Structure-function analysis of the volume-regulated anion channel VRAC using novel LRRC8 chimeras

项目总结 体积调节阴离子通道(Vrac)在脊椎动物细胞中广泛表达， 介导细胞体积调节所需的氯离子和有机溶质的外流，这是一种重要的生理过程 进程。VRAC分别由细胞膨胀和收缩激活和失活。他们还检测到 细胞内离子强度的变化，这改变了它们对细胞体积变化的敏感性。VRAC和 编码它们的基因与多种疾病有关，包括糖尿病、肥胖症、癌症和免疫力。 全基因组RNA干扰筛查导致2014年VRAC被编码 在Lrrc8基因家族的五个成员中，Lrrc8a-e.vrac/LRRC8通道是六异构体和 需要将基本亚基LRRC8A与一个或多个其他LRRC8蛋白共同组装。亚单位 组装顺序和化学计量比未知。 LRRC8A和LRRC8D通道的冷冻电子显微镜(EM)结构 最近确定的。然而，LRRC8A和LRRC8D同源分子在自然界中并不存在。此外， LRRC8A同分异构体通道具有非本地功能属性，而LRRC8D通道属性为 无法定义，因为它们不是运输到质膜的。因此，现有的低温电磁结构具有 了解vrac/LRRC8结构-功能关系的限制。直接翻译LRRC8A和 LRRC8D冷冻-EM结构信息的功能理解进一步受到未知因素的限制 以及可能可变的化学计量比和六异构体VRAC/LRRC8通道的组装。 我们的实验室在dk51610的资助下，对vrac进行了广泛的研究，并率先展示了 该频道的许多独特的功能属性。最近，我们描述了新的LRRC8嵌合体 通道构建，允许对具有生理功能的同源通道进行详细的分子研究 相关的功能性质和定义的化学计量比和组装。我们的嵌合体研究独一无二 证明了LRRC8A胞内环IL1具有独特的结构特征，2)细胞所需 体积感测，3)LRRC8的C末端是感测细胞内离子强度变化所必需的，4) LRRC8A IL1和C末端都是正确处理vrac/LRRC8通道的细胞所必需的。 此R01更新应用程序的首要目标是利用这些新的LRRC8嵌合体来更好地 阐明vrac/LRRC8通道结构-功能关系。我们将描述 LRRC8A在vrac/LRRC8通道调节中的C末端，并将检验LRRC8 IL1的假设 确定VRAC/LRRC8通道气孔属性并调节通道门控。我们还将确定 一个独特的LRRC8嵌合体在多种生理相关构象中的冷冻-EM结构。我们的研究 将为vrac/LRRC8通道的调节和功能提供新的见解，并将提供更高的 基于突变的详细结构-功能分析的信心基础。

PROJECT SUMMARY The volume-regulated anion channel (VRAC) is expressed ubiquitously in vertebrate cells where it mediates the efflux of Cl- and organic solutes required for cell volume regulation, an essential physiological process. VRACs are activated and inactivated by cell swelling and shrinkage, respectively. They also detect changes in intracellular ionic strength, which modifies their sensitivity to cell volume changes. VRACs and the genes that encode them are implicated in multiple diseases including diabetes, obesity, cancer and immunity. Whole genome RNA interference screening led to the demonstration in 2014 that VRACs are encoded by five members of the Lrrc8 gene family, Lrrc8a–e. VRAC/LRRC8 channels are hexaheteromers and require co-assembly of the essential subunit LRRC8A with one or more other LRRC8 proteins. Subunit assembly order and stoichiometry are unknown. Cryo-electron microscopy (EM) structures of homomeric LRRC8A and LRRC8D channels were recently determined. However, LRRC8A and LRRC8D homomers do not exist in Nature. Furthermore, LRRC8A homomeric channels have non-native functional properties and LRRC8D channel properties are undefinable because they are not trafficked to the plasma membrane. Existing cryo-EM structures thus have limitations for understanding VRAC/LRRC8 structure-function relationships. Directly translating LRRC8A and LRRC8D cryo-EM structural information into functional understanding is further constrained by the unknown and likely variable stoichiometry and assembly of hexaheteromeric VRAC/LRRC8 channels. Our laboratory, funded by DK51610, has studied VRAC extensively and was the first to demonstrate many of the channel's unique functional properties. Most recently, we described novel LRRC8 chimeric channel constructs that allow detailed molecular study of homomeric channels with physiologically relevant functional properties and defined stoichiometry and assembly. Our chimera studies uniquely demonstrated that 1) the LRRC8A intracellular loop, IL1, has unique structural features, 2) it is required for cell volume sensing, 3) the LRRC8 C-terminus is required for sensing changes in intracellular ionic strength and 4) both the LRRC8A IL1 and C-terminus are required for correct cellular processing of VRAC/LRRC8 channels. The overarching goal of this R01 renewal application is to utilize these novel LRRC8 chimeras to better elucidate VRAC/LRRC8 channel structure-function relationships. We will characterize the roles of the LRRC8A C-terminus in VRAC/LRRC8 channel regulation and will test the hypothesis that the LRRC8 IL1 determines VRAC/LRRC8 channel pore properties and regulates channel gating. We will also determine the cryo-EM structure of a unique LRRC8 chimera in multiple physiologically relevant conformations. Our studies will provide novel insights into the regulation and function of VRAC/LRRC8 channels and will provide a higher confidence foundation for detailed mutagenesis-based structure-function analyses.