Neuromodulation of Kv3.4 channels in nociceptors

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，许多病例对现有治疗仍然难治性。为了更有效地治疗神经性疼痛，有必要了解伤害感觉的分子基础以及从急性疼痛到持续性疼痛转变背后的不适应变化。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是如何导致疾病的，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。高压激活的a型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）时受到蛋白激酶c （PKC）的显著调节。基本上，Kv3.4 n端磷酸化将通道的快速失活的a型表型转化为非失活的延迟校正型表型。此外，Kv3.4通道加速伤害感受器动作电位的复极化，其方式取决于n端失活门的磷酸化状态。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起重要作用。我们假设，在从急性疼痛到持续疼痛的转变过程中，伤害感受器中发生的可塑性变化损害了Kv3.4通道调节AP复极化的能力，这将影响关键的下游过程，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模型，并将进行以下具体目的：1)研究Kv3.4通道在伤害感觉和神经性疼痛中的神经生理机制；2)探讨痛觉和神经性疼痛中pkc依赖性Kv3.4通道调控的信号机制。在损伤后的不同时间点，并相对于适当的对照，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、分子和电生理学方法来确定Kv3.4通道的磷酸化状态和膜斑块中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进行过表达和敲低。这些实验将开辟新的领域：1)阐明外周机制对脊髓损伤引起的神经性疼痛的贡献；2)阐明了包括gpcr、第二信使分子和PKC在内的Kv3.4通道信号微域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，这些策略可能有助于减轻神经性疼痛。

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.