ANO5:新的钙激活的氯离子通道的功能

The molecular role of Ca2+ activated Cl- channel(CACC) has been found recently as ANO(Anoctamin)1 and ANO2. As one of the member of the same family, ANO5， also named as TMEM16E, GDD1, LGMD2L mainly expressed in skeleten musal cells, has been first found by us as CACC too. It is reported that ANO5 mutations in patients, chondrocytes and osteoblasts.Mutations in ANO5 result in a spectrum of musculoskeletal disorders and abnormal bone mineralization and bone fragility.ANO5 resides predominantly in intracellular membrane vesicles and also in plasma membrane.ANO5 mutant patient show defective skeletal muscle membrane repair. According to our results ANO5 is also a Ca2+ activated Cl- channel, and it can only be activated under a unphysiology high Ca2+ condition. The hypothesis is ANO5 located in the endoplasmic vesicals can repair plasma membrane wound through exocytosis. We plan to use molecular biology technique and electromicroscope to know the expression and localization and traffic of ANO5 in the cell, and use patch lamp and Ussing chamber recording to detect over expressed of ANO5 to know the function in cell membrane wound repairing.

ANO1和 ANO2 最近被报道是钙激活的氯离子通道的分子基础，作为同一家族的ANO5,又名TMEM16E，GDD1，LGMD2L,主要分布于骨骼肌细胞以及成骨细胞和软骨细胞中。它的基因突变表现为骨骼肌病以及骨骼的矿物质化和变脆。在肌细胞内ANO5主要分布于胞内囊泡，在细胞膜也有表达。ANO5基因突变的病人表现为肌细胞膜损伤修复缺陷。经我们前期工作首次证明ANO5也是钙激活的氯离子通道，而且它只有在超生理条件的高钙作用下才可以被激活。我们认为ANO5很有可能通过胞吐作用，从胞内囊泡转移到细胞膜，进一步参与细胞膜的修复。本课题拟采用分子生物学技术，荧光显微镜技术观察ANO5在细胞内的表达定位和应对细胞膜损伤的反应，膜片钳技术结合基因过表达等方法研究ANO5对于细胞膜损伤修复的作用。

1. 内源性Ano5在骨骼肌的表达：采用免疫荧光技术观察发现，在小鼠发育的不同时期，内源性Ano5均可表达于骨骼肌中。在新生24 h内的小鼠骨骼肌中，Ano5可表达于骨骼肌细胞的胞质及胞膜；在1个月及6个月小鼠的骨骼肌，Ano5只表达于其细胞膜，并且随着小鼠年龄的增长，Ano5的表达逐渐减少。我们的研究结果发现，Ano5蛋白随着骨骼肌的发育表达逐渐减少，这也与肌肉营养不良的病人在后期才出现症状相符。.2. 内源性的Ano5在消化道上皮与肌层的表达：采用免疫荧光技术观察发现，内源性Ano5可表达于食管黏膜层、胃黏膜层、十二指肠黏膜层、结肠黏膜层与直肠黏膜层中；同时Ano5表达于食管上段肌层，但在胃肌层、十二指肠肌层、结肠肌层及直肠肌层中并未见表达。Ano5表达于食管上段骨骼肌，而不表达于消化管其它部位的平滑肌，这与limb-girdle muscular dystrophy 2L (LGMD2L)肌病病人出现食管上段吞咽困难相符。.3. Western blot and RT-PCR：采用WB 及 RT-PCR技术进一步研究发现，内源性Ano5可表达于食管黏膜层、胃黏膜层、十二指肠黏膜层、结肠黏膜层与直肠黏膜层中，同时Ano5表达于食管上段肌层，但在胃肌层、十二指肠肌层、结肠肌层及直肠肌层中并未见表达，这与免疫荧光的结果一致。为下一步在消化道黏膜损伤的情况下，研究Ano5的表达变化奠定基础。.4. Ano5在细胞膜损伤的变化： CFBE细胞含内源性的Ano5，用Ano5抗体可检测到Ano5在细胞将内呈点状均匀分布。在0.01% Triton X-100 处理10分钟后细胞浆周围Ano5阳性点形成线状围绕在细胞周围。说明含Ano5的囊泡在细胞膜受创伤的情况下转移向细胞膜。HEK293细胞含内源性Ano5，0.01% saponin处理1分钟，细胞浆Ano5的表达变少，而细胞膜Ano5表达增多，说明含Ano5的囊泡由胞浆转移到细胞膜，去填补细胞膜破损。

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