Sodium Channel Nav1.6 and Pathophysiology of Pain

描述(由申请人提供)： 这项提案的目的是证明钠通道Nav1.6在神经病理性和炎症性疼痛中的重要性，并确定开发新的有效疼痛疗法的具体目标和策略。钠通道的异常表达和突变与背根节(DRG)神经元的高兴奋性有关，背根节神经元是外周起源的炎症性和神经病理性疼痛的基础，这两者构成了神经病理性疼痛的主要障碍。Nav1.6是大型背根节神经元中的主要钠通道，在有髓纤维的轴突起始段和兰维尔成熟结节中含量丰富--这两个神经元是神经冲动传导所必需的。我们的初步数据显示，Nav1.6在大多数伤害性DRG神经元中也有表达，这些神经元产生了无髓纤维，这与我们之前发现的Nav1.6基因缺失小鼠坐骨神经C纤维复合动作电位传导受损的发现一致。在其他外周特异性钠通道被敲除的小鼠背根神经节神经元中持续存在Nav1.6表明，Nav1.6通道支持这些小鼠的疼痛信号传导。从大的背根节神经元发出的受损的AB传入的异位放电表明了Nav1.6在神经病理性疼痛中的重要贡献。因此，大量证据表明，在慢性疼痛中，Nav1.6参与了损伤诱导的DRG神经元的超兴奋性。研究表明，损伤会降低DRG神经元中Nav1.6转录本的水平。矛盾的是，我们的初步数据显示，在大鼠坐骨神经有髓轴突的结节上有显著的Nav1.6免疫染色，这与已发表的关于结扎的眶下神经的研究一致。我们还报道，Nav1.6是激活的MAP激酶p38的底物，损伤后在DRG神经元中激活，并受包括成纤维细胞生长因子家族成员和钙调蛋白在内的一系列胞浆蛋白的调节。因此，损伤介导的Nav1.6通道的调制也可能有助于改变神经元的超兴奋性。在这项提议中，我们将在细胞水平上研究NA1.6通道被敲除或敲除后，损伤对DRG神经元痛阈值和超兴奋性的影响，以检验以下假设：i)DRG神经元中Nav1.6的丢失可改善疼痛行为；ii)损伤可改变伤害性感受和大型DRG神经元的Nav1.6钠电流；iii)Nav1.6通道的调制有助于伤害性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)评估损伤对Nav1.6表达和调制的改变所介导的影响；v)将受损和备用DRG神经元在分子和细胞水平的变化与痛阈值的变化联系起来。

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.