Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB
Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB
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DOI：
10.1073/pnas.1805757115
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发表时间：
2018-07-31
影响因子：
11.1
通讯作者：
Tamkun, Michael M.
中科院分区：
文献类型：
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作者：
Johnson, Ben;Leek, Ashley N.;Tamkun, Michael M.
Kv2.1在神经细胞质膜上表现出两种不同的定位模式：一种是自由扩散的，通过电压依赖的K+电导调节电活动；另一种是定位于微米级的簇，这些簇包含密集的但不导电的通道。我们先前已经证实，这些簇代表内质网/质膜(ER/PM)连接，起到膜运输中心的作用，并且Kv2.1在原代神经元培养和转基因HEK细胞中都在形成这些膜接触位点方面发挥着结构性作用。聚集和ER/PM接触的形成由通道C末端的磷酸化调节，使细胞能够快速、动态地控制皮质ER和PM之间的物理关系本研究探讨了Kv2.1及其相关的Kv2.2通道与内质网相互作用的机制。在转基因的HEK细胞中使用基于邻近的生物素化技术，我们确定ER VAMP相关蛋白(VAP)是潜在的Kv2.1相互作用因子。Kv2.1和-2.2结合VAPA和VAPB的确认采用了共定位/重分布、siRNA敲除和基于Forster共振能量转移(FRET)的检测。使用包含Kv2.1C末端序列的CD4嵌合体来鉴定非规范的VAP结合基序。VAP最初被鉴定为海兔神经递质释放所需的蛋白质，现在已知是丰富的支架蛋白，参与整个内质网的膜接触部位的形成。VAP相互作用组包括AKAP、激酶、膜转运机制和调节内质网到质膜的非囊泡脂质运输的蛋白质因此，Kv2诱导的ER/PM接触部位的VAP浓度预计将对神经细胞生物学产生广泛的影响。
Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Forster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.