Ionic Mechanisms Underlying Dorsal Root Ganglion Excitability

描述(申请人提供)：神经性疼痛的痛感是复杂的，包括虚弱、感觉缺陷和麻木、反射变化、自发或对外部刺激产生的异常感觉、痛觉过敏和超敏。背根神经节(DRG)神经元兴奋性的紊乱是诱发神经病理性疼痛的关键，尤其是在糖尿病期间，糖尿病是神经病理性疼痛的最常见原因。在糖尿病过程中，p38丝裂原活化蛋白激酶(P38MAPK)信号系统被激活，当这一途径被抑制时，糖尿病引起的神经病理性疼痛得到缓解。然而，参与DRG神经元神经病变过程的主要离子电导尚不清楚。背根神经节神经元具有高水平的一种新的、未被研究的钾通道家族，称为钠激活钾通道(KNA)。我们以前的工作表明，KNA是外向钾电流的相当大的组成部分，负责DRG神经元的放电调节。当我们在实验中减少DRG神经元中这些通道的表达时，它会产生类似于神经病理性神经元的过度兴奋性。有两个基因编码这些通道，松弛和光滑。在异源表达系统中，SLICK和SLACK亚基可以共同组装形成具有非常慢的激活动力学的异构体通道系统，非常适合于控制激发调节。此外，同分异构体通道似乎受到Nedd4l依赖的泛素化的影响，这表明松弛/光滑异构体通道是天然KNA通道的首选配置。SLAK和SLICK在通道的钠结合/门控区域附近也有共同的p38MAPK磷酸化位点。糖尿病激活的p38MAPK信号后，KNA通道活性可能随之降低。由于糖尿病也影响转录活动，我们希望在神经元中发现KNA通道表达的长期变化。我们将利用电生理、生化、分子、疼痛行为分析和一只以前没有特征的光滑基因敲除小鼠，验证以下假设：异构体KNA通道抑制感觉神经元的过度兴奋性，神经病理性疼痛与DRG神经元中KNA通道活性降低有关。其具体目的是(1)研究p38MAPK对DRG KNA通道的调节；(2)研究DRG神经元KNA通道的亚基特性；(3)研究糖尿病时神经元KNA通道的活动，并与光滑基因敲除小鼠的疼痛行为进行比较。本研究项目将评估KNA通道在糖尿病神经病变中的作用。

DESCRIPTION (provided by applicant): Pain sensation in neuropathic pain is complex consisting of weakness, sensory deficits and numbness, reflex changes, abnormal sensations that occur either spontaneously or in reaction to external stimuli, hyperalgesia and allodynia. Perturbations in dorsal root ganglion (DRG) neuron excitability are key in precipitating neuropathic pain, especially during diabetes, the most common cause of neuropathic pain. During diabetes, the p38 mitogen-activated protein kinase (p38MAPK) signaling system is activated and when this pathway is inhibited, diabetes-induced neuropathic pain is attenuated. However, the major ion conductances involved in the neuropathic process of DRG neurons are unclear. DRG neurons possess high levels of a novel, understudied family of potassium channels called sodium-activated potassium channels (KNa). Our previous work has shown that KNa is a considerable component of the outward potassium current and is responsible for firing accommodation in DRG neurons. When we experimentally reduce the expression of these channels in DRG neurons, it produces hyperexcitability that resembles neuropathic neurons. There are two genes encoding these channels, Slack and Slick. In heterologous expression systems, the Slick and Slack subunits can co-assemble to form heteromeric channels systems with very slow activation kinetics ideal for controlling firing accommodation. Moreover, homomeric Slick channels appear to be subject to Nedd4l-dependent ubiquitination, suggesting that Slack/Slick heteromeric channels are the preferred configuration of native KNa channels. Slack and Slick also have p38MAPK consensus phosphorylation sites proximal to the sodium binding/gating region of the channels. A decrease in KNa channel activity likely ensues after diabetes-activated p38MAPK signaling. Since diabetes also affects transcriptional activities, we expect to find long-term changes in KNa channel expression in neurons. Using electrophysiological, biochemical, molecular, pain behavioral assays and a previously uncharacterized Slick knockout mouse, we will test the hypotheses: heteromeric KNa channels constrain sensory neuron hyperexcitability and neuropathic pain is associated with decreased KNa channel activity in DRG neurons. The specific aims are (1) To study the regulation of DRG KNa channels by p38MAPK (2) To investigate the subunit properties of KNa channels in DRG neurons (3) To study neuronal KNa channel activity during diabetes and compare pain behavior to Slick knockout mice. This research project will assess the involvement of KNa channels in the diabetic neuropathy.