Elucidation of mechanisms regulating Na pump isoform activities after neuronal excitation.

阐明神经元兴奋后调节钠泵亚型活性的机制。

基本信息

批准号：
11680759
负责人：
INOUE Nobuo
金额：
$ 2.37万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680759/
关键词：
Neuron cerebellar granule cells Na, K-pump Na, K-ATPase isoform ion transport phosphorylation 脱リン酸化

项目摘要

We investigated expression and regulation of Na pump isoforms in cultured cerebellar granule cells. The cells expressed three Na pump isoforms (α1, α2 and α3 isoforms), however the α1 isoform acted as a main ion pump under basal conditions. The ion pump activity of the α2/α3 isoforms increased remarkably after stimulation of the neurons with glutamate as reported previously in cultured cerebral neurons. The glutamate effects were mainly mediated by non-NMDA receptors. We examined mechanism of the differential regulation of the isoform activities in cultured cerebral neurons. An efficiency of K^+ transport of the α2/α3 isoform was lower than that of the α1 isoform under basal conditions. The low efficiency was due to inhibition of the α2/α3 isoform by physiological concentrations of extracellular potassium. The α2/α3 isoform activity was remarkably inhibited at more than 1 mM K^+, but not the a1 isoform. In contrast, the inhibition of the α2/α3 isoform by potassium was vanished after glutamate excitation of the neurons. Incubating the neurons with KN-93 (inhibitor of CaM kinase II) or W-7 (calmodulin antagonist) released the α2/α3 isoform from the inhibition by extracellular potassium. The transport efficiency of the α2/α3 isoform was raised without increasing the total K^+ uptake activity. Stimulation of the neurons with monensin (sodium ionophore) in the presence of KN-93 increased the total activity and mimicked the effects of glutamate excitation. These results suggest that glutamate excitation of the neurons increases the α2/α3 isoform activity by two mechanisms. First it releases the α2/α3 isoform from the inhibition by extracellular potassium, and second it activated the isoform by increasing intracellular sodium concentration. We also found α subunit of the Na pump was phosphorylated in situ under basal conditions, and an extent of the phosphorylation of the α subunit changed after stimulation the neurons with glutamate agonists.

我们研究了培养的小脑颗粒细胞中Na泵同工型的表达和调节。细胞表达三个Na泵同工型（α1，α2和α3同工型），但是在基本条件下，α1同工型充当主离子泵。如先前在培养的脑神经元中报道的神经元刺激神经元后，α2/α3同工型的离子泵活性显着增加。谷氨酸作用主要由非NMDA受体介导。我们检查了培养的脑神经元中同工型活性差异调节的机制。在基本条件下，α2/α3同工型的K^+转运效率低于α1同工型。低效率是由于细胞外钾的物理浓度抑制了α2/α3同工型。 α2/α3同工型活性在超过1 mM K^+下明显抑制，但不是A1同工型。相反，在神经元的谷氨酸兴奋后消失了钾对α2/α3同工型的抑制。将神经元与KN-93（CAM激酶II的抑制剂）或W-7（钙调蛋白拮抗剂）一起孵育α2/α3同工型从细胞外钾的抑制中释放出α2/α3同工型。提高了α2/α3同工型的转运效率，而不增加K^+摄取活性。在存在KN-93的情况下，用MONENSIN（钠离子载体）刺激神经元增加了总活性，并模仿了谷氨酸兴奋的作用。这些结果表明，神经元的谷氨酸兴奋会通过两种机制增加α2/α3同工型活性。首先，它从细胞外钾的抑制作用中释放出α2/α3同工型，其次，它通过增加细胞内钠浓度激活了同工型。我们还发现，Na泵的α亚基在基本条件下原位磷酸化，并且在用谷氨酸激动剂刺激神经元后，α亚基的磷酸化发生了变化。