Neuromodulation of Kv3.4 channels in nociceptors

描述(申请人提供)：持续性神经性疼痛影响全球数百万人，许多病例仍然对可用的治疗方法难以奏效。为了更有效地治疗神经性疼痛，有必要了解伤害性感受的分子基础以及从急性疼痛到持续性疼痛的不适应变化。背根神经节(DRG)神经元A-型钾电流的下调与神经病理性疼痛状态有关。然而，这种变化是如何导致疾病的尚不清楚，因为伤害性感受器中A-型K通道的具体作用和调节尚不清楚。A型高电压激活的Kv3.4通道在DRG伤害性感受器中高度表达，并在激活G蛋白偶联受体(GPCRs)时受到蛋白激酶C(PKC)的显著调节。基本上，Kv3.4 N末端的磷酸化将通道的快速失活的A型表型转换为非失活的延迟整流型表型。此外，Kv3.4通道以一种依赖于N末端失活门的磷酸化状态的方式加速伤害性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动态平衡可塑性的新机制中发挥作用。我们假设，在从急性疼痛到持续性疼痛的转变过程中，伤害性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，如钙信号和突触传递。为了探索这一假说，我们建立了神经病理性疼痛的脊髓损伤(SCI)模型，并将追求以下具体目标：1)研究Kv3.4通道在伤害性感受和神经病理性疼痛中的神经生理学机制；2)研究Kv3.4通道在伤害性感觉和神经病理性疼痛中依赖于PKC的信号机制。在损伤后的不同时间点，相对于适当的对照组，我们将监测疼痛行为，并应用膜片钳方法研究Kv3.4通道在DRG神经元中的活动和神经生理影响。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来过表达和敲除。这些实验将通过：1)阐明外周机制在脊髓损伤引起的神经病理性疼痛中的作用；2)阐明涉及Kv3.4通道信号微域的伤害性感受器稳态可塑性的新机制的运作基础，该微域包括GPCRs、第二信使分子和PKC；以及3)为开发可能有助于缓解神经病理性疼痛的新的、更有效的治疗策略奠定基础。

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.