氯離子通道為一種離子傳遞蛋白質，常見於各種型態的細胞中，其主要的功能包括：細胞體積的調節、膜間物質的傳遞、膜間電位的調控以及調節胞內酸化。研究發現各種不同的物種基因庫中均存在著氯離子通道蛋白的基因序列，由此證實其與細胞或個體的生存有著密不可分的關係。我們自黏菌的EST基因庫中釣出了一段氯離子通道基因序列，將其命名為clcA基因，clcA轉譯產生的ClcA蛋白共包含了863個氨基酸，並且經比對後發現ClcA與老鼠的氯離子通道蛋白RnCLC-7有最高的相似性，而RnCLC-7蛋白經研究指出其功能是在於蝕骨細胞的外酸化作用。因此，為了找出ClcA蛋白的功能，我們先利用南方墨點法分析出clcA為單一拷貝基因，利用反轉錄聚合酶連鎖反應得知clcA基因於發育期的16～20小時間具有最大表現量，之後利用同源重組作用，我們創造出clcA基因剔除突變株。此突變株於細菌培養基上展現出一種叢枝狀的多細胞結構，但在進行缺乏養分的發育實驗中，此一奇特表型卻不復見，只是延遲發育約6~8小時，而令人驚訝的是，我們將clcA-突變株細胞與細菌混和進行發育實驗後，又再度觀察到其特殊表型。雖然在進行胞內酸化測定及細菌吞噬作用能力分析試驗後，比較突變株與野生株的結果並無差別，但我們認為clcA-突變株的表型或缺失應與此種細菌的存在或其代謝物及分泌物有著直接的關係。另外，我們利用數位基因轉殖法釣出了另一段黏菌氯離子通道基因clcB，經由比對後發現其與植物的阿拉伯芥氯離子通道蛋白AtCLC-d有著最高的相似性，這似乎也應證了黏菌兼具動植物行為的特性。

Chloride channels are passive anion transport proteins involved in functions common to all cells, such as regulation of cell volume, transepithelial salt transport, electrical excitability and intracellular acidification. There is evidence from genome sequencing for multiple chloride channels in a wide rang of species ranging from bacteria to human. ClcA, a putative Dictyostelium chloride channel protein is identified from the EST database, composes of 863 amino acid residues and has 34% identities to mouse chloride channel protein CLC-7 that provides the chloride conductance required for an efficient proton pumping by the H+-ATPase of the osteoclast ruffled membrane and induced extracellular acidification. Southern blot analysis indicated clcA is a single copy gene. RT-PCR analysis indicated that clcA mRNA was expressed with a higher level during 16-20 hr of development. To understand the functions of ClcA, we created the clcA- cells, which contain a disrupted gene generated by homologous recombination. clcA- cells aggregate to form a multiple tip-like structure and shorter fruiting bodies on bacterial agar, but the strange structures were not observed in development. It just delayed about 6-8 hr. Surprisingly, we recreated the multiple tip-like phenotype of clcA- cells in development by mixing with bacteria, so we consider that the defects of clcA- cells may relate to the existence of bacteria. To identify the defects of acidification in clcA- cells, different NH4+ concentration development and measurement of intracellular pH were processed. We also used FACS to determine phagocytosis ability of wild-type and clcA- cells, but the results were shown that there was no difference in intracellular pH and phagocytosis between mutant and wild-type cells. Furthermore, using digital-cloning, we have identified another chloride channel gene, clcB. It indicated that there may be some functional redundance of chloride channel protein in Dictyostelium.

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