TMC gene function in sensory hair cells

描述（由申请人提供）：跨膜通道样基因1 （TMC1）突变是人类显性进行性听力损失（DFNA36）和隐性非综合征性感音神经性听力损失（DFNB7/B11）的基础（Kurima et al, 2002）。同样，Tmc1的半显性和隐性等位基因导致贝多芬（Bth）和耳聋（dn）突变小鼠的听力损失（Vreugde等人，2002；Kurima等人，2002）。Tmc1是Tmc基因家族的一员，该家族在哺乳动物中还包括其他7个类似基因（Keresztes et al., 2003）。Tmc1和密切相关的Tmc2在小鼠内耳的听觉和前庭毛细胞中表达。我们最近证明缺乏Tmc1和Tmc2的小鼠会失聪并遭受严重的前庭功能障碍。这些数据表明，Tmc1和Tmc2对正常毛细胞功能至关重要，但它们的确切分子功能尚不清楚。TMC1和TMC2蛋白可能是折叠或转运转导分子所需的细胞体成分，它们可能作为连接蛋白，与机械转导通道机械串联，或者它们可能是毛细胞转导通道本身的成孔亚基。为了调查可能的TMC功能，我们设计了一个系统和全面的研究策略，使我们能够区分这些不同的假设。我们将首先对单独表达Tmc1或Tmc2的突变小鼠的毛细胞转导特性进行彻底的生物物理表征。接下来，我们将把突变和嵌合形式的Tmc1和/或Tmc2引入缺乏这两种基因的毛细胞。我们之前的工作（Kawashima et al., 2011）证明了转染野生型Tmc1或Tmc2的Tmc1/Tmc2敲除小鼠的毛细胞中机械转导的恢复。因此，我们预测，类似的突变Tmc基因实验将为天然毛细胞的结构/功能研究提供一个新的平台，这将使我们能够识别Tmc蛋白的功能。最后，我们将TMC蛋白定位在Tmc1/Tmc2敲除小鼠的毛细胞中，这些小鼠转染了融合短多肽表位标签的野生型和突变型TMC构建物。针对表位标签的特异性抗体的免疫定位将提供比目前可用的定位数据更好的定位数据。综上所述，从拟议的研究中得到的数据将有助于确定毛细胞中TMC蛋白的功能，并可能支持它们作为毛细胞转导装置组成部分的作用。

DESCRIPTION (provided by applicant): Mutations in transmembrane channel-like gene 1 (TMC1) underlie dominant, progressive hearing loss (DFNA36) and recessive nonsyndromic sensorineural hearing loss (DFNB7/B11) in humans (Kurima et al., 2002). Similarly, semidominant and recessive alleles of Tmc1 cause hearing loss in Beethoven (Bth) and deafness (dn) mutant mice (Vreugde et al.,2002; Kurima et al., 2002). Tmc1 is a member of the Tmc gene family that includes seven other paralogs in mammals (Keresztes et al., 2003). Tmc1 and closely related Tmc2 are expressed in auditory and vestibular hair cells of the mouse inner ear. We have recently demonstrated that mice that lack Tmc1 and Tmc2 are deaf and suffer profound vestibular dysfunction. The data suggest that Tmc1 and Tmc2 are essential for normal hair cell function but their precise molecular function remains elusive. TMC1 and TMC2 proteins may be components of the cell body required for folding or trafficking of transduction molecules, they may function as linker proteins, mechanically in series with the mechanotransduction channel or they may be pore-forming subunits of the hair cell transduction channel itself. To investigate possible TMC functions we have designed a systematic and comprehensive research strategy that will allow us to distinguish amongst these various hypotheses. We will begin with a thorough biophysical characterization of the properties of hair cell transduction in mutant mice that express either Tmc1 or Tmc2 alone. Next, we will introduce mutant and chimeric forms of Tmc1 and/or Tmc2 into hair cells that lack both genes. Our previous work (Kawashima et al., 2011) demonstrated rescue of mechanotransduction in hair cells of Tmc1/Tmc2 knockout mice transfected with wild-type Tmc1 or Tmc2. Therefore, we predict that similar experiments with mutant Tmc genes will provide a novel platform for structure/function studies in native hair cells which will allow us to identify the function of TMC proteins. Lastly, e will localize TMC proteins in hair cells of Tmc1/Tmc2 knockout mice transfected with wild-type and mutant TMC constructs fused to short polypeptide epitope tags. Immunolocalization with antibodies specific to the epitope tag will provide localization data superior to that currently available. Taken together, the data that emerge from the proposed studies will help identify the function of TMC proteins in hair cells and may support their role as components of the hair cell transduction apparatus.