Discovering the function of a putative ion channel family linked to inherited diseases

发现与遗传性疾病相关的假定离子通道家族的功能

• 批准号: 9333887
• 负责人: Geoffrey W Abbott
• 金额: $ 27.04万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333887
• 关键词: Address; Affect; Arrhythmia; Auditory; Biochemical; Biological Assay; Biotinylation; Cell Line; Cell membrane; Cell surface; Colon; Complex; Corneal dystrophy; Data; Diabetes Mellitus; Disease; Drug or chemical Tissue Distribution; Electrophysiology (science); Endoplasmic Reticulum; Epilepsy; Epithelial Cells; Family; Fluorescence; Functional disorder; Future; Gene Family; Genes; Goals; Hair Cells; Home environment; Human; In Vitro; Inherited; Ion Channel; Ion Channel Protein; Ions; Knowledge; Link; Literature; Malignant Neoplasms; Measures; Molecular Mechanisms of Action; Mus; Mutation; Orphan; Pharmacology; Physiology; Potassium; Property; Protein Isoforms; Proteins; Regulation; Research; Rest; Role; Sequence Homology; Skin Cancer; Stimulus; Surface; System; Testing; Therapeutic; Tissues; Variant; base; deafness; drug development; gene function; genetic variant; human disease; in vitro Assay; in vivo; innovation; interest; mechanotransduction; member; mouse model; novel; positional cloning; prevent; screening; stoichiometry; therapeutic development; trafficking; voltage

There are eight known Transmembrane channel-like (TMC) isoforms in the human TMC gene family (TMC1-8), several of which are linked to inherited human diseases. The disease linkages, and the contrasting and generally wide tissue distribution of the TMC genes, indicate the importance of this gene family to human physiology and pathophysiology. Yet, despite numerous studies, the precise function of mammalian TMC proteins, which share no homology with other gene families, has remained enigmatic. Recent elegant studies relying upon recordings in auditory hair cells from mice with Tmc1 and/or Tmc2 genes deleted strongly suggest a role in hair cell mechanotransduction, explaining the linkage of these genes to inherited deafness. However, despite great interest, direct functional analysis of mammalian TMC gene function has been hampered by an inability to express TMC proteins at the cell surface in heterologous expression systems, to facilitate study of their putative roles as ion channels or their regulatory subunits. The TMC gene family therefore constitutes a rare entity: an orphan gene family of uncertain function, with several established disease linkages. Seeking to address this major gap in knowledge, we hypothesized that TMC proteins require other proteins to reach the cell surface. We developed a simple surface expression screen and discovered that TMC1 surface expression is specifically rescued by the KCNQ1 voltage-gated potassium (Kv) channel α subunit. We have also discovered that TMC1 inhibits the typical KCNQ1 current, instead forming a new current with novel attributes. The data define human TMC1 as a novel type of channel subunit, paving the way for long-awaited functional studies of mammalian TMC genes. We now propose to define mechanisms of TMC1 function and pathobiology, and to open up the rest of the TMC family to functional study by us and other groups, as quickly as possible, to facilitate future discovery of therapeutic approaches for TMC-linked human diseases. In Aim 1, we will test fundamental hypotheses regarding the functional role of TMC1 in complexes with KCNQ1, determining how TMC1 is activated and which functional properties of KCNQ1 are altered by TMC1 and vice versa. In Aim 2, with an innovative application of a high-throughput surface exposure assay followed by two different high-throughput functional assays (fluorescence-based and electrophysiological) we will test the hypothesis that all eight TMC proteins are ion channel subunits, and that they form complexes with other members of the forty-strong Kv α subunit gene family. The overall goal is to discover the basic functional mechanistic attributes of one of the few remaining enigmatic disease-linked human ion channel gene families.

人类TMC基因家族（TMC1-8）中有八个已知的跨膜通道样（TMC）同工型， 其中一些与遗传的人类疾病有关。疾病联系，对比和 通常，TMC基因的组织分布表明该基因家族对人的重要性 生理学和病理生理学。然而，多皮特众多研究，哺乳动物TMC的精确函数 与其他基因家族共享同源性的蛋白质仍然是神秘的。最近的优雅研究 依赖于TMC1和/或TMC2基因的小鼠的听觉毛细胞的录音很强 在毛细胞机械传导中的作用，解释了这些基因与遗传性耳聋的联系。然而， 尽管感兴趣，但哺乳动物TMC基因功能的直接功能分析受到了 无法在异源表达系统中的细胞表面表达TMC蛋白，以促进研究 他们作为离子渠道或监管亚基的推定角色。因此，TMC基因家族构成 稀有实体：一个不确定功能的孤儿基因家族，具有几种已建立的疾病联系。寻求 解决知识中的这一主要差距，我们假设TMC蛋白需要其他蛋白质才能达到 细胞表面。我们开发了一个简单的表面表达屏幕，发现TMC1表面表达 由KCNQ1电压门控钾（KV）通道α亚基特异性检索。我们也有 发现TMC1抑制了典型的KCNQ1电流，而是形成具有新型属性的新电流。 数据将人类TMC1定义为一种新型的通道亚基，为期待已久的功能贴上了道路 哺乳动物TMC基因的研究。现在，我们建议定义TMC1功能的机制和 病理生物学，并将TMC家族的其余部分开放给我们和其他群体的功能研究 尽可能促进未来发现与TMC连接的人类疾病的治疗方法。在AIM 1中， 我们将检验有关TMC1在KCNQ1复合物中的功能作用的基本假设， 确定TMC1如何激活以及KCNQ1的哪些功能特性被TMC1和VICE改变了 Versa。在AIM 2中，具有高通量表面暴露测定的创新应用，然后是两个 不同的高通量功能测定（基于荧光和电生理学）我们将测试 假设所有八种TMC蛋白都是离子通道亚基，并且它们与其他 40个KVα亚基基因家族的成员。总体目标是发现基本功能 剩下的少数剩下的神秘疾病与人离子通道基因家族之一的机械属性。