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

基本信息

批准号：
7968661
负责人：
Dax A Hoffman
金额：
$ 105.97万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/7968661
关键词：
Action Potentials Affect Alzheimer&apos s Disease Axon Back Biophysics Brain Brain region C-terminal Cells Chemosensitization Chinese Hamster Ovary Cell Clathrin Collaborations Complex Computer Simulation Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Data Dendrites Dendritic Spines Dependence Dominant-Negative Mutation Down-Regulation Emotions Endocytosis Epilepsy Exhibits Family Family member Fluorescence Forskolin Frequencies Gene Proteins Genes Genomics Head Hippocampus (Brain)Imaging Techniques Immunoblotting Infection Kinetics Knock-out Knockout Mice Knowledge Kv4.2 channel Label Learning Long-Term Potentiation Maintenance Measures Mediating Memory Modification Molecular Molecular Cloning Morphologic artifacts N-Methyl-D-Aspartate Receptors Nature Neuraxis Neurons Pattern Phase Phosphorylation Site Physiological Point Mutation Potassium Channel Property Proteins Protocols documentation Rattus Recovery Relative (related person)Role Shapes Signal Transduction Sindbis Virus Slice Small Interfering RNA Synapses Synaptic plasticity System Techniques Time United States National Institutes of Health Variant Vertebral column Voltage-Gated Potassium Channel Width Work calmodulin-dependent protein kinase II density electrical property experience hippocampal pyramidal neuron member neuronal cell body neurophysiology overexpression patch clamp phase change prevent research study trafficking voltage voltage clamp voltage gated channel

