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Analysis of localization and regulation of Na, K-pump isoforms in neurons.

神经元中 Na、K 泵亚型的定位和调节分析。

基本信息

批准号：
06680763
负责人：
INOUE Nobuo
金额：
$ 1.41万
依托单位：
Yokohama City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680763/
关键词：
Na, K-pump Na, K-ATPase isoform neuron localization regulation

项目摘要

Three molecular forms of alpha (alpha1, alpha2, and alpha3) subunits of the Na, K-pump isoforms were identified in cultured granule cells, prepared from 7-day old rat cerebella, by Western blot and histochemical analysis. The subunits were shown to localize on surface of soma and neurites of the cells with ankyrin and spectrin by using confocal laser microscopy. The amounts of these subunits were shown to increase during in vitro differentiation by culture. Na, K-ATPase activities of the Na pump isoforms increased correspondingly during culture, and total activity went up to 333 (]SY.+1.[) 32 (nmol/min・mg of protein) after 13 days. The activity was 4.8-fold, higher than basal level. The brain type (alpha2 and alpha3) isoform activity increased 8.9-fold, and the common type (alpha1) only 3.4-fold. By stimulating the differentiated cells with glutamate, the K+ uptake activities of Na pump isoforms were regulated differently and total activity increased about 20%. The brain type isoform activity increased moro than fourfold by the stimulation, but the common type decreased 23%. Therefore, the percentage of the brain type isoform activity increased from 12% to 43% by the stimulation. The regulation of Na, K-Pump activities by glutamate were abolished by calmodulin antagonist W-7 and did not occur in immature neurons. Number of Na, K-pump on surface of the cells estimated by <@D13@>D1H-ouabain binding study did not change by glutamate stimulation. These results suggest that calmodulin may function in the mechanism to regulate the Na, K-pump isoform activities and that the brain type Na, K-pump isofoum in differentiated neuron is important functionally to restore the ionic gradient after excitation.

通过蛋白质印迹和组织化学分析，在培养的颗粒细胞（从 7 日龄大鼠小脑中制备）中鉴定出 Na、K 泵亚型的 α（α1、α2 和 α3）亚基的三种分子形式。通过使用共焦激光显微镜，显示亚基定位于具有锚蛋白和血影蛋白的细胞体和神经突的表面。这些亚基的数量在体外培养分化过程中显示出增加。 Na泵亚型的Na、K-ATP酶活性在培养过程中相应增加，13天后总活性达到333 (]SY.+1.[) 32 (nmol/min·mg of Protein)。活性为基础水平的4.8倍。脑型（α2和α3）亚型活性增加了8.9倍，而普通型（α1）仅增加了3.4倍。通过用谷氨酸刺激分化细胞，Na泵亚型的K+摄取活性受到不同的调节，总活性增加了约20%。刺激后，大脑型亚型活性增加了四倍多，但普通型活性降低了 23%。因此，通过刺激，脑型亚型活性的百分比从12%增加到43%。谷氨酸对 Na、K 泵活性的调节被钙调蛋白拮抗剂 W-7 消除，并且在未成熟的神经元中不会发生。通过<@D13@>D1H-哇巴因结合研究估计的细胞表面Na、K-泵的数量没有因谷氨酸刺激而改变。这些结果表明钙调蛋白可能在调节Na，K-泵亚型活性的机制中发挥作用，并且分化神经元中的脑型Na，K-泵亚型对于恢复激发后的离子梯度具有重要功能。