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锥体神经元树突中运输。 我们先前报道说，神经元刺激导致Kv4.2通道从树突状刺到树突状轴的重新分布。 Kv4.2的这种活性依赖性的重新分布需要激活NMDA型谷氨酸受体和钙涌入，这两个要求与突触可塑性共享，这被认为是学习和记忆的基础。 鉴于CA1树突中KV4.2通道的不均匀分布，Mike Nestor进行了实验，以检验以下假设：KV4.2通道在基础活性期间沿着根尖树突的不同区域差异地运输，并且在CA1神经元中刺激。选择了近端（soma的50-150μm，原发性和倾斜）和远端（>200μm）顶端树突。光漂白（FRAP）技术后的荧光恢复用于测量EGFP标记的KV4.2（KV4.2G）的基础循环速率。我们发现，kv4.2通道的循环速率是从soma的50-150μm之间的一级和倾斜树突上的一个数量级。 KV4.2在200-250μm的SOMA时，通道循环显着增加。 Kv4.2突变体的表达缺乏蛋白激酶-A（KV4.2GS552A）的磷酸化位点的表达，从而消除了通道循环的这种距离依赖性变化。证明PKA磷酸化的基础是远端树突中的迁移率增加。 神经元刺激仅在远端部位显着增加KV4.2通道的循环。远端树突上Kv4.2循环的活性依赖性增加被KV4.2GS552A的表达阻断。 这些结果表明，依赖距离的kv4.2迁移率受PKA对Kv4.2活性依赖性磷酸化的调节。 KV4.2辅助亚基的功能作用：AKAP 研究研究员Lin Lin和Wei Sun今年发现了KV4.2的PKA调制的潜在来源，当时他们将A-激酶锚定蛋白（AKAP）固定为KV4.2的新辅助亚基。 AKAP靶向PKA到谷氨酸受体和离子通道复合物，以允许离散的局部信号传导。 我们确定KV4.2的C末端结构域与AKAP79/150的内部区域相互作用，该区域与其MAGUK结合结构域重叠。 AKAP79/150锚定的PKA活性显示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，破坏PKA锚定导致神经元兴奋性的降低，同时防止磷酸酶钙调酶脱磷酸化，从而增加兴奋性。 这些结果表明，AKAP79/150为KV4.2表达的动态PKA调节提供了一个平台，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末端相关，并调节通道生物物理特性，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介导的对KV4.2通道贩运的影响中的作用。 我们发现，尽管KV4.2和KCHIP4A之间的相互作用不需要Kv4.2S552的PKA磷酸化，但对于增强kchip4a产生的KV4.2通道复合物的稳定和膜表达是必不可少的。 Kv4.2与其他KCH​​IP同工型共表达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磷酸化通过其与KCHIP4A的特定相互作用在运输中起重要作用。 KV4.2辅助亚基的功能作用：DPP6 异源表达系统中的研究表明，KV4α-亚基与跨膜DPP6蛋白相互作用以调节通道运输和特性。 在CA1神经元中表达的DPP6辅助亚基蛋白最近在来自某些人群的大型拷贝数变体筛选中被鉴定为自闭症谱系障碍和ALS靶基因。 DPP6提高了KV4通道的开放概率，并增加了异源系统中的通道表面表达。 在DPP6的树突状录音中，研究生Wei Sun发现DPP6对于在CA1树突中观察到的A型K+电流梯度至关重要。 这种梯度损失导致了高度驱动的树突，对信息存储和编码产生了影响。 其他初步结果表明，DPP6在发育过程中的突触形成中至关重要。 我们目前正在研究这些结果，即树突状兴奋性可能是最近与DPP6基因相关的神经系统疾病的常见因素。 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）的神经元显示相对突触NR2B/NR2A亚基组成的降低，并且不表现出称为长期增强或LTP的突触可塑性形式。相反，通过表达KV4.2显性阴性或通过基因组敲除Kv4.2的表达减少IA，导致突触NR2B/NR2A的比例增加并增强LTP。 我们的数据表明，A型K+通道是突触复合物的组成部分，该突触复合物通过自发的NMDA受体激活来调节Ca2+信号传导，以控制突触NMDA受体的表达和可塑性。 进一步的进步包括研究KV4.2在控制体内和发育过程中突触NMDA受体表达中的作用。 突触NR2B馏分在发展上受到对突触可塑性，学习和记忆以及与学习障碍相关的疾病的影响。 Eunyoung Kim发现，体内注射病毒以改变KV4.2表达水平双向调节NR2B亚基在整个发育过程中的表达。 树突状内在可塑性中的记忆和疾病 我们先前已经显示，A型K+通道在调节突触可塑性后海马的CA1锥体神经元的内在兴奋性中的作用。 这种称为内在可塑性的非突触可塑性除了突触可塑性外，还代表神经元可用的信息存储机制。 Emilie Campanac试图通过使用GLUA1-缺乏小鼠来解离诱导突触和内在可塑性的信号。 在CA1锥体神经元中，GLUA1亚基是通过已知诱导长期增强的不同活性模式差异的。 在药物成瘾时也观察到了内在的可塑性。可卡因是一种令人上瘾的药物，具有精神刺激作用，归因于多巴胺转运蛋白的抑制，从而增加了多巴胺能传播。长期暴露于可卡因会导致位于内侧前额叶皮层（MPCF）和伏拟核的神经元的几个电压膜电导的神经调节。迄今为止，所有这些修饰都在体内表征。我们的目标是更精确地确定在树突中调节哪些电导。将成年雄性小鼠连续5天注射可卡因或盐水。可卡因注射后，MPCF的锥体神经元的内在兴奋性中没有明显差异。但是，我们确实在可卡因注射后发现了EPSP尖峰耦合曲线的左移。在存在GABAA受体抑制剂的情况下，消除了这种转移，表明可卡因后抑制作用降低。