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

在培养的大鼠脑神经细胞中表达Na泵的“共同型（α）”和“神经元型（α 2和α 3）”亚型。当细胞受到谷氨酸刺激或处于能量缺乏状态时，Na泵异构体的K^+摄取活性受到不同程度的调节：神经元型异构体的K^+摄取活性增加，α型异构体的K^+摄取活性降低。为了分析不同亚型对神经元K^+摄取活性的差异调节机制，我们采用将α 3亚基cDNA转染BALB/c 3T3细胞构建的T3-3-3细胞，同时表达原存在的α 1亚型和转染后的α 3亚型。在细胞的IC摄取活性中，a3亚型对细胞内Na^+的亲和力低于α 1亚型。膜片钳技术表明，α 3亚型的泵电流被膜去极化激活，而α 1亚型的泵电流不被膜去极化激活。这些结果表明，在谷氨酸激发后，两种异构体对K^+摄取活性的不同调节是由于两种异构体之间泵动动力学的差异。然而，成熟神经元兴奋后α - 1亚型的K^+摄取活性受到抑制，而未成熟神经元的K^+摄取活性则被激活。因此，细胞内信号转导的某些机制也可能参与了差异调节，并在培养成熟过程中发展起来。为了分析其作用机制，我们分别用二丁基cAMP、福斯克林或PMA处理t3 - 33细胞，但对其异构体活性没有或影响较小。在T3-3-3细胞的ATP酶活性中，alpha3亚型对ATP的亲和力小于alpha1亚型。因此，我们推测Na泵同工异构体在神经元能量缺乏状态下对活性的部分调控是由于不同同工异构体对ATP的亲和力不同。

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.