Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum
Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum
复制标题
深入了解小鼠小脑中独特电压传感器蛋白 (TMEM266) 的功能及其短形式
DOI：
10.1042/bcj20220033
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发表时间：
2022
影响因子：
4.1
通讯作者：
Okamura Y
中科院分区：
文献类型：
--
作者：
Kawai T;Narita H;Konno K;Akter S;Andriani RT;Iwasaki H;Nishikawa S;Yokoi N;Fukata Y;Fukata M;Wiriyasermkul P;Kongpracha P;Nagamori S;Takao K;Miyakawa T;Abe M;Sakimura K;Watanabe M;Nakagawa A;Okamura Y
电压传感蛋白一般由电压传感结构域和孔门结构域组成，形成电压门控离子通道。然而，有几种非常规的电压传感器蛋白缺乏孔门结构域，赋予它们独特的电压传感机制。TMEM266在小脑颗粒细胞中表达，是一种有趣的电压感应蛋白，它具有一个假定的细胞内线圈和一个功能未知的细胞质区域，而不是一个孔门结构域。在这里，我们通过进行共免疫沉淀实验来研究TMEM266的分子功能。我们意外地发现，TMEM266蛋白与小脑中仅具有电压传感器结构域和假定的细胞质线圈区的新型短形式剪接变体相互作用。TMEM266的线圈区晶体结构表明，这些线圈区在形成同型二聚体中起重要作用。体外表达实验支持短形式TMEM266 （sTMEM266）或全长TMEM266 （fTMEM266）形成同型二聚体的观点。我们还利用神经母细胞瘤细胞neuro2a对fTMEM266和sTMEM266进行了接近标记质谱分析，fTMEM266比sTMEM266显示出更多的相互作用分子，这表明fTMEM266的c端细胞质区与多种靶标结合。最后，tmem266缺陷动物在野外实验中表现出中度异常。本研究为TMEM266介导的新型电压传感机制提供了线索。
Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers.In vitroexpression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.