Live Imaging of Kv7.2/7.3 Cell Surface Dynamics at the Axon Initial Segment: High Steady-State Stability and Calpain-Dependent Excitotoxic Downregulation Revealed
Live Imaging of Kv7.2/7.3 Cell Surface Dynamics at the Axon Initial Segment: High Steady-State Stability and Calpain-Dependent Excitotoxic Downregulation Revealed
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DOI：
10.1523/jneurosci.2631-15.2016
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发表时间：
2016-02-17
影响因子：
5.3
通讯作者：
Olesen, Soren-Peter
中科院分区：
文献类型：
--
作者：
Benned-Jensen, Tau;Christensen, Rasmus Kordt;Olesen, Soren-Peter
电压门控性钾通道Kv7.2和Kv7.3位于轴突起始段（AIS），对动作电位的产生具有强控制作用。因此，它们的定位或细胞表面数量的变化可能会影响神经元信号传导。然而，没有什么是已知的细胞表面动力学Kv7.2/7.3在稳态或短期神经元刺激。这主要归因于它们的膜拓扑结构，这阻碍了细胞外表位标记。在这里，我们规避这一限制，融合一个额外的根皮素标记的螺旋的N末端的人Kv7.3。这七个跨膜嵌合体，命名为超级黄道phluorin（SEP）-TAC-7.3，功能和交通作为一个野生型（WT）通道。我们在分离的大鼠海马神经元中表达SEP-TAC-7.3，以使用实时成像检查AIS Kv7.2/7.3异聚体的侧向移动性、表面数量和定位。我们发现，它们是非常稳定的，并表现出非常低的表面流动性在稳态和神经元刺激。在后一种情况下，我们还发现，无论是本地化，也没有细胞表面数量的变化。然而，在高谷氨酸负荷下，我们观察到Kv7.2/7.3的快速不可逆内吞，这需要激活含NR 2B的NMDA受体、Ca 2+内流和钙蛋白酶激活。这种兴奋性毒性机制可能对锚蛋白G结合的AIS蛋白具有特异性，因为Nav1.2通道而不是AIS GABA（A）受体也被内吞。总之，我们已经，第一次，其特征在于使用一种新的嵌合策略的全长Kv 7通道的细胞表面动力学。这种方法可能也适用于其他Kv通道，因此对于该离子通道亚家族的额外表征具有价值。
The voltage-gated K+ channels Kv7.2 and Kv7.3 are located at the axon initial segment (AIS) and exert strong control over action potential generation. Therefore, changes in their localization or cell surface numbers are likely to influence neuronal signaling. However, nothing is known about the cell surface dynamics of Kv7.2/7.3 at steady state or during short-term neuronal stimulation. This is primarily attributable to their membrane topology, which hampers extracellular epitope tagging. Here we circumvent this limitation by fusing an extra phluorin-tagged helix to the N terminus of human Kv7.3. This seven transmembrane chimera, named super ecliptic phluorin (SEP)-TAC-7.3, functions and traffics as a wild-type (WT) channel. We expressed SEP-TAC-7.3 in dissociated rat hippocampal neurons to examine the lateral mobility, surface numbers, and localization of AIS Kv7.2/7.3 heteromers using live imaging. We discovered that they are extraordinarily stable and exhibit a very low surface mobility both during steady state and neuronal stimulation. In the latter case, we also found that neither localization nor cell surface numbers were changed. However, at high glutamate loads, we observed a rapid irreversible endocytosis of Kv7.2/7.3, which required the activation of NR2B-containingNMDAreceptors, Ca2+ influx, and calpain activation. This excitotoxic mechanism may be specific to ankyrin G-bound AIS proteins because Nav1.2 channels, but not AIS GABA(A) receptors, were also endocytosed. In conclusion, we have, for the first time, characterized the cell surface dynamics of a full-length Kv7 channel using a novel chimeric strategy. This approach is likely also applicable to other Kv channels and thus of value for the additional characterization of this ion channel subfamily.