CLC2氯離子通道交互作用蛋白質之鑑定

Abstract

CLC2氯離子通道廣泛地分布在各種組織，並且會受到過極化電位、細胞膨脹、細胞外微酸環境以及細胞內氯離子濃度增加而活化。近來研究發現，CLC2在大鼠海馬迴的錐狀細胞中，具有協助胞內氯離子向外排除之作用。此外，CLC2被推測在老年小鼠的神經保護機制中可能扮演角色。然而CLC2在神經細胞中確切的分子調控機轉仍有待釐清。因此，本篇論文的研究方向在尋找大鼠腦中與CLC2有交互作用之蛋白質，並探討這些蛋白質如何影響CLC2，進而對CLC2的基本性質與生理角色有更深入的了解。 本實驗室過去利用酵母菌雙雜交技術，篩選出大鼠腦部cDNA library中幾個可能與CLC2具有交互作用的蛋白質，本研究更進一步利用共同免疫沉澱法與GST pull-down確認其交互作用關係。結果顯示其中的四個蛋白質與CLC2具有交互作用。我們由免疫螢光染色實驗得知，共同表現交互作用蛋白質並不會影響CLC2在細胞內的分佈位置。然而我們目前尚無法確定交互作用蛋白質是否會影響CLC2總表現量，因此需要日後的實驗結果佐證。未來我們將利用電生理技術以及其他生化實驗方法，分析交互作用蛋白在CLC2的生物物理特性與細胞內的生合成所扮演之角色。

CLC2 channel is a ubiquitously expressed voltage-gated chloride channel that is activated by hyperpolarization, cell swelling, extracellular acidification, and rise of intracellular chloride concentration. In recent studies, CLC2 has been suggested to assist chloride extrusion in hippocampal pyramidal cells. In addition, CLC2 has been implicated to have neuroprotective roles in aging mice. However, the precise cellular regulatory properties of CLC2 channel in neurons remain elusive. Therefore, we aimed to understand the physiological roles and cellular signaling mechanisms of CLC2 channels by searching for novel CLC2-interacting proteins. Previous studies in our lab utilizing yeast-two hybrid screening of rat brain cDNA library has identified several potential CLC2-interacting proteins. In this study, we further confirmed these interactions by using co-immunoprecipitation and GST pull-down assay. Our data suggest that four of these candidate proteins display significant interaction with CLC2. Immunofluorescence analysis revealed that these candidate proteins failed to affect the subcellular localization of CLC2 in HEK293T cells. It is still unclear whether total CLC2 expression may be affected in the presence of candidate proteins and thus required further studies. In the future, we plan to apply electrophysiological and biochemical assays to determine whether these interacting candidates can affect the biophysical and biosynthetic properties of CLC2 channels.