Defining single-channel paracellular (tight junction) conductances using nanotechnology

使用纳米技术定义单通道旁细胞(紧密连接)电导

基本信息

  • 批准号:
    10593421
  • 负责人:
  • 金额:
    $ 26.34万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2023
  • 资助国家:
    美国
  • 起止时间:
    2023-02-01 至 2025-01-31
  • 项目状态:
    未结题

项目摘要

PROJECT SUMMARY/ABSTRACT Epithelia and endothelia form barriers that separate the internal and external milieus and maintain isolated compartments within organisms. These barriers are sealed by intercellular tight junctions that are assembled over claudin protein polymer networks. Beyond forming the barrier, claudins also create size- and charge- selective paracellular channels that accommodate ions and water. Because of these divergent functions, individual claudins are classified as sealing or pore-forming. Studies in mice and humans demonstrate that mutations of sealing or pore-forming claudins are causes of heritable disorders. Even without mutation, regulated changes in claudin isoform expression contribute to disease pathogenesis. For example, intestinal epithelial claudin-2 expression is upregulated in colitis, and we have shown that claudin-2 channel inactivation by genetic or pharmacological approaches markedly attenuates experimental immune-mediated colitis. Until recently, claudin channels were thought of as fixed conduits that allow continuous paracellular flux. Our development of the trans-tight junction patch clamp allowed the paradigm-altering discovery that claudin-2 channels open and close dynamically to create quantal paracellular conductance events (Weber et al, eLife, 2015). This observation generated many new questions with fundamental, translational, and therapeutic impact. It has not, however, been possible to address these questions using the trans-tight junction patch clamp method, which measures only a single, very small, area of junction and has proven too labor-intensive and technically difficult for application beyond our proof-of-principle analyses. For example, it has not, been possible to determine whether all claudin channels are dynamic; if different claudins create channels with distinct biophysical properties, e.g., open probability or conductance event size; or how these characteristics can be modulated by cellular regulatory processes and pharmacologic agents. This exploratory grant proposal seeks to apply nanotechnology to analysis of claudin channel function by creating a nanochip populated by an array of individually addressable, nanopillar-mounted electrodes. After culture of epithelial cell monolayers on these chips, electrodes within lateral intercellular spaces, just beneath the tight junctions, can be used to evaluate channel conductances. This approach obviates the difficult process of patching a GΩ seal across the paracellular space between adjacent cells. By eliminating the patch pipette and making concurrent analysis of multiple junctions and channels within a single monolayer possible, the tight junction-sensing nanochip will overcome the main limitations of the trans-tight junction patch clamp. This novel, enabling technology will lead to new fundamental, paradigm-changing discoveries and, ultimately, knowledge and tools needed for development of agents that regulate cellular tight junction barriers in order to treat disorders of epithelial barrier function at diverse sites including the gut, kidneys, and lungs.
项目摘要/摘要 上皮细胞和内皮细胞形成屏障,分隔内外环境, 生物体内的细胞。这些屏障由细胞间紧密连接封闭, claudin蛋白质聚合物网络。除了形成屏障外,claudins还创造了大小和电荷 选择性的细胞旁通道,容纳离子和水。由于这些不同的功能, 单独的紧密连接蛋白被分类为密封的或成孔的。对小鼠和人类的研究表明, 密封或成孔密封蛋白的突变是遗传性疾病的原因。即使没有突变, 密蛋白亚型表达的调节变化有助于疾病的发病机制。例如肠 结肠炎中上皮细胞claudin-2表达上调,我们已经证明claudin-2通道失活 通过遗传或药理学方法显著减弱实验性免疫介导的结肠炎。 直到最近,claudin通道被认为是固定的管道,允许连续的细胞旁流量。我们 反式紧密连接膜片钳的发展使得改变范式的发现, 通道动态地打开和关闭以产生量子细胞旁传导事件(Weber等,eLife, 2015年)。这一观察结果产生了许多新的问题,包括基本的,转化的和治疗的问题。 冲击然而,使用反式紧密连接补片不可能解决这些问题 钳位法,它只测量一个单一的,非常小的,结区,已被证明过于劳动密集型 并且在技术上难以应用于我们的原理验证分析之外。例如,它没有, 可以确定是否所有claudin通道都是动态的;如果不同的claudin创建通道, 不同的生物物理特性,例如,开放概率或电导事件大小;或者这些特征 可以通过细胞调节过程和药理学试剂来调节。 这项探索性的拨款提案旨在将纳米技术应用于claudin通道功能的分析, 产生由可单独寻址的纳米柱安装电极阵列填充的纳米芯片。后 在这些芯片上培养上皮细胞单层,电极位于细胞间隙的侧面,就在下面 紧密结可用于评估沟道电导。这种方法避免了困难的过程 在相邻细胞之间的细胞旁间隙贴上G-密封。通过消除贴片移液管 并且使得在单个单层内的多个连接和通道的同时分析成为可能, 紧密连接传感纳米芯片将克服跨紧密连接膜片钳的主要限制。 这种新颖的技术将带来新的基础性的、改变范式的发现,并最终, 开发调节细胞紧密连接屏障的试剂所需的知识和工具, 治疗包括肠道、肾脏和肺部在内的不同部位的上皮屏障功能障碍。

