Cloning and functional analysis of Cl-ATPase 51kDa subunit

CL-ATPase通过维持细胞内氯离子浓度而抑制由GABA引起的神经传递L*我们先前报道阿尔茨海默病患者脑内Cl-ATPase活性降低，小剂量淀粉样蛋白β(Aβ)抑制Cl-ATPase活性，导致细胞内氯离子水平升高。在……里面。在本研究中，(1)克隆了氯-ATPase 51 kDa亚基，(2)研究了磷脂酰肌醇-4-磷酸对AP诱导的氯-ATPase抑制的影响。(1)采用活性红柱纯化，提高了蛋白的回收率，并建立了检测氯-ATPase活性的灵敏方法。(2)低剂量(10 Nm)的A-β处理2天后，Cl-ATPase活性下降47%，而Na/K-ATPase*阴离子不敏感的ATPase活性无明显变化。磷脂酰肌醇(PI50-750 nM)和磷脂酰肌醇-4-磷酸依赖地恢复了被Aβ抑制的氯-三磷酸腺苷酶活性。加入75 nM PI或PI4P后，细胞内氯浓度由Aβ升高3倍恢复到正常水平。低剂量Aβ(10 Nm)加10μM谷氨酰胺作用于神经元可引起DNA断裂，细胞存活率下降，低密度脂蛋白释放量增加。75 nM的PI或PI4P修复了这些细胞损伤。人们普遍认为谷氨酸的神经毒性与阿尔茨海默病的发病有关。我们证明了细胞内氯离子浓度的升高和Cl-ATPase的降低可能参与了阿尔茨海默病神经退行性变的发病机制。此外，我们认为肌醇可能用于治疗或阻止阿尔茨海默病神经退行性变的进展。

Cl-ATPase enables the inhibitory neurotransmission by GABA by keeping the intracellular chloride concentration l* we previously reported that Cl-ATPase activity decreased in Alzheimer's disease brains and that low dose amyloid β(Aβ) inhibited Cl-ATPase activity leading to the elevation of intracellular chloride levels. In. the pres* study, (1)we performed the cloning of Cl-ATPase 51kDa subunit and (2)we examined the effects phosphatidylinositol 4 phosphate on Ap-induced inhibition of Cl-ATPase.(1)We introduced reactive red column for the purification of Cl-ATPase to increase the recovery of the protein and set up sensitive assay for Cl-ATPase activity. We have identified several candidate proteins by mass spectrometric analysis on prot* spots separated two-dimensional electrophoresis.(2)Low dose(10nM) of Aβ treatment for 2 days decreased Cl-ATPase activity by 47%, while did not affect Na/K-ATPase * anion insentive ATPase activities. Phosphatidylinositol(PI 50-750nM) and phosphatidylinositol-4-phosphate do* dependency recovered the Cl-ATPase activity that was suppressed by Aβ. Intracellular Cl concentration that was increa* three times by Aβ returned to normal by adding 75nM PI or PI4P. Low dose of Aβ(10nM) plus 10μM glutam* administration to neurons induced DNA fragmentation and reduced cell viability as shown by increased LDL release and WST assay. Seventy-five nM PI or PI4P recovered these cell damages. It is widely accepted that glutamate neurotoxicity involved in the pathogenesis of Alzheimer's disease. We demonstrated that elevation of intracellular chloride concentration * to the reduction of Cl-ATPase was possibly involved in the pathogenesis of neurodegeneration in Alzheimer's disease, * addition, we suggested that inositol might be used for the treatment or prevention of the progression of neurodegeneraton Alzheimer's disease.