Functional characterization of the dark matter ion channel polycystin2-like2

项目摘要 离子通道是多跨膜蛋白，每秒转运约106至107个离子穿过 膜。人类基因组编码约400种不同的离子通道（占基因组的1.5%）， 受精、增殖、发育、学习和记忆。虽然它是经过充分验证的 离子通道是许多疾病的核心，批准的药物只适用于一小部分人， 这类蛋白质。扩大离子通道作为潜在药物靶点的主要瓶颈是 事实上，几种离子通道在其生物学功能和调节方面的特征很差。 离子通道的亚细胞区室化到细胞器膜如内质网， 内体或初级纤毛进一步使详细的生物物理表征复杂化。因此，A 大量的离子通道有资格作为人类基因组的暗物质，其功能未知。一个这样 暗物质离子通道是PKD2-L2，多囊蛋白通道家族的三个成员之一。突变 其他多囊蛋白成员引起多种人类疾病，从先天性心脏病， 多器官（肝、肾和胰腺）囊肿形成的偏侧性缺陷。多囊蛋白通道是 富含初级纤毛，顶端膜的触角状突起，但分子机制是 对哪些多囊蛋白通道被调节并因此对纤毛信号传导起作用仍然知之甚少。的 该项目的中心目标是功能性表征PKD2-L2含有多囊蛋白通道，并确定 PKD2-L2如何参与纤毛信号传导。有两个具体目标。第一个目标的特点是 含有PKD2-L2的同聚和异聚多囊蛋白通道的生物物理性质。第二个目的 是确定同源和异源PKD2-L2通道的亚细胞定位。申请人的 初步观察包括几种新的未发表的方法。该项目的完成将是一个关键 进一步了解初级纤毛内离子通道信号的基本原理。我们的 长期目标是了解纤毛离子通道失调如何引起人类纤毛病，并建立 PKD2-L2作为治疗靶点。

PROJECT SUMMARY Ion channels are multispan transmembrane proteins that transport ~106 to 107 ions per second across membranes. The human genome encodes ~400 different ion channel (1.5% of the genome) that control such diverse processes as fertilization, proliferation, development, learning and memory. Although it is well validated that ion channels are at the core of many diseases, approved drugs are available for only a small percentage of this protein class. A major bottleneck in expanding the palette of ion channels as potential drug targets is the fact that several ion channels are only poorly characterized with respect to their biological function and regulation. Subcellular compartmentalization of ion channels to organellar membranes such as endoplasmic reticulum, endosomes or primary cilia further complicates a detailed biophysical characterization. As a consequence, a significant number of ion channels qualify as dark matter of the human genome with unknown function. One such dark matter ion channel is PKD2-L2, one of three members of the polycystin channel family. Mutations in the other polycystin members cause a plethora of human diseases, ranging from congenital heart disease and laterality defects to cyst formation in multiple organs (liver, kidney and pancreas). Polycystin channels are enriched in primary cilia, antenna-shaped protrusions of the apical membrane but the molecular mechanisms by which polycystin channels are regulated and thus contribute to ciliary signaling remains poorly understood. The central goal of this project is to functionally characterize PKD2-L2 containing polycystin channels and determine how PKD2-L2 contributes to ciliary signaling. There are two specific aims. The first aim characterizes the biophysical properties of homomeric and heteromeric polycystin channels containing PKD2-L2. The second aim is to determine the subcellular localization of homomeric and heteromeric PKD2-L2 channels. The applicants’ preliminary observations include several novel unpublished methods. Completion of this project will be a critical step towards understanding the fundamental principles of ion channel signaling within primary cilia. Our long‐ term goal is to understand how dysregulation of ciliary ion channels cause human ciliopathies and establish PKD2-L2 as a therapeutic target.