Regulation of Kv2.1 K(+) conductance by cell surface channel density.
Regulation of Kv2.1 K(+) conductance by cell surface channel density.
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DOI：
10.1523/jneurosci.3008-12.2013
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发表时间：
2013-01-16
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影响因子：
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通讯作者：
Tamkun MM
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文献类型：
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作者：
Fox PD;Loftus RJ;Tamkun MM
Kv2.1电压门控性钾离子通道在海马神经元胞索马、近端树突和轴突起始段的细胞膜上自由扩散，并以微米级簇的形式集中分布。在转染的HEK细胞中，簇微域内的Kv2.1通道是不导电的。使用TIRF显微镜，将HEK细胞表面上GFP标记的Kv2.1通道的数量与通过相同细胞的全细胞电压钳测量的K+通道电导进行比较。这种方法表明，随着通道密度的增加，非聚集通道停止传导。在观察到的最高密度下，所有通道中只有4%是导电的。突变Kv2.1通道，未能集群也具有17%的导电K+在较高的表面密度的非导电状态。非传导状态是Kv2.1特异性的，因为Kv1.4总是传导的，而不管细胞表面表达水平如何。抗Kv2.1免疫荧光强度，标准化为转染的HEK细胞中的Kv2.1表面密度，用于测定培养的大鼠海马神经元中内源性Kv2.1的表达水平。将内源性Kv2.1水平与通过全细胞电压钳确定的传导通道的数量进行比较。在培养14天和20天的神经元中，分别只有13%和27%的内源性Kv2.1传导。总之，这些数据表明，非导电状态主要取决于表面密度，而不是集群的位置，这种非导电状态也存在于培养的海马神经元中发现的原生Kv2.1。Kv2.1蛋白相对于K+电导的这种过量进一步支持Kv2.1在可兴奋组织中的非传导作用。
The Kv2.1 voltage-gated K+ channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are non-conducting. Using TIRF microscopy the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared to K+ channel conductance measured by whole-cell voltage-clamp of the same cell. This approach indicated that as channel density increases non-clustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the non-conducting state with 17% conducting K+ at higher surface densities. The non-conducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immuno-fluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared to the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 days, respectively. Together these data indicate that the non-conducting state depends primarily on surface density as opposed to cluster location and that this non-conducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K+ conductance further supports a non-conducting role for Kv2.1 in excitable tissues.