Sodium Channel Nav1.6 and Pathophysiology of Pain

描述（由申请人提供）： 该提案的目标是证明钠通道Nav1.6在神经性疼痛和炎症性疼痛中的重要性，并确定开发新的有效疼痛治疗方法的特定靶点和策略。钠通道的表达失调和突变与背根神经节（DRG）神经元的过度兴奋有关，背根神经节（DRG）神经元的过度兴奋是外周源性炎性疼痛和神经性疼痛的基础，这两者构成了大多数神经性疼痛病症。Nav1.6是大DRG神经元中的主要钠通道，在轴突起始段和有髓纤维中的成熟Ranvier节点丰富，这两个神经元隔室对于神经冲动的传导是必需的。我们的初步数据显示，Nav1.6也在大多数伤害性DRG神经元中表达，这些神经元产生无髓纤维，这与我们先前在Nav1.6缺失小鼠的坐骨神经中发现的受损的C纤维复合动作电位传导一致。敲除其他外周特异性钠通道的小鼠DRG神经元中Nav1.6的持续存在表明Nav1.6通道支持这些小鼠中疼痛信号的传导。异位放电的受伤的Ab传入神经元发出的大DRG神经元表明Nav1.6，神经病理性疼痛的重要贡献。因此，大量证据表明，Nav1.6有助于慢性疼痛中DRG神经元的损伤诱导的超兴奋性。研究表明，损伤降低了DRG神经元中Nav1.6转录物的水平。有趣的是，我们的初步数据显示，在大鼠坐骨神经中横断的有髓轴突的节点处有显著的Nav1.6免疫染色，与结扎的眶下神经的类似的已发表研究一致。我们还报道了Nav1.6是激活的MAP激酶p38的底物，其在损伤后在DRG神经元中被激活，并且由包括FGF家族和钙调蛋白的成员在内的大量胞质蛋白调节。因此，损伤介导的Nav1.6通道调节也可能导致神经元过度兴奋性改变。在本研究中，我们将在细胞水平上研究损伤介导的Nav1.6通道敲低或敲除后对DRG神经元痛阈和过度兴奋性的影响，以验证以下假设：i）DRG神经元中Nav1.6的缺失改善疼痛行为; ii）损伤改变伤害性和大DRG神经元中Nav1.6钠电流; iii）Nav1.6通道的调节有助于促伤害感受因子的信号转导。我们还将使用分子和免疫学方法以及电压钳和电流钳记录来：iv）评估损伤介导的对Nav1.6表达和调制的改变调节的影响;和v）将损伤和备用DRG神经元中分子和细胞水平的变化与疼痛阈值的变化相关联。

DESCRIPTION (provided by applicant): The goal of this proposal is to demonstrate the importance of sodium channel Nav1.6 in neuropathic and inflammatory pain, and to identify specific targets and strategies for the development of new and effective pain therapeutics. Dysregulated expression and mutations of sodium channels have been linked to hyperexcitability of dorsal root ganglia (DRG) neurons which underlies peripherally-originated inflammatory and neuropathic pain, both of which constitute the majority of neuropathic pain disorders. Nav1.6, the predominant sodium channel in large DRG neurons, is abundant at axon initial segments and mature nodes of Ranvier in myelinated fibers-the two neuronal compartments essential for conduction of nerve impulses. Our preliminary data shows that Nav1.6 is also expressed in most nociceptive DRG neurons which give rise to unmyelinated fibers, consistent with our previous finding of impaired C-fiber compound action potential conduction in sciatic nerve of Nav1.6-null mice. The continued presence of Nav1.6 in DRG neurons from mice knocked-out of other peripheral-specific sodium channels suggests that Nav1.6 channels support the conduction of pain signals in these mice. Ectopic firing by injured Ab afferents emanating from large DRG neurons suggests an important contribution of Nav1.6, to neuropathic pain. Thus, substantial evidence suggests that Nav1.6 contributes to injury-induced hyperexcitability of DRG neurons in chronic pain. Studies have shown that injury reduces levels of Nav1.6 transcripts in DRG neurons. Paradoxically, our preliminary data show significant Nav1.6 immunostaining at nodes of transected myelinated axons in rat sciatic nerve, consistent with similar published studies of ligated infraorbital nerves. We have also reported that Nav1.6 is a substrate for activated MAP kinase p38, which is activated in DRG neurons after injury, and is modulated by a host of cytosolic proteins including members of the FGF family and calmodulin. Thus, injury- mediated modulation of Nav1.6 channels may also contribute to altered neuronal hyperexcitability. In this proposal, we will carry out studies of injury-mediated effects on pain thresholds and hyperexcitability at the cellular level in DRG neurons following knock-down or knock-out of Nav1.6 channels to test the hypotheses that: i) Loss of Nav1.6 in DRG neurons ameliorates pain behavior; ii) Injury alters Nav1.6 sodium currents in nociceptive and large DRG neurons; iii) Modulation of Nav1.6 channels contributes to signal transduction of pro-nociceptive factors. We will also use molecular and immunological methods, and voltage-clamp and current-clamp recordings to: iv) Assess injury-mediated effects on altered regulation of expression and modulation of Nav1.6; and v) Correlate changes at the molecular and cellular levels in injured and spared DRG neurons to changes in pain thresholds.