Potassium Channels and Dendritic Function in Hippocampal Pyramidal Neurons

海马锥体神经元的钾通道和树突功能

• 批准号: 8351173
• 负责人: Dax A Hoffman
• 金额: $ 113.59万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8351173
• 关键词: A kinase anchoring protein; Action Potentials; Adult; Affect; Alzheimer' s Disease; Apical; Axon; Back; Binding; Biophysics; Brain; Brain region; C-terminal; Calcium; Cells; Central Nervous System Diseases; Chronic; Cocaine; Code; Complex; Coupling; Cyclic AMP-Dependent Protein Kinases; Data; Dendrites; Dendritic Spines; Development; Disease; Distal; Dominant-Negative Mutation; Drug Addiction; Emotions; Epilepsy; Excitatory Postsynaptic Potentials; Exhibits; Fluorescence Recovery After Photobleaching; Frequencies; Gene Targeting; Genes; Genomics; Glutamate Receptor; Goals; Hippocampus (Brain); Impairment; Information Storage; Injection of therapeutic agent; Investigation; Ion Channel; Knock-out; Knockout Mice; Knowledge; Kv channel-interacting protein 1; Kv4 channel; Kv4.2 channel; Learning; Left; Long-Term Potentiation; Measures; Medial; Mediating; Membrane; Memory; Modification; Molecular; Mus; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; N-terminal; Nature; Neuraxis; Neurons; Nucleus Accumbens; Pattern; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Play; Population; Potassium Channel; Prefrontal Cortex; Probability; Property; Protein Dephosphorylation; Protein Isoforms; Protein Subunits; Proteins; Receptor Activation; Recruitment Activity; Regulation; Relative (related person); Reporting; Research; Role; Saline; Shapes; Signal Transduction; Site; Source; Surface; Synapses; Synaptic plasticity; System; Techniques; Testing; The Sun; Variant; Virus; Work; autism spectrum disorder; calcineurin phosphatase; cocaine exposure; density; dopamine transporter; electrical property; experience; graduate student; hippocampal pyramidal neuron; in vivo; inhibitor/antagonist; male; mutant; nervous system disorder; neuronal cell body; neuronal excitability; neurophysiology; novel; patch clamp; prevent; protein expression; psychostimulant; receptor; receptor expression; research study; synaptogenesis; trafficking; transmission process; voltage; voltage gated channel

Kv4.2 trafficking in CA1 pyramidal neuron dendrites. We previously reported that neuronal stimulation results in a redistribution of Kv4.2 channels away from dendritic spines to the dendritic shaft. This activity-dependent redistribution of Kv4.2 required activation of NMDA-type glutamate receptors and calcium influx, two requirements shared with synaptic plasticity, which is thought to underlie learning and memory. Given the nonuniform distribution of Kv4.2 channels in CA1 dendrites, Mike Nestor performed experiments to test the hypothesis that Kv4.2 channels are differentially trafficked at different regions along the apical dendrite during basal activity and upon stimulation in CA1 neurons. Proximal (50-150 μm from the soma, primary and oblique) and distal (>200 μm) apical dendrites were selected. The fluorescence recovery after photobleaching (FRAP) technique was used to measure basal cycling rates of EGFP-tagged Kv4.2 (Kv4.2g). We found that the cycling rate of Kv4.2 channels was one order of magnitude slower at both primary and oblique dendrites between 50-150 μm from the soma. Kv4.2 channel cycling increased significantly at 200-250 μm from the soma. Expression of a Kv4.2 mutant lacking a phosphorylation site for protein kinase-A (Kv4.2gS552A) abolished this distance-dependent change in channel cycling; demonstrating that phosphorylation by PKA underlies the increased mobility in distal dendrites. Neuronal stimulation increased cycling of Kv4.2 channels significantly at distal sites only. This activity-dependent increase in Kv4.2 cycling at distal dendrites was blocked by expression of Kv4.2gS552A. These results indicate that distance-dependent Kv4.2 mobility is regulated by activity-dependent phosphorylation of Kv4.2 by PKA. Functional role of the Kv4.2 auxiliary subunits: AKAPs A potential source of PKA modulation of Kv4.2 was uncovered this year by research fellow Lin Lin and Wei Sun when they identified A-kinase anchoring proteins (AKAPs) as novel accessory subunits for Kv4.2. AKAPs target PKA to glutamate receptor and ion channel complexes to allow for discrete, local signaling. We determined that the C-terminal domain of Kv4.2 interacts with an internal region of AKAP79/150 that overlaps with its MAGUK binding domain. AKAP79/150-anchored PKA activity was shown to control Kv4.2 surface expression in heterologous cells and hippocampal neurons. Consistent with these findings, disrupting PKA anchoring leads to a decrease in neuronal excitability while preventing dephosphorylation by the phosphatase calcineurin results in increased excitability. These results demonstrate that AKAP79/150 provides a platform for dynamic PKA regulation of Kv4.2 