Neuromodulation of Kv3.4 channels in nociceptors

伤害感受器 Kv3.4 通道的神经调节

• 批准号: 8510863
• 负责人: MANUEL L COVARRUBIAS
• 金额: $ 23.25万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8510863
• 关键词: Action Potentials; Acute; Affect; Agonist; Animal Model; Axon; Cells; Complex; Development; Disease; Down-Regulation; Electrophysiology (science); Etiology; G-Protein-Coupled Receptors; Health; Immunohistochemistry; In Vitro; Injury; Laminectomy; Light; Link; Membrane; Methods; Modeling; Molecular; Molecular Target; Monitor; N-terminal; Neurons; Nociception; Nociceptors; Pain; Pathway interactions; Peptides; Peripheral; Persistent pain; Phenotype; Phospho-Specific Antibodies; Phosphorylation; Plastics; Play; Potassium Channel; Presynaptic Terminals; Process; Property; Protein Isoforms; Protein Kinase C; Proteins; Proteomics; Rattus; Refractory; Relative (related person); Reporting; Role; Second Messenger Systems; Shapes; Signal Transduction; Site; Small Interfering RNA; Spinal Ganglia; Spinal cord injury; Staging; Structure; Surface; Synaptic Transmission; Testing; Therapeutic; Time; Viral Vector; Western Blotting; World Health; base; effective therapy; immunoreactivity; in vivo; inhibitor/antagonist; interest; knock-down; mRNA Expression; neuronal cell body; neurophysiology; neuroregulation; novel; operation; overexpression; pain behavior; painful neuropathy; patch clamp; public health relevance; research study; second messenger; socioeconomics; tool; voltage

DESCRIPTION (provided by applicant): Persistent neuropathic pain affects millions of people worldwide and many cases remain refractory to available therapies. To treat neuropathic pain more effectively, it is necessary to understand the molecular basis of nociception and the maladaptive changes underlying the transition from acute to persistent pain. The down- regulation of A-type K+ currents in dorsal root ganglion (DRG) neurons has been implicated in the neuropathic pain state. However, it is not clear how this change contributes to disease because the specific roles and modulation of A-type K+ channels in nociceptors are not understood. The A-type high voltage-activated Kv3.4 channel is highly expressed in DRG nociceptors and is dramatically modulated by protein kinase-C (PKC) upon activating G-protein coupled receptors (GPCRs). Basically, phosphorylation of the Kv3.4 N-terminus converts the channel's fast-inactivating A-type phenotype into a non-inactivating delayed-rectifier-type phenotype. Furthermore, Kv3.4 channels accelerate the repolarization of the nociceptor action potential in a manner that depends on the phosphorylation status of the N-terminal inactivation gate. Kv3.4 channels might thus be instrumental in a novel mechanism of homeostatic plasticity involving second messenger signaling complex. We hypothesize that plastic changes occurring in nociceptors during the transition from acute to persistent pain compromise the ability of Kv3.4 channels to regulate the repolarization of the AP, which will impact critical downstream processes, such as Ca2+ signaling and synaptic transmission. To explore this hypothesis, we implemented a spinal cord injury (SCI) model of neuropathic pain and will pursue the following specific aims: 1) To investigate the neurophysiological mechanisms implicating Kv3.4 channels in nociception and neuropathic pain; and 2) To investigate the signaling mechanisms implicating PKC-dependent modulation of Kv3.4 channels in nociception and neuropathic pain. At various time points after the injury, and relative to appropriate controls, we will monitor pain behaviors and apply patch-clamp methods to investigate the activity and neurophysiological impact of Kv3.4 channels in DRG neurons. Also, we will combine immunological, molecular, and electrophysiological approaches to determine the phosphorylation status of Kv3.4 channels and the activity of PKC in membrane patches. To manipulate the expression of Kv3.4 channels in vivo, we will use viral vectors and siRNA to overexpress and knockdown. These experiments will break new ground by 1) shedding light on the contribution of peripheral mechanisms to neuropathic pain resulting from SCI; 2) elucidating the basis of operation of a novel mechanism of nociceptor homeostatic plasticity involving a Kv3.4 channel signaling microdomain that includes GPCRs, second messenger molecules and PKC; and 3) setting the stage to develop new and more effective therapeutic strategies that may help alleviate neuropathic pain.

描述（由申请人提供）：持续的神经性疼痛会影响全球数百万的人，许多病例仍然对可用疗法难治性。为了更有效地治疗神经性疼痛，有必要了解伤害感受的分子基础以及从急性到持续性疼痛过渡的障碍变化。背根神经节（DRG）神经元中A型K+电流的下调与神经性疼痛状态有关。但是，尚不清楚这种变化如何促进疾病，因为尚不了解伤害感受器中A型K+通道的特定作用和调节。 A型高电压激活的KV3.4通道在DRG伤害感受器中高度表达，并且在激活G蛋白偶联受体（GPCR）时，蛋白激酶-C（PKC）大大调节了蛋白激酶-C（PKC）。基本上，Kv3.4 N末端的磷酸化将通道的快速灭活A型表型转换为非灭活的延迟型型型型表型。此外，KV3.4通道以依赖于N末端失活门的磷酸化状态的方式加速了伤害感受器动作电位的复极化。因此，KV3.4通道可能会在涉及第二信使信号复合物的稳态可塑性机理中发挥作用。我们假设在从急性到持续性疼痛的过渡期间，伤害感受器发生的塑性变化损害了Kv3.4通道调节AP的重极化的能力，这将影响关键的下游过程，例如CA2+信号传导和突触传播。为了探讨这一假设，我们实施了神经性疼痛的脊髓损伤（SCI）模型，并将追求以下特定目的：1）研究涉及伤害感受和神经性疼痛的KV3.4通道的神经生理机制； 2）研究涉及在伤害感受和神经性疼痛中对PKC依赖性调节的信号传导机制。在受伤后的各个时间点，相对于适当的对照，我们将监测疼痛行为，并应用贴片钳方法来研究DRG神经元中KV3.4通道的活性和神经生理影响。此外，我们将结合免疫学，分子和电生理方法，以确定KV3.4通道的磷酸化状态以及PKC在膜片斑块中的活性。为了操纵体内Kv3.4通道的表达，我们将使用病毒载体和siRNA过表达和敲低。这些实验将在1）阐明周围机制对SCI引起的神经性疼痛的贡献； 2）阐明一种新型伤害感受器稳态可塑性的操作基础，该机制涉及KV3.4通道信号传导微域（包括GPCR，第二信使分子和PKC）； 3）为开发新的，更有效的治疗策略而建立舞台，以帮助减轻神经性疼痛。