Shao Dedong, Tan Jinru, Fan Xiaozhi, Shu Yilai, Qu Qianhui, Tang Yi-Quan
State Key Laboratory of Brain Function and Disorders, MOE Frontiers Center for Brain Science, Institutes of Brain Science, ENT Institute and Otorhinolaryngology Department of Eye & ENT Hospital, Fudan University, Shanghai 200032, China.
NHC Key Laboratory of Hearing Medicine, Fudan University, Shanghai 200032, China.
Int J Mol Sci. 2025 Jul 1;26(13):6356. doi: 10.3390/ijms26136356.
Transmembrane channel-like (TMC) proteins are essential for hearing and balance; however, the molecular mechanisms that regulate their proper folding and membrane targeting remain poorly understood. Here, we establish as a genetically tractable model to dissect TMC-1 trafficking by combining CRISPR knock-in strains, super-resolution microscopy, and genome-wide forward genetic screening. We show that TMC-1 robustly localizes to the plasma membrane in both neurons and muscle cells and identify a conserved valine (V803) in transmembrane domain 9 (TM9) as critical for its biogenesis and trafficking. Structural analyses guided by AlphaMissense and AlphaFold uncover two evolutionarily conserved functional hotspots, one in the extracellular loop adjacent to TM9 and the other in the TMC signature motif, which are interconnected by an evolutionarily conserved disulfide bond. Disrupting this bond in worm TMC-1 abolishes its cell-surface localization and destabilizes the mechanotransduction channel complex. Together, these findings provide a structural framework for interpreting deafness-causing mutations in human TMC1 and highlight disulfide-bond-linked hotspots as key molecular determinants of TMC protein biogenesis and trafficking.
跨膜通道样（TMC）蛋白对听觉和平衡至关重要；然而，调节其正确折叠和膜定位的分子机制仍知之甚少。在这里，我们通过结合CRISPR敲入菌株、超分辨率显微镜和全基因组正向遗传筛选，建立了一个易于遗传操作的模型来剖析TMC-1的运输过程。我们发现TMC-1在神经元和肌肉细胞中都能稳定地定位于质膜，并确定跨膜结构域9（TM9）中的一个保守缬氨酸（V803）对其生物合成和运输至关重要。由AlphaMissense和AlphaFold指导的结构分析揭示了两个进化上保守的功能热点，一个在与TM9相邻的细胞外环中，另一个在TMC特征基序中，它们通过一个进化上保守的二硫键相互连接。破坏线虫TMC-1中的这个二硫键会消除其细胞表面定位，并使机械转导通道复合物不稳定。总之，这些发现为解释人类TMC1中导致耳聋的突变提供了一个结构框架，并突出了二硫键连接的热点作为TMC蛋白生物合成和运输的关键分子决定因素。
