Mechanisms and specificity of sodium channel trafficking: Developing a novel analgesic strategy

项目总结： 钠通道转运的机制和特异性：开发一种新的止痛策略。 疼痛的负担是巨大的，目前的疼痛治疗往往无效并使人上瘾。 人们迫切需要替代方案。电压门控钠通道Nav1.7在疼痛中优先表达- 感觉神经元。Nav1.7的突变可导致从剧烈疼痛(功能获得)到 在人类中完全无痛(功能丧失)，这表明抑制它可以在没有 中枢神经系统副作用或成瘾潜力。然而，正在进行的开发Nav1.7电导抑制剂的努力 细胞膜还没有产生新的治疗方法。我们提出了一种替代策略来抑制 Nav1.7功能；通过调节来往于细胞表面的通道数量来减少通道数量 细胞膜。要实现这一目标，需要确定和调整具体的机制 调解Nav1.7的贩运。该项目将调查Nav1.7是否通过特定机制进行交易。 无论NAV是通过专用机制还是与其他轴突蛋白一起通过 不同的功能是一个根本的问题。Nav1.7和NaV1.8在功能上是相关的，因为它们 有助于神经元去极化并促进疼痛。相比之下，电压门控钾(KV)通道 反对神经兴奋，抑制疼痛。这一提议将检验离子通道与 不同的生理功能根据它们的功能相互独立地传输。 以前使用荧光蛋白标签观察钠通道转运的尝试都失败了 因为细胞质和细胞膜上的大量钠离子通道掩盖了 单个小泡携带较少通道的信号。为了克服这一点，我们开发了光脉冲追踪器 轴突长距离(Opal)成像，它利用带有自我标记的功能性人体NAV通道 蛋白质(HaloTag和SnapTag)和微流体室选择性地标记正在被 活跃地贩卖轴突。这种方法可以实时显示钠通道的囊泡分类，轴突 第一次在远端感觉轴突中运输和内吞作用。 在拟议的实验中，我们将依次检查轴突运输的两个主要方面：目标1将 调查顺行运输到远端终端的情况，目标2将询问内吞和逆行 贩卖人口。在每个目标中，我们将1)确定导航是否通过活的共同作用被分类为特定的囊泡- 用标记的水泡标记进行定位成像，2)确定不同但功能相关的NAV 异构体一起传输，以及3)确定功能相反的NAV和KV通道 一起或分开贩卖的。总之，这些实验将解释轴突囊泡的逻辑。 运输，并可能提供新的治疗疼痛的靶点。

Project Summary: Mechanisms and specificity of sodium channel trafficking: Developing a novel analgesic strategy. The burden of pain is significant and current pain treatments are often ineffective and addictive. Alternatives are urgently needed. Voltage-gated sodium channel NaV1.7 is preferentially expressed in pain- sensing neurons. Mutations in NaV1.7 can cause disorders ranging from intense pain (gain-of-function) to complete painlessness (loss-of-function) in humans, suggesting that its inhibition could provide analgesia without CNS side-effects or addictive potential. However, ongoing efforts to develop inhibitors of NaV1.7 conductance at the cell membrane have not yet resulted in new therapies. We propose an alternative strategy for inhibition of NaV1.7 function; reducing the number of channels at the cell surface by modulating their trafficking to and from the cell membrane. Achieving this goal would require identifying and modulating mechanisms that specifically mediate NaV1.7 trafficking. This project will investigate whether NaV1.7 is trafficked by specific mechanisms. Whether NaVs are trafficked by dedicated mechanisms or together with other axonal proteins with different functions is a fundamental question. NaV1.7 and NaV1.8 are functionally related, as they both contribute to neuronal depolarization and promote pain. In contrast, voltage-gated potassium (KV) channels oppose neuronal excitation and suppress pain. This proposal will test the hypothesis that ion channels with different physiological functions are trafficked separately from each other according to their functions. Previous attempts to observe sodium channel trafficking using fluorescent protein tags have failed because the substantial pool of sodium channels in the cytoplasm and at the cell membrane conceal the weak signal of individual vesicles carrying few channels. To overcome this, we developed Optical Pulse-chase Axonal Long-distance (OPAL) imaging, which utilizes functional human NaV channels tagged with self-labeling proteins (HaloTag and SNAPTag) and microfluidic chambers to selectively label channels that are being actively trafficked in axons. This method allows live visualization of sodium channel vesicular sorting, axonal transport, and endocytosis in distal sensory axons for the first time. In the proposed experiments, we will examine two major aspects of axonal trafficking in turn: Aim 1 will investigate anterograde trafficking to distal terminals and Aim 2 will interrogate endocytosis and retrograde trafficking. In each Aim, we will 1) Determine whether NaVs are sorted into specific vesicles by live co- localization imaging with tagged vesicle markers, 2) Determine whether different but functionally related NaV isoforms are trafficked together, and 3) Determine whether functionally opposite NaV and KV channels are trafficked together or separately. Together, these experiments will explain the logic of axonal vesicular transport and potentially provide new therapeutic targets for pain.