项目摘要

Kv4.2 control of firing patterns in hippocampal CA1 pyramidal neurons. Although recent molecular cloning studies have found several families of voltage-gated K channel genes expressed in the mammalian brain, at present, information regarding the relationship between the protein products of these genes and their various neuronal functions is lacking. Our lab has used a combination of molecular, electrophysiological, imaging techniques to show that the voltage gated potassium channel subunit Kv4.2 controls AP half-width, frequency-dependent AP broadening and dendritic action potential propagation. More recently, we examined the role of A-type K+ channels in regulating intrinsic excitability of CA1 pyramidal neurons of the hippocampus after synapse-specific long-term potentiation (LTP) induction (potentially a cellular form of memory). In electrophysiological recordings we found that LTP induced a potentiation of excitability which was accompanied by a two-phased change in A-type K+ channel activity recorded in nucleated patches from organotypic slices of rat hippocampus. Induction of LTP resulted in an immediate but short lasting hyperpolarization of the voltage-dependence of steady-state A-type K+ channel inactivation along with a progressive, long-lasting decrease in peak A-current density. Blocking clathrin-mediated endocytosis prevented the A-current decrease and most measures of intrinsic plasticity. These results suggest that two temporally distinct but overlapping mechanisms of A-channel downregulation together contribute to the plasticity of intrinsic excitability. This change in intrinsic plasticity resulted in a global enhancement of EPSP-spike coupling. Kv4.2 trafficking in CA1 pyramidal neuron dendrites. Using a modified Sindbis virus system to overexpress EGFP-labeled Kv4.2 (Kv4.2g) in cultured hippocampal neurons, we found that the EGFP fluorescence in dendritic spines of Kv4.2g expressing neurons appeared brighter than that from the adjacent dendritic shaft. The ratio of spine head to dendritic shaft fluorescence in Kv4.2g expressing neurons was approximately two-fold greater than in neurons expressing EGFP. Kv4.2 expression in spines was further shown using electronmicroscopy in collaboration with Ron Petralia here at the NIH. We found stimulation (AMPA) to result in an activity-dependent redistribution of Kv4.2g away from spines to the dendritic shaft and a punctate accumulation of Kv4.2g within the soma. This AMPA-induced redistribution of Kv4.2g occurred within 15 min of stimulation and was reversible, indicating that the treatment was not excitotoxic. Controls showed that this activity-dependent Kv4.2 internalization occurs natively and is not an artifact of overexpression. More recently we examined the role of protein kinase A (PKA) in Kv4.2 activity-dependent trafficking. In hippocampal neurons, PKA activation with forskolin or 8-Br-cAMP induced Kv4.2 internalization from dendritic spines, whereas PKA inhibition prevented AMPA-induced internalization. Furthermore, introduction of a point mutation at the C-terminal PKA phosphorylation site of Kv4.2 (S552A) prevented the AMPA-induced internalization of Kv4.2. We continue to investigate the mechanisms of Kv4.2 expression and trafficking. Role of voltage-gated potassium channels in synaptic plasticity. Using the Sindbis virus system to infect organotypic slice cultures with Kv4.2g and Kv4.2g(W362F), we have begun investigating the role of Kv4.2 in LTP using a depolarization pairing protocol. For the first 10 min after pairing, potentiation is similar in all three groups, achieving 100% increase in EPSC size. After this period, however, Kv4.2 overexpressing neurons fail to maintain potentiation such that EPSC size is back to baseline after 25 min. Conversely, expression of Kv4.2g(W362F) results in a potentiation, which reaches a greater level 40-50 min after initiation, compared to controls. These data indicate that Kv4.2 channels modulate the degree of LTP by influencing the induction of a late phase of potentiation or by controlling the mechanisms of LTP maintenance. We are currently characterizing the mechanisms of Kv4.2's effect on LTP. The effect of changes in IA on the ability to induce subsequent synaptic plasticity (meta-plasticity) has not yet been investigated. We have found that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced IA showed a decrease in relative synaptic NR2B/NR2A subunit composition and, as noted above do not exhibit 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. Bidirectional synaptic remodeling was mimicked in experiments manipulating intracellular Ca2+ and dependent on spontaneous activation of NMDA receptors and active CaMKII. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDAR activation to control synaptic NMDAR expression and plasticity. Functional role of Kv4.2 auxiliary subunits Kv4 currents in heterologous cells display slower kinetics of inactivation and recovery from inactivation than that typically recorded in neurons. Reconciliation of these results came with the finding of neuron specific auxiliary subunit expression of KChIPs and DPLs. In hippocampal CA1 pyramidal neurons, DPPX (also called DPP6) is the prominent DPL family member. To investigate the physiological role of DPPX in CA1 neurons, in collaboration with Dr. Bernardo Rudy, we developed short-interfering RNAs (siRNAs) to suppress the expression of all DPPX variants. The reduction of DPPX proteins in CHO cells transfected with DPPX siRNA (siDPPX) was more than 95% complete, as quantified by immunoblotting. To investigate whether DPPX alters the kinetics of A-type currents in a native system, we conducted voltage-clamp experiments in outside-out patches from CA1 pyramidal neurons in hippocampal organotypic slices infected with siDPPX. After allowing 2-3 days post-infection for DPPX knockdown we found, in accordance with heterologous studies, that siDPPX results in a delayed recovery from inactivation, slowed time-to-peak, and rightward shifted the steady-state inactivation and activation curves. To determine the physiological effect of kinetic modifications by siDPPX, we carried out current-clamp experiments in siDPPX expressing cells. Compared to negative control siRNA neurons, siDPPX-infected neurons exhibited delayed time to AP onset, increased AP threshold, decreased firing frequency, increased AP half-width and reduced fast AHP amplitudes. Thus siDPPX had contrasting effects, decreasing excitability subthreshold and increasing excitability suprathreshold. Computer simulations supported our experimental results and demonstrated how DPPX remodeling of A-channel properties can result in opposing sub- and suprathreshold effects on excitably. We are currently investigating the dendritic role of DPPX in knockout mice and have developed siRNAs targeting members of the KChIP family of auxiliary subunits for similar studies.

