Structural and functional investigation of the human CLC-7/OSTM1 complex

氯离子通道(ClC)家族的成员存在于所有门中，哺乳动物中有9个成员。氯-质子逆向转运体ClC-7属于ClC家族，是溶酶体、晚期内体以及破骨细胞分泌酸性的质膜区褶皱边缘的主要ClC转运体。人类或小鼠的ClC-7基因突变会导致骨化病和溶酶体储存疾病。氯离子转运蛋白ClC-7揭示了ClC家族的几个典型特征，包括它的二聚体拓扑结构和离子转运途径的结构。此外，该转运体遵循经典的CLC转运体比率，即两个Cl-对一个H+。然而，CLC-7有一些非常耐人寻味的特征，这些特征使它有别于CLC家族的其他成员。例如，ClC-7的电压依赖性激活和失活要比其他ClC转运体慢得多。此外，ClC-7是唯一一个需要辅助蛋白-骨化相关膜蛋白1(OSTM1)的成员，既要稳定又要发挥功能。ClC-7和OSTM1共同定位于溶酶体和破骨细胞的褶皱边缘。OSTM1是一种1型跨膜蛋白，含有一个大的、高度糖基化的N-末端、一个跨膜的螺旋和短的C-末端。CLC-7-、OSTM1-和CLC-7/OSTM1缺陷小鼠寿命短，并发展成严重的骨石化、视网膜变性、溶酶体储存病和神经变性。到目前为止，其辅助蛋白OSTM1对ClC-7的调控机制仍是一个悬而未决的问题。我提出了一种结合结构、电生理和生化工具的多学科方法来研究ClC-7/OSTM1。在我在宿主实验室的头几个月里，我能够建立从鸡体中分离纯化ClC-7的方法。然后，我用冷冻电子显微镜(Cryo-EM)对纯化的样品进行了分析，发现有300多万个均匀分布的单一颗粒。接下来，我的目标是研究单独的人ClC-7(Aim 1)和CLC-7与OSTM1的复合体(Aim 2)的功能和结构。将该复合体的冷冻-EM结构与ClC-7的分离形式进行比较，将有助于识别重要的调控区域，并有助于揭示ClC-7蛋白的独特调控性质。然后，我将使用脂质重组的ClC-7和ClC-7/OSTM1样本，通过基于荧光的通量分析和电生理测量，进一步研究ClC-7的调控机制。这些研究将最终确定ClC-7/OSTM1相互作用和调控的机制。除了ClC-7/OSTM1的分子特征外，这些研究还将促进我们对ClC-7/OSTM1复合体的生理和病理作用的理解。

Members of the chloride channel (CLC) family occur in all phyla, with nine members present in mammals. The chloride-proton antiporter CLC-7 belongs to the CLC family and is the major CLC transporter in lysosomes, late endosomes and in the ruffled border, the acid-secreting plasma membrane domain of osteoclasts. Mutations of CLC-7 in humans or mice cause osteopetrosis and lysosomal storage diseases. The Cl- transporter CLC-7 reveals several features typical for the CLC family including its dimeric topology and the architecture of the ion translocation pathway. Also, the transporter obeys the classical CLC transporter ratio of two Cl- to one H+. However, CLC-7 has some very intriguing features which distinguish it from the rest of the CLC family. For instance, the voltage-dependent activation and deactivation of CLC-7 is much slower than other CLC transporters. Furthermore, CLC-7 is the only member that requires an accessory protein, osteopetrosis-associated membrane protein 1 (OSTM1), for both stability and function. CLC-7 and OSTM1 are co-localized in lysosomes and in the ruffled border of osteoclasts. OSTM1 is a type 1 transmembrane protein containing a large, highly glycosylated N-terminus and single membrane-spanning helix and short C-terminus. CLC-7-, OSTM1- and CLC-7/OSTM1-deficient mice have short life spans and develop severe osteopetrosis, retinal degeneration, lysosomal storage diseases, and neurodegeneration. So far, the mechanism of CLC-7 regulation by its accessory protein OSTM1 remains an open question. I propose a multidisciplinary approach combining structural, electrophysiological and biochemical tools for investigation of CLC-7/OSTM1. During my first months in the host laboratory, I was able to establish the protocol for purification of CLC-7 from Gallus gallus. I then analyzed the purified sample by cryo-electron microscopy (cryo-EM), which revealed over 3,000,000 well-distributed single particles. Next, I aim to investigate the function and structure of human CLC-7 alone (Aim 1) and CLC-7 in complex with OSTM1 (Aim 2). Comparison of the cryo-EM structure of the complex with the isolated form of CLC-7 will facilitate identification of important regulatory regions, and will help unravel the unique regulatory nature of the CLC-7 protein. I will then further investigate the regulatory mechanism of CLC-7 by fluorescence-based flux assays and electrophysiological measurements, using lipid-reconstituted samples of CLC-7 and CLC-7/OSTM1. These investigations will ultimately determine the mechanism of CLC-7/OSTM1 interaction and regulation. Beyond the molecular characterization of CLC-7/OSTM1, these studies will promote our understanding of the physiological and pathological roles of the CLC-7/OSTM1 complex.