expression, fundamentally impacting neuronal excitability. Functional role of the Kv4.2 auxiliary subunits: KChIP4a KChIPs (KChIP1-4), associate with the N-terminal of Kv4.2 and modulate the channels biophysical properties, turnover rate and surface expression. We investigated the role of Kv4.2 C-terminal PKA phosphorylation site S552 in the KChIP4a-mediated effects on Kv4.2 channel trafficking. We found that while interaction between Kv4.2 and KChIP4a does not require PKA phosphorylation of Kv4.2S552, phosphorylation of this site is necessary for both enhanced stabilization and membrane expression of Kv4.2 channel complexes produced by KChIP4a. Enhanced surface expression and protein stability conferred by co-expression of Kv4.2 with other KChIP isoforms did not require PKA phosphorylation of Kv4.2 S552. These data demonstrate that PKA phosphorylation of Kv4.2 plays an important role in the trafficking of Kv4.2 through its specific interaction with KChIP4a. Functional role of the Kv4.2 auxiliary subunits: DPP6 Studies in heterologous expression systems have shown that Kv4 α-subunits interact with transmembrane DPP6 proteins to regulate channel trafficking and properties. The DPP6 auxiliary subunit protein, which is expressed in CA1 neurons, has recently been identified in large copy-number variants screens from some populations as an Autism Spectrum Disorder and ALS target gene. DPP6 enhances the opening probability of Kv4 channels and increases channel surface expression in heterologous systems. In dendritic recordings from DPP6 knock out mice, graduate student Wei Sun discovered that DPP6 is critical for generating the A-type K+ current gradient observed in CA1 dendrites. The loss this gradient led to hyper-excitable dendrites, with implications for information storage and coding. Additional, preliminary results show a critical role for DPP6 in synapse formation during development. We are currently investigating the possibility, suggested from these results, that dendritic excitability might be a common factor altered in neurological disorders recently associated with the DPP6 gene. Role of Kv4.2 channels in synaptic plasticity and development We have found that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced A-type K+ current (IA) showed a decrease in relative synaptic NR2B/NR2A subunit composition and do not exhibit a form of synaptic plasticity called long-term potentiation or LTP. Conversely, reducing IA by expression of a Kv4.2 dominant negative or through genomic knockout of Kv4.2 led to an increased fraction of synaptic NR2B/NR2A and enhanced LTP. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDA receptor activation to control synaptic NMDA receptor expression and plasticity. Additional advances included an investigation into the role of Kv4.2 in controlling the expression of synaptic NMDA receptors in vivo and during development. Synaptic NR2B fraction is developmentally regulated with implications for synaptic plasticity and learning and memory as well as diseases associated with learning impairments. Eunyoung Kim has found that in vivo injection of virus to alter Kv4.2 expression levels bidirectionally regulates NR2B subunit expression throughout development. Dendritic intrinsic plasticity in memory and disease We have shown previously, a role of A-type K+ channels in regulating intrinsic excitability of CA1 pyramidal neurons of the hippocampus after the induction of synaptic plasticity. This non-synaptic plasticity, called intrinsic plasticity, might represent an information storage mechanism available to neurons in addition to synaptic plasticity. Emilie Campanac is attempting to dissociate the signals involved in the induction of synaptic and intrinsic plasticity by the use of GluA1- lacking mice. In CA1 pyramidal neurons, the GluA1 subunit is differentially recruited by different patterns of activity known to induce long-term potentiation. Intrinsic plasticity has also been observed upon drug addiction. Cocaine is an addictive drug with psychostimulant effects that are attributed to inhibition of the dopamine transporter, which increases dopaminergic transmission. Chronic exposure to cocaine leads to neurodaptations in several voltage membrane conductances of neurons localized in the medial prefrontal cortex (mPCF) and nucleus accumbens. To date, all of these modifications have been characterized in the soma. Our goal is to identify more precisely which conductances are regulated in dendrites. Adult male mice were injected for 5 consecutive days with cocaine or saline. No significant difference was observed in intrinsic excitability in pyramidal neurons of the mPCF after cocaine injection. We did, however, find a left shift in the EPSP-Spike coupling curve after cocaine injection. This shift is abolished in the presence of an inhibitor of GABAA receptors suggesting a decrease of inhibition after cocaine.

