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：开发一种新的镇痛策略。 疼痛的负担是显着的，目前的疼痛治疗往往是无效的和上瘾的。 迫切需要替代办法。电压门控钠通道NaV1.7优先在疼痛中表达- 感觉神经元NaV1.7的突变可导致从剧烈疼痛（功能获得）到 完全无痛（功能丧失），表明其抑制可以提供镇痛， CNS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， 细胞膜尚未导致新的疗法。我们提出了一种替代策略来抑制 NaV1.7功能;通过调节细胞表面通道的往返运输，减少细胞表面通道的数量 细胞膜。实现这一目标需要确定和调整机制， 介导NaV1.7贩运。该项目将调查NaV1.7是否通过特定机制进行贩运。 NaV是否通过专门的机制或与其他轴突蛋白一起运输， 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 对抗神经元兴奋并抑制疼痛。该提议将检验离子通道与 不同的生理功能根据它们的功能彼此分开地进行交易。 以前试图用荧光蛋白标签观察钠通道运输的尝试都失败了 因为细胞质和细胞膜上大量的钠离子通道掩盖了细胞内钠离子通道的薄弱。 携带很少通道的单个囊泡的信号。为了克服这一点，我们开发了光学脉冲追踪 轴突长距离（OPAL）成像，利用标记有自标记的功能性人类NaV通道 蛋白质（HaloTag和SNAPTag）和微流体室，以选择性地标记被 活跃在轴突中该方法允许钠通道囊泡分选、轴突生长和神经生长的实时可视化。 运输和内吞作用的远端感觉轴突的第一次。 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 研究顺行运输到远端终末，Aim 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NaV是否通过活的共沉淀被分选到特定的囊泡中， 用标记的囊泡标记物进行定位成像，2）确定不同但功能相关的NaV 同种型一起运输，和3）确定功能上相反的NaV和KV通道是否 一起或单独贩卖。总之，这些实验将解释轴突囊泡的逻辑 运输和潜在地提供新的疼痛治疗靶点。