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

Kv2.1 诱导的内质网/质膜接触位点的神经细胞生物学

• 批准号: 9973443
• 负责人: Michael Blake Hoppa
• 金额: $ 41.93万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9973443
• 关键词: Actins; Action Potentials; Address; Adrenergic Receptor; Affect; Axon; Binding; Biology; Brain; Brain Hypoxia-Ischemia; C-terminal; Cell membrane; Cell surface; Cells; Cellular biology; Chimera organism; Coupled; Data; Dendrites; Developmental Delay Disorders; Diffusion; Dominant-Negative Mutation; Endoplasmic Reticulum; Estrogen receptor positive; Exocytosis; Glutamates; Goals; Hippocampus (Brain); Homeostasis; Human; Hyperactive behavior; Image; Ions; Ischemic Stroke; Lead; Length; Link; Lipids; Literature; Mammals; Mediating; Membrane; Membrane Potentials; Membrane Proteins; Molecular; Monitor; Mutation; Neurons; Nonsense Codon; Organelles; Pattern; Phosphatidylinositol 4,5-Diphosphate; Physiology; Play; Point Mutation; Population; Potassium Channel; Presynaptic Terminals; Property; Proteins; Rattus; Recovery; Regulation; Research; Resolution; Role; SNAP receptor; Scaffolding Protein; Signal Transduction; Signaling Protein; Sirolimus; Site; Stroke; Structure; Surface; Synaptic Vesicles; System; Testing; Transfection; Vesicle; Voltage-Gated Potassium Channel; Work; base; cell type; epileptic encephalopathies; experimental study; human disease; knock-down; large-conductance calcium-activated potassium channels; mutant; neuronal cell body; novel; presynaptic; prevent; reconstitution; research study; sensor; small hairpin RNA; stem; stroke therapy; stroke-like episode; treatment strategy; voltage

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.

KV2.1 K+通道是最丰富的表达和广泛分布的电压门控的K+通道 哺乳动物。我们以前的研究表明，除了充当延迟的整流器K+通道外 并调节质膜电位，这是一种非导电，多数Kv2.1形成内质的人群 网状/质膜（ER/PM）接触位点。在海马神经元中Kv2.1通道与 皮质内质网在soma表面产生微米大小的kv2.1簇，近端 树突和轴突初始段。文献中的数据表明ER/PM连接调节神经元爆发 发射，直接从ER到细胞表面和质膜PIP2水平直接转移。 我们的初步数据表明，KV2.1诱导的ER/PM连接，但没有其他ER/PM连接，改变ER Ca2+稳态，质膜组织和胞吐作用。有趣的是，KV2.1与 皮质ER受神经元活性和中风样侮辱（例如缺氧，缺血和过量）的调节 谷氨酸，表明在多动症或 神经元侮辱。因此，拟议的研究研究了Kv2.1的新型非导电函数1）是 神经元生理学的中心和2）受神经元活动，侮辱和中风调节。三个 具体目的将解决Kv2.1改变ER Ca2+稳态和的分子机制 膜蛋白在体细胞/PM连接处的定位和突触前ER/PM接触的胞吐作用。目的 1。检验以下假设：Kv2.1诱导的ER/PM接触站点通过 提供局部的K+电导。初步数据表明，神经元中ER Ca2+补充有所增强 表达kv2.1。目的2。检验Kv2.1和皮质肌动蛋白的一致作用的假设 控制Ca2+信号蛋白在ER/PM连接附近的定位。初步数据 表示Kv2.1诱导的ER/PM连接会影响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。检验以下假设：突触囊泡胞吐作用由kv2.1调节。 突触前端子的ER/PM连接处的通道。初步数据表明两者 内源性和转染的KV2.1位于突触前末端，基于shRNA的敲低 KV2.1将谷氨酸能囊泡胞吐作用抑制50％，而不会影响动作潜力。而Kv2.1 引起人类癫痫性脑病的点突变抗通道电导，这是点突变体的子集 与发育延迟有关的，在C端频道中引起过早的停止密码子，不应 影响电导。相反，这些突变被预测仅防止Kv2.1与皮质ER结合。 因此，影响人体的基础的电导和皮质ER重塑作用的突变 疾病。该提案中的研究将大大提高我们对Kv2.1-的作用的理解。 包含ER/PM接触位点在神经元生理中发挥作用。