Kv4.2 在 CA1 锥体神经元树突中的运输。 我们之前报道过神经元刺激导致 Kv4.2 通道从树突棘重新分布到树突轴。 Kv4.2 的这种活动依赖性重新分布需要 NMDA 型谷氨酸受体的激活和钙流入，这两个要求与突触可塑性相同，而突触可塑性被认为是学习和记忆的基础。 鉴于 CA1 树突中 Kv4.2 通道的分布不均匀，Mike Nestor 进行了实验来检验以下假设：在基础活动期间和 CA1 神经元受到刺激时，Kv4.2 通道在沿顶端树突的不同区域有差异性传输。选择近端（距离体体50-150μm，初级和斜向）和远端（>200μm）顶端树突。光漂白后荧光恢复 (FRAP) 技术用于测量 EGFP 标记的 Kv4.2 (Kv4.2g) 的基础循环率。我们发现，在距体体 50-150 μm 之间的初生树突和斜树突处，Kv4.2 通道的循环速率要慢一个数量级。 Kv4.2 通道循环在距离体细胞 200-250 μm 处显着增加。 缺乏蛋白激酶 A 磷酸化位点的 Kv4.2 突变体 (Kv4.2gS552A) 的表达消除了通道循环中这种距离依赖性的变化；证明 PKA 磷酸化是远端树突活动性增加的基础。 神经元刺激仅在远端显着增加 Kv4.2 通道的循环。远端树突 Kv4.2 循环的这种活性依赖性增加被 Kv4.2gS552A 的表达阻断。 这些结果表明，距离依赖性 Kv4.2 迁移性是通过 PKA 对 Kv4.2 的活性依赖性磷酸化来调节的。 Kv4.2 辅助亚基的功能作用：AKAP Lin Lin 和 Wei Sun 研究员今年发现了 Kv4.2 的 PKA 调节的潜在来源，他们将 A 激酶锚定蛋白 (AKAP) 鉴定为 Kv4.2 的新型辅助亚基。 AKAP 将 PKA 靶向谷氨酸受体和离子通道复合物，以实现离散的局部信号传导。 我们确定 Kv4.2 的 C 端结构域与 AKAP79/150 的内部区域相互作用，该内部区域与其 MAGUK 结合结构域重叠。 AKAP79/150 锚定的 PKA 活性可控制异源细胞和海马神经元中的 Kv4.2 表面表达。 与这些发现一致的是，破坏 PKA 锚定会导致神经元兴奋性降低，同时防止磷酸酶钙调神经磷酸酶的去磷酸化会导致兴奋性增加。 这些结果表明 AKAP79/150 为 Kv4.2 表达的动态 PKA 调节提供了平台，从根本上影响神经元兴奋性。 Kv4.2 辅助亚基的功能作用：KChIP4a KChIPs (KChIP1-4) 与 Kv4.2 的 N 末端结合，调节通道的生物物理特性、周转率和表面表达。 我们研究了 Kv4.2 C 末端 PKA 磷酸化位点 S552 在 KChIP4a 介导的 Kv4.2 通道运输效应中的作用。 我们发现，虽然 Kv4.2 和 KChIP4a 之间的相互作用不需要 Kv4.2S552 的 PKA 磷酸化，但该位点的磷酸化对于增强 KChIP4a 产生的 Kv4.2 通道复合物的稳定性和膜表达是必需的。 Kv4.2 与其他 KChIP 同种型共表达所带来的增强的表面表达和蛋白质稳定性不需要 Kv4.2 S552 的 PKA 磷酸化。 