Regulation of Kv2 potassium channel diversity and function in brain by electrically silent subunits

Kv2家族的电压门控性K+通道(Kv2.1和Kv2.2)在脑内高度表达，具有重要的 以及哺乳动物神经元的多种功能。首先，Kv2通道作为调节神经元动作电位和膜兴奋性的电压激活通道发挥着重要的典型作用，而基因突变或 有针对性地删除这些通道会导致多动和癫痫发作。第二，Kv2频道高度 聚集在神经元胞体和近端树突上，在组织中具有单独的结构作用 称为内质网-质膜(ER-PM)连接的特殊微域，其作用是 脑神经元中钙和脂质信号的重要中枢。KV2频道通过与 Er VAP蛋白通过Kv2 C末端尾部的一个高度保守的PRC结构域。KV2频道被认为是 主要由Kv2.1和Kv2.2亚基组成，它们可以共同组装形成同四聚体或异四聚体 频道。Kv2频道分集的另一个潜在来源--但在很大程度上尚未开发--来自电力 沉默或附属K+通道(KVS)亚单位基因。这个范围广泛、高度保守的家庭被称为 因为它们需要与Kv2.1或Kv2.2共同组装才能形成功能 在异种细胞中表达的离子传导通道。基因表达分析表明 然而，KVS亚单位家族成员在不同的脑区和神经元亚型中表达不同 目前还没有关于大脑中天然KVS-KV2通道的蛋白质水平的信息。在预赛中 基于质谱学的蛋白质组学实验，我们鉴定了几个与Kv2.1相互作用的KVS亚基 并产生针对这些亚单位的特异性抗体。使用这些抗体，我们已经使主要的 发现一个KVS亚基是Kv2通道的一个非常常见的成分，并且它与 皮层神经元亚群中ER-PM连接的KV2。因此，这项探索性研究的目的是 鉴定这些KVS家族成员的神经元表达和亚细胞定位，并确定 它们对哺乳动物脑神经元Kv2通道多样性的贡献。这项探索性工作将提供 KVS亚基选择性表达如何影响大脑和脑内Kv2通道多样性的重要新见解 它如何调节Kv2通道的功能和在不同神经元群体中的定位。

Voltage-gated K+ channels of the Kv2 family (Kv2.1 and 2.2) are highly expressed in brain and have important and diverse functions in mammalian neurons. First, Kv2 channels play important canonical roles as voltage-activated channels that regulate neuronal action potentials and membrane excitability, and genetic mutations or targeted deletions of these channels cause hyperactivity and seizures. Second, Kv2 channels are highly clustered on neuronal cell bodies and proximal dendrites and have a separate structural role in organizing specialized microdomains called endoplasmic reticulum - plasma membrane (ER-PM) junctions, which serve as important hubs for Ca and lipid signaling in brain neurons. Kv2 channels organize these sites by interacting with ER VAP proteins via a highly conserved PRC domain in the Kv2 C-terminal tail. Kv2 channels are thought to be largely composed of Kv2.1 and Kv2.2 subunits, which can co-assemble to form homo- or hetero-tetrameric channels. Another potential - but largely unexplored - source of Kv2 channel diversity comes from electrically silent or accessory K+ channel (KvS) subunit genes. This extensive and highly conserved family was termed “electrically silent” because they require co-assembly together with Kv2.1 or Kv2.2 in order to form functional ion-conducting channels when expressed in heterologous cells. Gene expression analyses demonstrate that KvS subunit family members are differentially expressed in distinct brain regions and neuron subtypes, however there is currently no information available on native KvS-Kv2 channels in brain at the protein level. In preliminary mass spectrometry-based proteomics experiments, we identified several KvS subunits that interact with Kv2.1 in brain and generated specific antibodies to these subunits. Using these antibodies, we have made the major discovery that one KvS subunit is a remarkably common constituent of Kv2 channels and that it colocalizes with Kv2 at ER-PM junctions in subsets of cortical neurons. The objective of this exploratory study, therefore, is to characterize the neuronal expression and subcellular localization of these KvS family members, and to define their contribution to Kv2 channel diversity in mammalian brain neurons. This exploratory work will provide important new insight into how selective expression of KvS subunits impacts Kv2 channel diversity in brain and how it modulates Kv2 channel function and localization in different neuronal populations.