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

Kv2.1 K+通道是细胞中表达最丰富和分布最广泛的电压门控性K+通道， 哺乳动物我们以前的研究表明，除了作为一个延迟整流钾通道， 和调节质膜电位，Kv2.1的非传导性的大多数群体形成内质网， 内质网/质膜（ER/PM）接触部位。在海马神经元中，Kv2.1通道结合至 皮质内质网在索马表面产生微米大小的Kv2.1簇， 树突和轴突起始段。文献数据表明ER/PM连接调节神经元爆发 发射，非囊泡脂质直接从ER转移到细胞表面，和质膜PIP 2水平。 我们的初步数据表明，Kv2.1诱导的ER/PM连接，而不是其他ER/PM连接，改变ER Ca 2+稳态、质膜组织和胞吐作用。有趣的是，Kv2.1与 皮质ER受神经元活动和中风样损伤如缺氧、缺血和过量 谷氨酸，表明与这些微结构域相关的功能在过度活跃或 神经元损伤因此，拟议的研究检查了Kv2.1的一种新的非导电功能，即：1） 神经元生理学的中枢，以及2）受神经元活动、损伤和中风的调节。三 具体目标将阐述Kv2.1改变ER Ca 2+稳态的分子机制， 在体细胞ER/PM连接处的膜蛋白定位和在突触前ER/PM接触处的胞吐作用。目的 1.为了检验Kv2.1诱导的ER/PM接触位点通过以下方式增强钙池操作的Ca 2+内流的假设： 提供局部K+电导。初步数据表明，ER Ca 2+再填充在神经元中增强， 表达Kv2.1。目标2.为了验证Kv2.1和皮层肌动蛋白的协同作用 控制ER/PM连接附近的Ca 2+信号蛋白的定位。初步数据 表明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。 肾上腺素能受体目标3：为了验证突触囊泡胞吐作用受Kv2.1调节的假设， 在突触前末梢的ER/PM连接处的通道。初步数据显示， 内源性和转染的Kv2.1定位于突触前末端，基于shRNA的Kv2.1基因敲低， Kv2.1抑制50%的多巴胺能囊泡胞吐作用，而不影响动作电位。当Kv2.1 引起人类癫痫性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末端诱导了不应该 影响电导。相反，预测这些突变仅阻止Kv2.1与皮质ER结合。 因此，影响Kv2.1的传导和皮质ER重塑作用的突变是人类免疫缺陷的基础。 疾病本提案中的研究将大大促进我们对Kv2.1- 含有ER/PM接触位点的蛋白质在神经元生理学中起作用。

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.