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Analysis of regulational mechanism of Na pump isoform activities using cells transfected neuron-type isoform.

利用转染神经元型亚型的细胞分析钠泵亚型活性的调节机制。

基本信息

批准号：
09680768
负责人：
INOUE Nobuo
金额：
$ 2.11万
依托单位：
Yokohama City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680768/
关键词：
Na, K-pump Na, K-ATPase isoform neuron regulation gene transfection

项目摘要

Na pump isoforms, "common-type (alpha)" and "neuron-type (alpha2 and alpha3)" isoforms, were expressed in cultured rat cercbral neurons. When the cells were excited with glutamate or maintained in energy-deficient states, the K^+ uptake activities of Na pump isoforms were regulated differently : that of neuron-type isoform increased but that of alpha isoform decreased. To analyze mechanism(s) of the differential regulation of K^+ uptake activities of the isoforms in the neurons, we used T3-3-3 cells which were established by transfecing alpha3 subunit cDNA into BALB/c 3T3 cells and expressed both preexisting alpha1 isoform and transfected alpha3 isoform, In the IC uptake activity of the cells, affinity of a3 isoform for intracellular Na^+ was lower than that of alpha1 isoform. Patch-clamp technique demonstrated that pump current of alpha3 isoform was activated by membrane depolarization but that of alpha1 isoform was not. These findings suggest that the differential regulation of K^+ uptake activities of the isoforms after excitation with glutamate results from the difference in pump kinetics between the isoforms. However, the inhibition of the K^+ uptake activity of alpha1 isoform after excitation occurred in mature neurons, in contrast activation of the activity occurred in immature neurons. Therefore, some mechanism(s) in intracellular signal transduction may also be involved in the differential regulation and developed during maturation in culture. To analyze the mechanism, the T3-3-3 cells were treated with dibutyryl cAMP or forskolin or PMA, however, no or less effect on the isoform activities was detected. Affinity of alpha3 isoform for ATP was smaller than that of alpha1 isoform in the ATPase activity of the T3-3-3 cells. Therefore, it is speculated that a part of the regulation of the Na pump isoform activities in energy-deficient states of the neurons is due to differences in affinities for ATP between the isoforms.

Na泵亚型“普通型(α)”和“神经元型(α2和α3)”亚型在培养的大鼠脑神经元中表达。当细胞被谷氨酸兴奋或维持在能量缺乏状态时，Na泵亚型的K^+摄取活性受到不同的调节：神经元型亚型的K^+摄取活性增加，而α亚型的K^+摄取活性降低。为了分析神经元中K^+摄取活性差异调节的机制，我们使用了通过将α3亚基cDNA转染到BALB/c 3T3细胞中而建立的T3-3-3细胞，该细胞表达预先存在的α1同种型和转染的α3同种型。在细胞的IC摄取活性中，a3同种型对细胞内Na^+低于α1亚型。膜片钳技术表明，α3 异构体的泵浦电流是由膜去极化激活的，但 α1 异构体的泵浦电流则不然。这些发现表明，谷氨酸激发后同工型 K^+ 摄取活性的差异调节是由于同工型之间泵动力学的差异造成的。然而，兴奋后α1亚型的K^+摄取活性的抑制发生在成熟神经元中，相反，该活性的激活发生在未成熟神经元中。因此，细胞内信号转导的一些机制也可能参与差异调节并在培养成熟过程中发展。为了分析其机制，用二丁酰cAMP或毛喉素或PMA处理T3-3-3细胞，然而，没有检测到对亚型活性的影响或影响较小。在T3-3-3细胞的ATP酶活性中，α3亚型对ATP的亲和力小于α1亚型。因此，推测神经元能量缺乏状态下Na泵同种型活性的部分调节是由于同种型之间对ATP的亲和力的差异造成的。