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

钠通道运输的机制和特异性：开发新型镇痛策略

• 批准号: 10231702
• 负责人: Grant Philip Higerd
• 金额: $ 3.09万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10231702
• 关键词: Absence of pain sensation; Analgesics; Axon; Axonal Transport; Biological Assay; Brain; Cell membrane; Cell surface; Color; Cytoplasm; Data; Disease; Distal; Endocytosis; Endosomes; Epidemic; Goals; Heart; Human; Image; Individual; Ion Channel; Label; Lead; Link; Logic; Mediating; Methods; Microfluidics; Microscopy; Molecular; Movement; Mutation; Neurons; Optics; Organelles; Pain; Pain management; Painless; Pharmacological Treatment; Physicians; Physiologic pulse; Physiological; Potassium; Presynaptic Terminals; Protein Isoforms; Proteins; Research; Resolution; Scientist; Sensory; Signal Transduction; Sodium Channel; Sorting - Cell Movement; Specificity; Surface; Testing; Therapeutic; Time; Training; Vesicle; Video Microscopy; Visualization; career; disability; experimental study; gain of function; ineffective therapies; inhibitor/antagonist; innovation; intense pain; loss of function; neuronal excitability; new therapeutic target; novel; novel therapeutics; opioid use; prevent; side effect; trafficking; vesicle transport; voltage

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.

项目摘要： 钠通道运输的机制和特异性：制定一种新型的镇痛策略。 疼痛的负担很大，当前的疼痛治疗通常无效且令人上瘾。 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 感知神经元。 NAV1.7中的突变会导致疾病，范围从强烈的疼痛（功能获得）到 人类完全无痛（功能丧失），表明其抑制作用可以提供镇痛 中枢神经系统副作用或上瘾的潜力。但是，正在进行的努力开发NAV1.7电导抑制剂的努力 细胞膜尚未导致新疗法。我们提出了一种抑制的替代策略 NAV1.7功能;通过调节贩运往返的贩运来减少细胞表面的通道数量 细胞膜。实现这一目标将需要识别和调节机制 中介NAV1.7贩运。该项目将调查NAV1.7是否通过特定机制贩运。 NAV是通过专用机制或与其他轴突蛋白一起贩运的 不同的功能是一个基本问题。 NAV1.7和NAV1.8在功能上相关，因为它们都 有助于神经元去极化并促进疼痛。相反，电压门控钾（KV）通道 反对神经元激发并抑制疼痛。该提议将检验以下假设。 不同的生理功能根据其功能分别彼此交通。 以前尝试使用荧光蛋白标签观察钠通道运输的尝试失败了 因为细胞质中的大量钠通道池掩盖了弱的细胞膜 带有很少通道的个别囊泡的信号。为了克服这一点，我们开发了光学脉冲练习 轴突长距离（蛋白石）成像，它利用具有自标签标签的功能性人类导航通道 蛋白质（Halotag和Snaptag）和微流体室，可有选择地标记正在 积极贩运轴突。该方法允许实时可视化钠通道囊泡分选，轴突 第一次转运和远端感觉轴突的内吞作用。 在拟议的实验中，我们将研究轴突贩运的两个主要方面：AIM 1将 研究向远端末端的地前运输，AIM 2将询问内吞和逆行 贩运。在每个目标中，我们将通过实时共同确定是否将NAV分类为特定的囊泡 带有标记的囊泡标记的本地化成像，2）确定是否不同但功能相关的NAV是否不同 同工型一起被贩运，3）确定功能相反的NAV和KV通道是否为 一起贩运或分别贩运。这些实验将共同解释轴突囊泡的逻辑 运输并可能为疼痛提供新的治疗靶标。