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) 中有八种已知的跨膜通道 (TMC) 亚型， 其中一些与人类遗传疾病有关，以及对比和对比。 TMC基因普遍广泛的组织分布，表明该基因家族对人类的重要性 然而，尽管有大量的研究，哺乳动物 TMC 的精确功能。 与其他基因家族没有同源性的蛋白质在最近的研究中仍然是个谜。 根据删除 Tmc1 和/或 Tmc2 基因的小鼠听觉毛细胞的记录，强烈建议 在毛细胞机械转导中的作用，解释了这些基因与遗传性耳聋的联系。 尽管人们对此非常感兴趣，但对哺乳动物 TMC 基因功能的直接功能分析一直受到以下因素的阻碍： 无法在异源表达系统的细胞表面表达 TMC 蛋白，以方便研究 因此，它们作为离子通道或其调节亚基的假定作用构成了一个 罕见实体：功能不确定的孤儿基因家族，与几种已确定的疾病相关。 为了解决这一知识上的重大差距，我们发现 TMC 蛋白需要其他蛋白才能到达 我们开发了一个简单的表面表达筛选，发现 TMC1 表面表达。 是由 KCNQ1 电压门控钾 (Kv) 通道 α 亚基专门拯救的。 发现 TMC1 抑制典型的 KCNQ1 电流，而是形成具有新颖属性的新电流。 这些数据将人类 TMC1 定义为一种新型通道亚基，为期待已久的功能性通道亚基铺平了道路。 我们现在建议定义 TMC1 功能和哺乳动物 TMC 基因的研究。 病理生物学，并尽快开放 TMC 家族的其他成员，供我们和其他小组进行功能研究 尽可能促进未来发现 TMC 相关人类疾病的治疗方法。 我们将测试有关 TMC1 在与 KCNQ1 复合物中的功能作用的基本假设， 确定 TMC1 如何被激活以及 TMC1 改变 KCNQ1 的哪些功能特性，反之亦然 反之亦然，在目标 2 中，创新应用了高通量表面暴露测定，然后进行了两次测定。 不同的高通量功能测定（基于荧光和电生理）我们将测试 假设所有八种 TMC 蛋白都是离子通道亚基，并且它们与其他蛋白形成复合物 四十强 Kv α 亚基基因家族的成员总体目标是发现基本功能。 为数不多的与疾病相关的神秘疾病相关人类离子通道基因家族之一的机械属性。