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

我们研究了钠泵异构体在培养的小脑颗粒细胞中的表达和调控。细胞表达3种钠泵亚型(α-1、α-2和α-3)，但在基础条件下，α-1亚型是主要的离子泵。谷氨酸刺激培养的大脑神经元后，α-2/α-3异构体的离子泵活性显著增加。谷氨酸的作用主要由非NMDA受体介导。我们研究了培养的大脑神经元异构体活性的差异性调节机制。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胞外钾抑制了α-2/α-3亚型。当K~(2+)浓度大于1 mM时，α-2/α-3亚型活性受到显著抑制，而A1亚型活性无明显变化。而谷氨酸刺激后，钾对α-2/α-3亚型的抑制作用消失。用KN-93(CaM激酶II抑制剂)或W-7(钙调蛋白拮抗剂)孵育神经元，可使α2/α3亚型从细胞外钾的抑制中释放出来。α-2/α-3异构体的转运效率在不增加总K~+吸收活性的情况下得到提高。在KN-93存在的情况下，用莫能菌素(钠离子载体)刺激神经元，可增加神经元的总活性，并模拟谷氨酸兴奋的效应。这些结果表明，谷氨酸兴奋神经元可通过两种机制增加α2/α3亚型的活性。首先，它使α-2/α-3亚型从细胞外钾的抑制中释放出来，其次，它通过增加细胞内钠离子浓度来激活该亚型。我们还发现，在基础条件下，Na泵的α亚基被原位磷酸化，谷氨酸激动剂刺激神经元后，α亚基的磷酸化程度发生了变化。

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.