项目成果

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JERROLD R. TURNER其他文献

JERROLD R. TURNER的其他文献

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{{ truncateString('JERROLD R. TURNER', 18)}}的其他基金

Advanced Multi-color Confocal and FRAP-SAC Microscope
先进的多色共焦和 FRAP-SAC 显微镜
  • 批准号:
    7792767
  • 财政年份:
    2010
  • 资助金额:
    $ 26.34万
  • 项目类别:
Cell Imaging
细胞成像
  • 批准号:
    7030417
  • 财政年份:
    2006
  • 资助金额:
    $ 26.34万
  • 项目类别:
Mechanisms and consequences of cytokine-induced tight junction barrier regulation
细胞因子诱导的紧密连接屏障调节的机制和后果
  • 批准号:
    8111221
  • 财政年份:
    2005
  • 资助金额:
    $ 26.34万
  • 项目类别:
Regulation of Paracellular Permeability by IFNg and TNFa
IFNg 和 TNFa 对细胞旁通透性的调节
  • 批准号:
    6924157
  • 财政年份:
    2005
  • 资助金额:
    $ 26.34万
  • 项目类别:
Mechanisms and consequences of cytokine-induced tight junction barrier regulation
细胞因子诱导的紧密连接屏障调节的机制和后果
  • 批准号:
    7996729
  • 财政年份:
    2005
  • 资助金额:
    $ 26.34万
  • 项目类别:
Perijunctional myosin light chain kinase recruitment: A novel, non-enzymatic target for therapeutic intestinal barrier restoration
接合周围肌球蛋白轻链激酶募集:用于治疗性肠屏障恢复的新型非酶靶点
  • 批准号:
    10441427
  • 财政年份:
    2005
  • 资助金额:
    $ 26.34万
  • 项目类别:
Regulation of Paracellular Permeability by IFNgamma and TNFa
IFNγ 和 TNFa 对细胞旁通透性的调节
  • 批准号:
    7252409
  • 财政年份:
    2005
  • 资助金额:
    $ 26.34万
  • 项目类别:
The Myosin Light Chain Kinase-Phosphatase Axis in GI Homeostasis and Disease
胃肠道稳态和疾病中的肌球蛋白轻链激酶-磷酸酶轴
  • 批准号:
    8725914
  • 财政年份:
    2005
  • 资助金额:
    $ 26.34万
  • 项目类别:
Regulation of Paracellular Permeability by IFNy and TNFa
IFNγ和TNFa对细胞旁通透性的调节
  • 批准号:
    7027748
  • 财政年份:
    2005
  • 资助金额:
    $ 26.34万
  • 项目类别:
Regulation of Paracellular Permeability by IFNgamma and TNFa
IFNγ 和 TNFa 对细胞旁通透性的调节
  • 批准号:
    7460826
  • 财政年份:
    2005
  • 资助金额:
    $ 26.34万
  • 项目类别:

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探针型纳米线传感器,用于无标记、原位、超灵敏地检测贴壁培养中单细胞的生物标志物
  • 批准号:
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  • 财政年份:
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  • 财政年份:
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