CRMP2, Nav1.7 sodium channel, and chronic pain

摘要 慢性疼痛的患病率高得惊人，给社会造成了巨大的负担 缺乏有效的治疗方法。这项应用解决了如何间接调制神经元的兴奋性 疼痛条件可以通过改变Nav1.7钠通道的表达和功能来实现。这个 科学前提是，由于Nav1.7频道的直接封锁没有成功，目标是 Nav1.7的调节剂可能提供治疗优势，允许分级的止痛反应。拉杰什博士 这个项目的首席研究员Khanna首先发现Nav1.7在表面的表达是 受一种名为轴突崩溃反应中介蛋白2(CRMP2)的蛋白质的调节，而CRMP2的一个突变体 缺乏小泛素样修饰物(SUMO)的翻译后修饰(去SUMO)会减少 Nav1.7的表面表达和电流。重要的是，相关的Nav1.1、Nav1.3、NaV1.5、Nav1.6、Nav1.8和 Nav1.9通道不受影响。在初步研究中，我们证明了CRMP2 SUMO化的丢失 增加与内吞蛋白的结合，潜在地解释了Nav1.7从表面上的去除。这个 周围神经损伤后，苏木糖基化CRMP2组分明显增加。令人兴奋的是，在体内 转染CRMP2-K374A相扑缺失型质粒或模拟CRMP2 SUMO化基序的多肽， 进入脊髓逆转神经病理性疼痛模型中的机械性异位痛觉。总而言之，这些发现 有力地支持了CRMP2 SUMO化缺失降低Nav1.7在血浆中定位的假设 膜，从而降低伤害性神经元的兴奋性和对热和机械的阈值 急性和慢性疼痛的刺激物。我们将在三个具体目标上检验这一假设。在目标1中，我们将测试 CRMP2在Nav1.7电流和神经元兴奋性上的一般作用 转基因K374A Crmp2敲入小鼠模型中CRMP2的SUMO化位点(K374)已 替换为丙氨酸突变；这只小鼠是由托马斯·多奇曼博士制造的，他是这一研究的联合研究员 他是亚利桑那大学基因工程小鼠模型核心设施的项目主任。这个 Nav1.7表面运输和内化发生的机制尚不清楚，将在 本提案的目标2。目标3将评估CRMP2苏氨酸化状态对急性疼痛的贡献 阈值以及在使用Nav1.7水平为 增加；这些研究将与Todd Vanderah博士合作进行，Todd Vanderah博士是这方面的联合调查员 在临床前疼痛模型方面拥有深厚专业知识的项目。最后，在这些小鼠身上，我们还将测量CRMP2- 依赖于非目标对记忆、运动/镇静以及与Nav1.7相关的行为的影响，包括 闻。这项拟议的研究将极大地提高我们对细胞内运输蛋白如何 可以在疾病/伤害中进行修饰，为揭示修饰和损伤的机理奠定了坚实的基础 NAV1.7在慢性疼痛中的贩运，并为疼痛研究提供新的和选择性的治疗靶点。

Abstract Chronic pain conditions cause an immense burden on society due to their astonishingly high prevalence and lack of effective treatments. This application addresses how indirect modulation of the excitability of neurons in pain conditions can be achieved by altering the expression and function of the Nav1.7 sodium channel. The scientific premise is that because direct blockade of Nav1.7 channels has been unsuccessful, targeting regulators of Nav1.7 may offer therapeutic advantages allowing for a graded analgesic response. Dr. Rajesh Khanna, Principal Investigator on this project, first discovered that expression of Nav1.7 at the surface is regulated by a protein, axonal collapsin response mediator protein 2 (CRMP2), and that a mutant of CRMP2 lacking the small ubiquitin-like modifier (SUMO) post-translational modification (deSUMOylation) reduces Nav1.7 surface expression and currents. Importantly, the related Nav1.1, Nav1.3, Nav1.5, Nav1.6, Nav1.8, and Nav1.9 channels are unaffected. In preliminary studies, we demonstrate that loss of CRMP2 SUMOylation increases binding to endocytic proteins, potentially accounting for removal of Nav1.7 from the surface. The fraction of SUMOylated CRMP2 increases significantly following peripheral nerve injury. Excitingly, in vivo transfection of a CRMP2-K374A SUMO-null plasmid or a peptide mimicking the CRMP2 SUMOylation motif, into the spinal cord reversed mechanical allodynia in a model of neuropathic pain. Together, these findings strongly support the hypothesis that loss of CRMP2 SUMOylation reduces Nav1.7 localization at the plasma membrane, thereby decreasing nociceptive neuron excitability and thresholds to thermal and mechanical stimuli in acute and chronic pain. We will test this hypothesis in three specific aims. In Aim 1, we will test the general role of CRMP2 SUMOylation on Nav1.7 currents and neuronal excitability using a recently created new transgenic K374A Crmp2 knock-in mouse model where the SUMOylation site (K374) of CRMP2 has been replaced with an alanine mutation; this mouse was made by Dr. Thomas Doetschman, a co-Investigator on this project and Director of the Genetically Engineered Mouse Models Core facility at the University of Arizona. The mechanism by which Nav1.7 surface trafficking and internalization occur is unknown and will be examined in Aim 2 of this proposal. Aim 3 will evaluate the contribution of the CRMP2 SUMOylation state to acute pain thresholds as well as after experimentally induced pain thresholds using models in which Nav1.7 levels are increased; these studies will be performed in collaboration with Dr. Todd Vanderah, a co-Investigator on this project with deep expertise in preclinical pain modeling. Finally, in these mice, we will also measure CRMP2- dependent off-target effects on memory, locomotion/sedation, as well as behaviors linked to Nav1.7, including smell. The proposed study will considerably improve our understanding of how intracellular trafficking proteins can be modified in diseases/injuries, lay a solid foundation for unraveling mechanisms of the modification and trafficking of Nav1.7 in chronic pain, and offer novel and selective therapeutic targets for pain research.