这些数据表明，Kv4.2 的 PKA 磷酸化通过与 KChIP4a 的特异性相互作用，在 Kv4.2 的运输中发挥着重要作用。 Kv4.2辅助亚基的功能作用：DPP6 异源表达系统的研究表明，Kv4 α 亚基与跨膜 DPP6 蛋白相互作用，调节通道运输和特性。 DPP6 辅助亚基蛋白在 CA1 神经元中表达，最近在一些人群的大拷贝数变异筛选中被鉴定为自闭症谱系障碍和 ALS 靶基因。 DPP6 增强 Kv4 通道的开放概率并增加异源系统中的通道表面表达。 在 DPP6 敲除小鼠的树突记录中，研究生 Wei Sun 发现 DPP6 对于产生在 CA1 树突中观察到的 A 型 K+ 电流梯度至关重要。 该梯度的损失导致树突过度兴奋，这对信息存储和编码产生影响。 此外，初步结果显示 DPP6 在发育过程中突触形成中发挥着关键作用。 我们目前正在研究这种可能性，从这些结果表明，树突兴奋性可能是最近与 DPP6 基因相关的神经系统疾病中改变的一个常见因素。 Kv4.2 通道在突触可塑性和发育中的作用 我们发现改变功能性 Kv4.2 表达水平会导致 CA1 突触的快速、双向重塑。表现出增强的 A 型 K+ 电流 (IA) 的神经元表现出相对突触 NR2B/NR2A 亚基组成的减少，并且不表现出称为长时程增强或 LTP 的突触可塑性形式。相反，通过表达 Kv4.2 显性失活或通过基因组敲除 Kv4.2 来减少 IA 会导致突触 NR2B/NR2A 比例增加和 LTP 增强。 我们的数据表明，A 型 K+ 通道是突触复合体的组成部分，突触复合体通过自发 NMDA 受体激活来调节 Ca2+ 信号传导，从而控制突触 NMDA 受体的表达和可塑性。 其他进展包括对 Kv4.2 在体内和发育过程中控制突触 NMDA 受体表达的作用的研究。 突触 NR2B 部分受到发育调节，对突触可塑性、学习和记忆以及与学习障碍相关的疾病有影响。 Eunyoung Kim 发现体内注射病毒来改变 Kv4.2 表达水平可以在整个发育过程中双向调节 NR2B 亚基表达。 记忆和疾病中的树突内在可塑性 我们之前已经证明，A 型 K+ 通道在诱导突触可塑性后调节海马 CA1 锥体神经元的内在兴奋性中的作用。 这种非突触可塑性，称为内在可塑性，可能代表了除了突触可塑性之外神经元可用的信息存储机制。 Emilie Campanac 正尝试通过使用缺乏 GluA1 的小鼠来分离参与突触诱导和内在可塑性的信号。 在 CA1 锥体神经元中，GluA1 亚基通过已知可诱导长时程增强的不同活动模式进行差异性募集。 药物成瘾时也观察到了内在的可塑性。可卡因是一种具有精神兴奋作用的成瘾药物，其归因于抑制多巴胺转运蛋白，从而增加多巴胺能传递。长期接触可卡因会导致位于内侧前额皮质 (mPCF) 和伏核的神经元的几种电压膜电导发生神经适应。迄今为止，所有这些修饰都已在体细胞中得到表征。我们的目标是更准确地确定树突中哪些电导受到调节。成年雄性小鼠连续5天注射可卡因或盐水。注射可卡因后，mPCF 锥体神经元的内在兴奋性没有观察到显着差异。然而，我们确实发现注射可卡因后 EPSP-Spike 耦合曲线左移。当 GABAA 受体抑制剂存在时，这种转变被消除，表明可卡因后抑制作用减弱。