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)和隐性非综合征感音神经性听力损失(DFNB7/B11)的基础(Kurima等人，2002年)。同样，TMC1的半显性和隐性等位基因会导致贝多芬(BTH)和耳聋(DN)突变小鼠的听力损失(Vreugde等人，2002年；Kurima等人，2002年)。TMC1是TMC基因家族的成员，该家族包括哺乳动物中的其他七个近亲基因(Keresztes等人，2003年)。TMC1及其亲缘关系密切的TMC2表达于小鼠内耳的听觉和前庭毛细胞。我们最近证明，缺乏TMC1和TMC2的小鼠是聋人，并遭受严重的前庭功能障碍。这些数据表明，TMC1和TMC2对于正常的毛细胞功能是必不可少的，但它们的确切分子功能仍然难以捉摸。TMC1和TMC2蛋白可能是折叠或运输转导分子所需的细胞体的组成部分，它们可能作为连接蛋白发挥作用，机械地与机械转导通道串联，或者它们可能是毛细胞转导通道本身的成孔亚单位。为了研究TMC可能的功能，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说。我们将首先对只表达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，我们将把突变型和嵌合型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等人，2011年)展示了对野生型TMC1或TMC2基因敲除的TMC1/TMC2基因敲除小鼠毛细胞的机械转导的拯救作用。因此，我们预测，对突变的TMC基因进行类似的实验将为研究天然毛细胞的结构/功能提供一个新的平台，这将使我们能够识别TMC蛋白的功能。最后，e将在TMC1/TMC2基因敲除小鼠的毛细胞中定位TMC蛋白，这些小鼠被野生型和突变型TMC构建物融合到短多肽表位标签中。用针对表位标签的抗体进行免疫定位将提供比目前可用的更好的定位数据。综上所述，拟议研究中出现的数据将有助于确定TMC蛋白在毛细胞中的功能，并可能支持它们作为毛细胞转导装置的组成部分的作用。

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.