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

在人类TMC基因家族(TMC1-8)中有八种已知的跨膜通道样(TMC)亚型， 其中几种与遗传性人类疾病有关。疾病之间的联系，以及对比和 TMC基因在组织中的广泛分布，表明该基因家族对人类的重要性 生理学和病理生理学。然而，尽管有大量研究，哺乳动物TMC的确切功能 蛋白质与其他基因家族没有同源性，一直是个谜。最近的优雅研究 根据TMC1和/或TMC2基因缺失小鼠的听觉毛细胞记录，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。然而， 尽管引起了极大的兴趣，但哺乳动物TMC基因功能的直接功能分析一直受到 在异源表达系统中不能在细胞表面表达TMC蛋白，以便于研究 它们被认为是离子通道或它们的调节亚单位。因此，TMC基因家族构成了一个 稀有实体：一个功能不确定的孤儿基因家族，有几个已确定的疾病联系。寻求 为了解决这一重大的知识差距，我们假设TMC蛋白质需要其他蛋白质才能到达 细胞表面。我们开发了一个简单的表面表达屏幕，发现TMC1的表面表达 被KCNQ1电压门控钾(Kv)通道α亚基特异性拯救。我们还有 发现TMC1抑制典型的KCNQ1电流，而形成具有新属性的新电流。 这些数据将人类TMC1定义为一种新型的通道亚单位，为期待已久的功能铺平了道路 哺乳动物TMC基因的研究。我们现在建议定义TMC1的功能和机制 病理生物学，并向我们和其他小组开放TMC家族的其他成员进行功能研究 尽可能地促进未来发现与TMC相关的人类疾病的治疗方法。在目标1中， 我们将测试有关TMC1在KCNQ1络合物中的功能作用的基本假设， 确定TMC1如何被激活以及KCNQ1的哪些功能属性被TMC1和USE改变 反过来说。在目标2中，创新地应用了高通量表面暴露分析，然后是两个 不同的高通量功能分析(基于荧光和电生理)，我们将测试 假设所有八种TMC蛋白质都是离子通道亚基，并且它们与其他蛋白质形成复合体 Kvα亚单位基因家族成员。总体目标是发现基本的功能 为数不多的与神秘疾病相关的人类离子通道基因家族之一的机制属性。

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.