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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.

我们研究了钠泵亚型在培养的小脑颗粒细胞中的表达和调节。细胞表达三种Na泵亚型（α1、α2和α3亚型），但α1亚型在基础条件下充当主要离子泵。在培养的脑神经元中，谷氨酸刺激神经元后，α2/α3亚型的离子泵活性显著增加。谷氨酸作用主要由非NMDA受体介导。我们研究了在培养的大脑神经元的异构体活动的差异调节的机制。在基础条件下，α2/α3亚型的K^+转运效率低于α1亚型。低效率是由于生理浓度的细胞外钾抑制α2/α3亚型。当K^+浓度超过1 mM时，α2/α3亚型的活性受到显著抑制，但α 1亚型的活性不受抑制。而谷氨酸兴奋神经元后，钾对α2/α3亚型的抑制作用消失。用KN-93（CaM激酶Ⅱ抑制剂）或W-7（钙调素拮抗剂）孵育神经元，可解除细胞外钾对α2/α3亚型的抑制。α2/α3亚型的转运效率提高，而总的K^+吸收活性没有增加。在KN-93存在下，用莫能菌素（钠离子载体）刺激神经元增加了总活性，并模仿谷氨酸兴奋的作用。这些结果表明，谷氨酸兴奋神经元通过两种机制增加α2/α3亚型活性。首先，它从细胞外钾的抑制中释放α2/α3亚型，其次，它通过增加细胞内钠浓度来激活亚型。在基础状态下，Na泵α亚基在原位磷酸化，谷氨酸受体激动剂刺激神经元后，α亚基磷酸化程度发生变化。