Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability
Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability
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DOI：
10.1073/pnas.1106201108
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发表时间：
2011-11-01
影响因子：
11.1
通讯作者：
Evans, A. Mark
中科院分区：
文献类型：
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作者：
Ikematsu, Naoko;Dallas, Mark L.;Evans, A. Mark
可兴奋细胞中动作电位的激发加速了 ATP 周转。电压门控钾通道 Kv2.1 调节中枢神经元的动作电位频率，而无处不在的细胞能量传感器 AMP 激活蛋白激酶 (AMPK) 会因 ATP 耗尽而被激活，并通过关闭能量消耗过程来保护细胞。我们发现，用 AMPK 激活剂 A-769662 处理表达 Kv2.1 的 HEK293 细胞会导致通道激活和失活的电流-电压关系发生超极化转变。我们鉴定了 AMPK 在 Kv2.1 上直接磷酸化的两个位点（S440 和 S537），并使用磷酸特异性抗体和定量质谱法表明，在 A-769662 处理的细胞中这两个位点的磷酸化均增加。在用 S440A 表达 Kv2.1 的细胞中，A-769662 的作用被消除，但用 S537A 取代则不会，表明 S440 的磷酸化是造成这些作用的原因。通过贴片移液管引入细胞后，重组 AMPK 具有活性，但通过硫代磷酸化具有磷酸酶抗性，观察到相同的电压门控变化。离子霉素引起的 Kv2.1 门控变化与 A-769662 引起的变化非常相似，但通过涉及 Kv2.1 去磷酸化的不同机制发挥作用。在培养的大鼠海马神经元中，A-769662 会引起与 HEK293 细胞中类似的电压门控超极化转变，这种效应可通过使用 Kv2.1 抗体进行细胞内透析而消除。当通过贴片移液管将活性硫代磷酸化 AMPK 引入培养的神经元时，观察到诱发动作电位的频率逐渐、随时间变化。我们的结果表明，在代谢应激条件下，神经元中 AMPK 的激活通过降低神经元兴奋性从而保存能量来发挥保护作用。
Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.