Neuronal Cell Biology of Kv2.1-induced Endoplasmic Reticulum/Plasma Membrane Contact sites

Kv2.1K+通道是体内表达最丰富、分布最广的电压门控性K+通道 哺乳动物。我们之前的研究表明，除了作为延迟整流钾通道发挥作用外， 调节质膜电位，Kv2.1的非传导性多数群体形成内质 网状/质膜(ER/PM)接触部位。在海马神经元中，Kv2.1通道与 皮质内质网在近端胞体表面产生微米大小的Kv2.1簇 树突和轴突的起始段。文献中的数据表明，ER/PM连接调节神经元爆发 激发后，非囊泡的脂质直接从内质网转移到细胞表面，质膜上的PIP2水平升高。 我们的初步数据显示，Kv2.1诱导的ER/PM连接，而不是其他ER/PM连接，改变了ER 钙离子稳态、质膜组织和胞吐作用。有趣的是，Kv2.1与 皮质内质网受神经元活动和中风样损伤的调节，如缺氧、缺血和过度 谷氨酸，表明与这些微域相连的功能在多动或 神经性侮辱。因此，拟议的研究检验了Kv2.1的一种新的非导电功能，即1)是 神经生理学的中枢；2)受神经元活动、伤害和中风的调节。三位一体 具体目标将阐述Kv2.1改变ER钙稳态和 体细胞ER/PM连接的膜蛋白定位和突触前ER/PM接触的胞吐作用。目标 1.检验Kv2.1诱导的ER/PM接触部位通过以下途径促进商店操作的钙离子内流的假设 提供局部K+电导。初步数据表明，神经元内内质网钙离子再充盈增强 表达Kv2.1。目的2.验证Kv2.1和皮质肌动蛋白协同作用的假说 控制钙信号蛋白在ER/PM连接附近的定位。初步数据 提示Kv2.1诱导的ER/PM连接影响Cav1.2、BK K+通道和b2的细胞表面分布 肾上腺素能受体。目的3.验证突触囊泡胞吐受Kv2.1调控的假说 突触前终末ER/PM交界处的通道。初步数据显示， 内源性和转染型Kv2.1定位于突触前终末，基于shRNA的敲除 Kv2.1可在不影响动作电位的情况下抑制50%的谷氨酸能囊泡胞吐。而Kv2.1 导致人类癫痫脑病的点突变改变了通道电导，这是点突变的一个子集 与发育迟缓有关的基因在通道C末端诱导不应该提前停止的密码子 影响电导。相反，这些突变被预测只会阻止Kv2.1与皮质内质网结合。 因此，影响Kv2.1电导和皮层内质网重塑作用的突变是人类的基础 疾病。这项建议中的研究将极大地促进我们对Kv2.1- 含有ER/PM的接触部位在神经生理学中发挥作用。

The Kv2.1 K+ channel is the most abundantly expressed and widely distributed voltage-gated K+ channel in mammals. Our previous research demonstrates that in addition to functioning as a delayed rectifier K+ channel and regulating plasma membrane potential, a non-conducting, majority population of Kv2.1 forms endoplasmic reticulum/plasma membrane (ER/PM) contact sites. In hippocampal neurons Kv2.1 channel binding to the cortical endoplasmic reticulum generates micron-sized Kv2.1 clusters on the surface of the soma, proximal dendrites and axon initial segment. Data in the literature indicate that ER/PM junctions regulate neuronal burst firing, the non-vesicular lipid transfer directly from the ER to the cell surface, and plasma membrane PIP2 levels. Our preliminary data show that the Kv2.1-induced ER/PM junctions, but not other ER/PM junctions, alter ER Ca2+ homeostasis, plasma membrane organization, and exocytosis. Interestingly, Kv2.1 interaction with the cortical ER is regulated by neuronal activity and stroke-like insults such as hypoxia, ischemia and excess glutamate, indicating that the functions linked to these microdomains are remodeled following hyperactivity or neuronal insult. Thus, the proposed research examines a novel non-conducting function of Kv2.1 that 1) is central to neuronal physiology and 2) is regulated by neuronal activity, insult and stroke. The three Specific Aims will address the molecular mechanisms by which Kv2.1 alters ER Ca2+ homeostasis and membrane protein localization at somatic ER/PM junctions and exocytosis at presynaptic ER/PM contacts. Aim 1. To test the hypothesis that Kv2.1-induced ER/PM contact sites enhance store-operated Ca2+ entry by providing localized K+ conductance. Preliminary data suggest that ER Ca2+ refilling is enhanced in neurons expressing Kv2.1. Aim 2. To test the hypothesis that the concerted action of Kv2.1 and cortical actin controls the localization of Ca2+ signaling proteins in the vicinity of ER/PM junctions. Preliminary data indicate Kv2.1-induced ER/PM junctions influence the cell surface distribution of Cav1.2, BK K+ channels and b2 adrenergic receptors. Aim 3. To test the hypothesis that synaptic vesicle exocytosis is modulated by Kv2.1 channels at the ER/PM junction in presynaptic terminals. Preliminary data demonstrate that both endogenous and transfected Kv2.1 is localized at presynaptic terminals and that shRNA-based knockdown of Kv2.1 suppresses glutamatergic vesicle exocytosis by 50% without affecting the action potential. While Kv2.1 point mutations that cause human epileptic encephalopathy alter channel conductance, a subset of point mutants that are linked to developmental delay induce premature stop codons in the channel C-terminus that should not affect conductance. Instead, these mutations are predicted to only prevent Kv2.1 binding to the cortical ER. Thus, mutations affecting both the conductance and cortical ER remodeling roles of Kv2.1 underlie human disease. The research in this proposal will substantially advance our understanding of the role that Kv2.1- containing ER/PM contact sites play in neuronal physiology.