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全球数百万人，许多病例对现有疗法仍难治。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转变为持续性疼痛的适应不良变化。背根神经节（DRG）神经元中A型K+电流的下调与神经病理性疼痛状态有关。然而，目前尚不清楚这种变化如何导致疾病，因为A型K+通道在伤害感受器中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伤害感受器中高度表达，并且在激活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末端的磷酸化将通道的快速失活A型表型转化为非失活延迟整流型表型。此外，Kv3.4通道以依赖于N-末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通道可能有助于一种新的机制，稳态可塑性，涉及第二信使信号复合物。我们假设，在从急性到持续性疼痛的过渡过程中，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Ca 2+信号传导和突触传递。为了探索这一假说，我们实施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机制; 2）研究Kv3.4通道在伤害性感受和神经病理性疼痛中的PKC依赖性调节的信号传导机制。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，并应用膜片钳方法研究DRG神经元Kv3.4通道的活性和神经生理学影响。此外，我们将结合联合收割机免疫学，分子和电生理学的方法来确定磷酸化状态的Kv3.4通道和PKC的活性在膜补丁。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1）阐明外周机制对SCI引起的神经病理性疼痛的贡献，2）阐明伤害感受器稳态可塑性的新机制的运作基础，该机制涉及Kv3.4通道信号微域，该微域包括GPCR、第二信使分子和PKC;和3）为开发新的和更有效的治疗策略奠定基础，所述治疗策略可以帮助减轻神经性疼痛。

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.