mGluR3激活对海马星形胶质细胞双孔钾通道TWIK-1膜转运的调节和机制研究

TWIK-1, a two-pore domain potassium channel, is the most abundant potassium channel expressed in mature cortical and hippocampal astrocytes. However, the function of TWIK-1 in native astrocytes remains unclear. Our preliminary data showed that TWIK-1 is preferentially located in cytoplasm, but not membrane of hippocampal astrocytes. In heterologous system, activation of Gi/Go coupled receptor leads to an increasing membrane insertion of TWIK-1. Mature astrocytes in vivo only express Gi/Go coupled metabotropic glutamate receptor 3 (mGluR3). While the function of mGluR3 remains unknown, this receptor certainly can sense and transduce neuronal glutamate signaling into astrocyte. Therefore, it is sensible to hypothesize that variation of extracellular glutamate dynamically transmits neuronal activity to astrocytes by regulating TWIK-1 membrane expression. We here propose to investigate the role of mGluR3 activation on TWIK-1 membrane expression and its functional impact on astrocyte physiology in hippocampal slices. We will achieve these aims by using of wild type and TWIK-1 knockout mice as animal model. Immunofluorescence staining will be combined with western blot to quantify the ratio of TWIK-1 on membrane versus cytoplasm. Whole-cell patch clamp will be combined with living cell imaging technique to record membrane properties and relative Na+ to K+ permeability upon mGluR3 activation. As for the signaling pathway mechanisms, small G protein ARF6 and Rab11b are critical for TWIK-1 internalization and recycling to membrane, therefore their activities will be examined before and after mGluR3 activation. This proposal should be the first to address the functional relationship between astrocytic mGluR3 and TWIK-1 in the context of regulation of TWIK-1 membrane insertion in native cells. The resultant data should provide insights into the physiological role of TWIK-1 channel in the still mysterious function of astrocyte in the adult brain and pathophysiological relevance of TWIK-1 in glutamate induced excitotoxicity in disease states.

双孔钾通道TWIK-1是脑成熟星形胶质细胞表达量最高的钾通道，功能尚不明确。最近申报者首次观察到TWIK-1主要位于星形胶质细胞胞质，胞膜表达很少。体外研究发现Gi/Go偶联受体活化可促进TWIK-1转位到细胞膜，而Gi/Go偶联的代谢型谷氨酸受体3（mGluR3）是成熟星形胶质细胞唯一高表达的mGluR类型，结合前期结果我们推测mGluR3激活可能调控星形胶质细胞TWIK-1膜转位。本项目拟以野生型和TWIK-1 基因敲除小鼠脑片为对象，免疫荧光染色和蛋白印迹法检测海马星形胶质细胞mGluR3激活是否促进TWIK-1蛋白从胞质向胞膜转运；以脑片膜片钳联合活细胞成像，观察膜电生理特性和膜对Na+通透性的变化；并分析膜蛋白内化和再循环关键信号分子在TWIK-1膜表达中的作用。本项目将首次揭示脑TWIK-1通道的生理功能和调节机制，阐明谷氨酸兴奋性毒性产生的新机理，为拮抗脑损伤提供新分子靶点。

项目背景：双孔钾通道TWIK-1是脑成熟星形胶质细胞（星胶）表达量最高的钾通道，功能尚不明确。近期申报者观察到TWIK-1主要位于星胶胞质，胞膜表达很少。体外研究发现Gi/Go偶联受体活化可促进TWIK-1转位到细胞膜，而Gi/Go偶联的代谢型谷氨酸受体3（mGluR3）是成熟星胶唯一高表达的mGluR类型，我们推测mGluR3激活可能调控星胶TWIK-1膜转位。研究内容：本项目以野生型和TWIK-1 基因敲除小鼠为对象，采用脑片膜片钳联、免疫荧光染色和蛋白印迹法等多种技术，研究了1）mGluR3激活是否促进星胶TWIK-1通道从胞质转运到胞膜，以及涉及的信号转导机制；2）TWIK-1通道上膜后的可能作用。主要研究结果：1）免疫荧光染色显示海马星胶mGluR3激活明显促进TWIK-1蛋白从胞质向胞膜转运； 2）TWIK-1是一种阳离子非选择性通道，可以通透Na+。mGluR3激活后野生型小鼠星胶膜电位轻度去极化，而TWIK-1 KO星胶的膜电位保持不变，从功能角度提示mGluR3激活促进星胶TWIK-1通道膜转位；3）mGluR3激活增加星胶摄取细胞外NH4+的能力，增幅达30%，而TWIK-1 KO小鼠的星胶没有此变化，提示TWIK-1通道上膜后的可能作用之一是增加对胞外NH4+的摄取；4）mGluR3激活增强小G蛋白Rab11和SNARE复合体介导的内体再循环过程，可能是海马星胶TWIK-1通道转运至细胞膜的信号机制。科学意义：1）TWIK-1在膜上的表达很可能是一个可被双向调控的过程，静息时主要贮存于胞质；当脑内环境稳态出现波动时，如谷氨酸堆积，TWIK-1将转位到细胞膜。2）星胶内谷氨酰胺合成的两条关键途径都需要NH4+参与，因此TWIK-1上膜后摄取细胞外NH4+可能参与星胶主导的谷氨酸-谷氨酰胺循环，及时清除神经元释放至细胞外的谷氨酸，并为神经元补充新的谷氨酸，满足神经元活动的需要。本项目的完成揭示了星胶TWIK-1通道的重要生理功能和调节机制，为阐明谷氨酸兴奋性毒性的产生提供新机理，为拮抗脑损伤提供新分子靶点。