Neuromodulation of Kv3.4 channels in nociceptors

伤害感受器 Kv3.4 通道的神经调节

• 批准号: 8920366
• 负责人: MANUEL L COVARRUBIAS
• 金额: $ 1.66万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-03 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8920366
• 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）：持续性神经性疼痛影响着全世界数百万人，并且许多病例对现有疗法仍然无效。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疼痛到持续性疼痛转变的适应不良变化。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。然而，目前尚不清楚这种变化如何导致疾病，因为伤害感受器中 A 型 K+ 通道的具体作用和调节尚不清楚。 A 型高压激活 Kv3.4 通道在 DRG 伤害感受器中高表达，并在激活 G 蛋白偶联受体 (GPCR) 后受到蛋白激酶 C (PKC) 的显着调节。基本上，Kv3.4 N 末端的磷酸化会将通道的快速失活 A 型表型转化为非失活延迟整流型表型。此外，Kv3.4 通道以取决于 N 端失活门的磷酸化状态的方式加速伤害感受器动作电位的复极化。因此，Kv3.4 通道可能在涉及第二信使信号复合体的稳态可塑性新机制中发挥重要作用。我们假设，在从急性疼痛到持续性疼痛的转变过程中，伤害感受器中发生的塑性变化会损害 Kv3.4 通道调节 AP 复极化的能力，这将影响关键的下游过程，例如 Ca2+ 信号传导和突触传递。为了探索这一假设，我们建立了神经性疼痛的脊髓损伤（SCI）模型，并将追求以下具体目标：1）研究涉及伤害感受和神经性疼痛的 Kv3.4 通道的神经生理学机制； 2) 研究伤害感受和神经性疼痛中 Kv3.4 通道的 PKC 依赖性调节所涉及的信号传导机制。在损伤后的不同时间点，相对于适当的对照，我们将监测疼痛行为并应用膜片钳方法来研究 DRG 神经元中 Kv3.4 通道的活动和神经生理学影响。此外，我们将结合免疫学、分子学和电生理学方法来确定 Kv3.4 通道的磷酸化状态和膜片中 PKC 的活性。为了在体内操纵Kv3.4通道的表达，我们将使用病毒载体和siRNA来过表达和敲低。这些实验将通过以下方式开辟新天地：1）揭示外周机制对 SCI 引起的神经性疼痛的影响； 2) 阐明涉及 Kv3.4 通道信号微域（包括 GPCR、第二信使分子和 PKC）的伤害感受器稳态可塑性新机制的运作基础； 3）为开发新的、更有效的治疗策略奠定基础，这可能有助于减轻神经性疼痛。