Electrically silent KvS subunits associate with native Kv2 channels in brain and impact diverse properties of channel function.
Electrically silent KvS subunits associate with native Kv2 channels in brain and impact diverse properties of channel function.
复制标题
电沉默 KvS 亚基与大脑中的天然 Kv2 通道相关联，并影响通道功能的多种特性。
DOI：
10.1101/2024.01.25.577135
复制
发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Trimmer,JamesS
中科院分区：
文献类型：
--
作者：
Ferns,Michael;vanderList,Deborah;Vierra,NicholasC;Lacey,Taylor;Murray,Karl;Kirmiz,Michael;Stewart,RobertG;Sack,JonT;Trimmer,JamesS
Kv 2家族的电压门控K+通道在脑中高度表达，并且在调节神经元兴奋性和组织内质网-质膜（ER-PM）连接中起双重作用。在异源细胞中的研究表明，两个成孔α亚基Kv2.1和Kv2.2与“电沉默”KvS亚基组装形成具有不同生物物理特性的异源四聚体通道。在这里，使用基于质谱的蛋白质组学，我们确定了五个KvS亚基作为从小鼠大脑免疫纯化的天然Kv2.1通道的组成部分，最丰富的是Kv5.1。我们发现Kv5.1与Kv2.1共免疫沉淀，并且在较小程度上与来自脑裂解物的Kv2.2共免疫沉淀，并且Kv5.1蛋白水平在Kv2.1敲除小鼠中降低了70%，在Kv2.1/2.2双敲除小鼠中降低了95%。啮齿动物脑切片的多重免疫荧光标记显示，在新皮质中，Kv5.1免疫标记在大部分Kv2.1和Kv2.2阳性第2/3层神经元中以及较小比例的第5层和第6层神经元中明显。在亚细胞水平上，Kv5.1与Kv2.1和Kv2.2在皮层神经元的ER-PM连接处共聚集，尽管含有Kv5.1的通道的聚集相对于同源Kv 2通道减少。我们还发现，在异源细胞中，与Kv5.1共表达减少了聚集，并改变了Kv2.1通道的药理学特性。总之，这些研究结果表明，Kv5.1电沉默亚基是天然脑Kv 2通道的一个重要组成部分，其纳入异源通道可以影响Kv 2通道功能的各个方面。
Voltage-gated K+ channels of the Kv2 family are highly expressed in brain and play dual roles in regulating neuronal excitability and in organizing endoplasmic reticulum - plasma membrane (ER-PM) junctions. Studies in heterologous cells suggest that the two pore-forming alpha subunits Kv2.1 and Kv2.2 assemble with “electrically silent” KvS subunits to form heterotetrameric channels with distinct biophysical properties. Here, using mass spectrometry-based proteomics, we identified five KvS subunits as components of native Kv2.1 channels immunopurified from mouse brain, the most abundant being Kv5.1. We found that Kv5.1 co-immunoprecipitates with Kv2.1 and to a lesser extent with Kv2.2 from brain lysates, and that Kv5.1 protein levels are decreased by 70% in Kv2.1 knockout mice and 95% in Kv2.1/2.2 double knockout mice. Multiplex immunofluorescent labelling of rodent brain sections revealed that in neocortex Kv5.1 immunolabeling is apparent in a large percentage of Kv2.1 and Kv2.2-positive layer 2/3 neurons, and in a smaller percentage of layer 5 and 6 neurons. At the subcellular level, Kv5.1 is co-clustered with Kv2.1 and Kv2.2 at ER-PM junctions in cortical neurons, although clustering of Kv5.1-containing channels is reduced relative to homomeric Kv2 channels. We also found that in heterologous cells coexpression with Kv5.1 reduces the clustering and alters the pharmacological properties of Kv2.1 channels. Together, these findings demonstrate that the Kv5.1 electrically silent subunit is a component of a substantial fraction of native brain Kv2 channels, and that its incorporation into heteromeric channels can impact diverse aspects of Kv2 channel function.