Kv4.2 对海马 CA1 锥体神经元放电模式的控制。尽管最近的分子克隆研究发现了在哺乳动物大脑中表达的多个电压门控K通道基因家族，但目前缺乏有关这些基因的蛋白质产物与其各种神经元功能之间关系的信息。我们的实验室结合了分子、电生理学、成像技术，证明电压门控钾通道亚基 Kv4.2 控制 AP 半宽度、频率依赖性 AP 展宽和树突动作电位传播。最近，我们研究了 A 型 K+ 通道在突触特异性长时程增强 (LTP) 诱导（可能是一种细胞形式的记忆）后调节海马 CA1 锥体神经元内在兴奋性的作用。在电生理记录中，我们发现 LTP 诱导兴奋性增强，伴随着大鼠海马器官切片有核斑块中记录的 A 型 K+ 通道活性的两相变化。 LTP 的诱导导致稳态 A 型 K+ 通道失活的电压依赖性立即但短暂的超极化，同时峰值 A 电流密度逐渐、持久降低。阻断网格蛋白介导的内吞作用可防止 A 电流降低和大多数内在可塑性测量。这些结果表明，A 通道下调的两种时间上不同但重叠的机制共同促进了内在兴奋性的可塑性。这种内在可塑性的变化导致 EPSP-尖峰耦合的整体增强。 Kv4.2 在 CA1 锥体神经元树突中的运输。使用改良的辛德比斯病毒系统在培养的海马神经元中过表达 EGFP 标记的 Kv4.2 (Kv4.2g)，我们发现表达 Kv4.2g 的神经元的树突棘中的 EGFP 荧光看起来比相邻树突轴的 EGFP 荧光更亮。表达 Kv4.2g 的神经元中棘头与树突轴荧光的比率大约是表达 EGFP 的神经元中的两倍。与 NIH 的 Ron Petralia 合作，使用电子显微镜进一步显示了脊柱中的 Kv4.2 表达。我们发现刺激 (AMPA) 会导致 Kv4.2g 从棘到树突轴的活性依赖性重新分布，以及 Kv4.2g 在胞体内的点状积累。 AMPA 诱导的 Kv4.2g 重新分布发生在刺激后 15 分钟内，并且是可逆的，表明该治疗不具有兴奋毒性。对照表明，这种活性依赖性 Kv4.2 内化是天然发生的，而不是过度表达的产物。最近，我们研究了蛋白激酶 A (PKA) 在 Kv4.2 活性依赖性运输中的作用。在海马神经元中，毛喉素或 8-Br-cAMP 激活 PKA 诱导树突棘 Kv4.2 内化，而 PKA 抑制则阻止 AMPA 诱导的内化。此外，在 Kv4.2 (S552A) 的 C 端 PKA 磷酸化位点引入点突变可阻止 AMPA 诱导的 Kv4.2 内化。我们继续研究 Kv4.2 表达和贩运的机制。电压门控钾通道在突触可塑性中的作用。使用 Sindbis 病毒系统用 Kv4.2g 和 Kv4.2g(W362F) 感染器官切片培养物，我们已开始使用去极化配对方案研究 Kv4.2 在 LTP 中的作用。配对后的前 10 分钟，所有三组的增强效果相似，EPSC 大小增加了 100%。然而，在此期间之后，Kv4.2 过度表达的神经元无法维持增强作用，使得 EPSC 大小在 25 分钟后回到基线。相反，Kv4.2g(W362F) 的表达会导致增强，与对照相比，在启动后 40-50 分钟达到更高的水平。这些数据表明，Kv4.2 通道通过影响增强后期的诱导或通过控制 LTP 维持机制来调节 LTP 程度。我们目前正在描述 Kv4.2 对 LTP 影响的机制。 IA 变化对诱导后续突触可塑性（元可塑性）能力的影响尚未得到研究。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 IA 增强的神经元表现出相对突触 NR2B/NR2A 亚基组成的减少，并且如上所述，不表现出 LTP。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。双向突触重塑在操纵细胞内 Ca2+ 的实验中被模仿，并且依赖于 NMDA 受体和活性 CaMKII 的自发激活。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 NMDAR 激活来调节 Ca2+ 信号传导，从而控制突触 NMDAR 表达和可塑性。 Kv4.2辅助亚基的功能作用异源细胞中的 Kv4 电流显示出比神经元中通常记录的更慢的失活和失活恢复动力学。随着 KChIP 和 DPL 神经元特异性辅助亚基表达的发现，这些结果得到了协调。在海马 CA1 锥体神经元中，DPPX（也称为 DPP6）是重要的 DPL 家族成员。为了研究 DPPX 在 CA1 神经元中的生理作用，我们与 Bernardo Rudy 博士合作，开发了短干扰 RNA (siRNA) 来抑制所有 DPPX 变体的表达。通过免疫印迹定量，转染 DPPX siRNA (siDPPX) 的 CHO 细胞中 DPPX 蛋白的减少完成度超过 95%。为了研究 DPPX 是否改变本地系统中 A 型电流的动力学，我们对感染 siDPPX 的海马器官切片中的 CA1 锥体神经元的外侧斑块进行了电压钳实验。在感染后 2-3 天进行 DPPX 敲低后，根据异源研究，我们发现 siDPPX 导致失活恢复延迟，减慢达峰时间，并使稳态失活和激活曲线右移。为了确定 siDPPX 动力学修饰的生理效应，我们在 siDPPX 表达细胞中进行了电流钳实验。与阴性对照 siRNA 神经元相比，siDPPX 感染的神经元表现出 AP 起始时间延迟、AP 阈值增加、放电频率降低、AP 半宽度增加和快速 AHP 振幅降低。因此，siDPPX 具有相反的效果，降低兴奋性阈下和增加兴奋性阈上。计算机模拟支持了我们的实验结果，并证明了 A 通道特性的 DPPX 重塑如何对兴奋性产生相反的阈下和阈上效应。我们目前正在研究 DPPX 在基因敲除小鼠中的树突作用，并开发了针对辅助亚基 KChIP 家族成员的 siRNA，用于类似的研究。