记录和冷冻-EM，我们首次揭示了 三聚体组装，离子传导途径，阴离子选择性的基础，pH依赖的构象变化， 以及这一新通道的pH传感机制。一个新频道的发现代表着一项突破 开辟了一个新领域。未来5年，我们将重点关注PAC渠道的多元化监管机制 以及它在我们最近发现的囊泡酸化中令人惊讶的生理功能。长期的 此Mira计划的目标是应用多学科方法，包括高吞吐量功能 基因组学、膜片钳电生理学、结构生物学、成像和小鼠遗传学 氯离子通道生物学领域。

PROJECT SUMMARY/ABSTRACT Chloride is the most abundant free anion in animal cells. Chloride channels play a wide range of functions including cell volume regulation, fluid secretion, regulation of excitability, and acidification of intracellular organelles. Their physiological role is impressively illustrated by many genetic diseases involving chloride dysregulation, such as cystic fibrosis, myotonia, and epilepsy. However, despite recent progress, chloride channels have long suffered as poor cousins in the aristocratic family of ion channels. For decades, the field has been dominated by sodium, potassium and calcium channels. Indeed, there are still many electrophysiologically well-characterized chloride channels without molecular identity. This gap makes it impossible to elucidate their precise function and how their dysfunction leads to disease. In the previous R35 MIRA ESI funding period, we performed an unbiased RNAi screen and identified PAC, a novel membrane protein with no sequence similarity to other ion channels, as the long sought-after acid or proton-activated chloride (PAC) channel. By mediating chloride influx and subsequent cell swelling, PAC currents have been implicated in acid-induced cell injury. We generated PAC knockout mice and demonstrated that PAC plays a key role in acid-induced cell death in vitro and ischemic brain injury in vivo. Thus, PAC is a potential drug target for stroke and other acidosis-associated diseases. By combining mutagenesis, patch-clamp recording, and cryo-EM, we revealed for the first time the trimeric assembly, ion conducting pathway, the basis of anion selectivity, pH-dependent conformational change, and pH-sensing mechanism of this new channel. Discovery of a novel channel represents a breakthrough that opens up a new field. In the next 5 years, we will focus on the diverse regulatory mechanisms of the PAC channel and its surprising physiological function in vesicular acidification that we have recently discovered. The long-term goal of this MIRA program is to apply a multi-disciplinary approach including high-throughput functional genomics, patch-clamp electrophysiology, structural biology, imaging, and mouse genetics to the underexplored area of chloride channel biology.