Cloning and functional analysis of Cl-ATPase 51kDa subunit

Cl-ATP酶通过维持细胞内氯离子浓度而使GABA抑制神经传递。我们以前报道过阿尔茨海默病脑中Cl-ATP酶活性降低，低剂量淀粉样蛋白β（Aβ）抑制Cl-ATP酶活性，导致细胞内氯离子水平升高。In.在前期研究的基础上，（1）克隆了Cl-ATP酶51 kDa亚基，（2）研究了磷脂酰肌醇4-磷酸对AP诱导的Cl-ATP酶抑制的影响。(1)We采用活性红柱纯化Cl-ATP酶，提高了蛋白质的回收率，建立了灵敏的Cl-ATP酶活性测定方法。我们已经确定了几个候选蛋白质的质谱分析蛋白质分离的双向电泳。（2）低剂量（10 nM）Aβ处理2天，使Cl-ATP酶活性降低47%，而对Na/K-ATP酶 * 阴离子不敏感的ATP酶活性无影响。磷脂酰肌醇（PI 50- 750 nM）和磷脂酰肌醇-4-磷酸依赖性恢复了被Aβ抑制的Cl-ATP酶活性。加入75 nM PI或PI 4P后，Aβ使细胞内Cl浓度增加3倍，细胞内Cl浓度恢复正常。低剂量Aβ（10 nM）加10μM glutam* 给予神经元可诱导DNA断裂并降低细胞活力，如LDL释放增加和WST试验所示。75 nM PI或PI 4P恢复了这些细胞损伤。谷氨酸神经毒性参与阿尔茨海默病的发病机制已被广泛接受。我们证明了细胞内氯离子浓度的升高和Cl-ATP酶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，我们还提出了肌醇可能用于治疗或预防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.