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

• 批准号: 10593421
• 负责人: JERROLD R. TURNER
• 金额: $ 26.34万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10593421
• 关键词: Address; Adherent Culture; Antigens; Applications Grants; Area; Attenuated; Bacteria; Behavior; Biological; Biology; Biophysics; Cations; Cells; Cellular biology; Characteristics; Charge; Classification; Colitis; Data; Detection; Development; Devices; Disease; Electrodes; Electrophysiology (science); Endothelium; Epithelial Cells; Epithelium; Event; Extracellular Space; Foundations; Frequencies; Functional disorder; Future; Genetic; Goals; Health; Hereditary Disease; Hospitals; Human; Immune; Individual; Infectious colitis; Intercellular Junctions; Ion Channel; Ions; Ischemia; Kidney; Kinetics; Knowledge; Lateral; Life; Lung; Measurement; Measures; Mediating; Methods; Microelectrodes; Molecular; Mus; Mutation; NanoPillar; Nanochip Analytical Device; Nanotechnology; Nephritis; Nephrolithiasis; Organism; Pathogenesis; Permeability; Physiological; Polymers; Population; Predisposition; Principal Investigator; Probability; Process; Protein Isoforms; Proteins; Regulation; Research Personnel; Resolution; Risk; Site; Site-Directed Mutagenesis; Skin; Structure; Surface; Technology; Testing; Therapeutic; Tight Junctions; Time; Tissues; Virus; Water; Woman; Work; X-Ray Crystallography; biophysical properties; design; drug development; fabrication; gastrointestinal; improved; in silico; innovation; interest; intestinal epithelium; medical schools; microbial; microelectronics; millisecond; molecular modeling; monolayer; mutant; novel; patch clamp; pharmacologic; prevent; seal; skills; success; targeted treatment; tool; water channel

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 上皮和内皮形成障碍，将内部和外部环境分开并保持孤立 生物体内的隔室。这些障碍通过组装的细胞间紧密连接来密封 超过claudin蛋白聚合物网络。除了形成障碍外，克劳丁还会创建尺寸和电荷 - 接受离子和水的选择性旁细胞通道。由于这些不同的功能， 单个claudins被归类为密封或孔形成。对小鼠和人类的研究表明 密封或形成孔隙的claudins的突变是遗传性疾病的原因。即使没有突变， Claudin同工型表达的调节变化有助于疾病发病机理。例如，肠道 上皮Claudin-2表达在结肠炎中进行了更新，我们已经证明了Claudin-2通道失活 通过遗传或药物方法显着减弱了实验性免疫介导的结肠炎。 直到最近，Claudin通道仍被认为是允许连续副细胞通量的固定导管。我们的 跨紧密连接夹夹的开发使Claudin-2的改变范式可以发现 通道开放和动态关闭以创建数量细胞电导事件（Weber等，Elife， 2015）。该观察结果通过基本，翻译和疗法产生了许多新问题 影响。但是，没有可能使用跨距交界补丁解决这些问题 夹具方法，仅测量一个非常小的连接区域，并且证明劳动密集型 除了我们的原则分析证明，在技术上很难进行应用。例如，它没有 可以确定所有Claudin通道是否都是动态的；如果不同的claudins使用 不同的生物物理特性，例如开放概率或电导事件大小；或这些特征如何 可以通过细胞调节过程和药物来调节。 该探索性赠款提案旨在将纳米技术应用于Claudin Channel功能的分析 创建由一系列可寻址的，纳米式式电极的纳米芯片。后 这些芯片上的上皮细胞单层的培养，侧面间空间内的电极，就在 紧密的连接可以用于评估通道电导。这种方法避免了困难的过程 在相邻细胞之间的副细胞空间上修补GΩ密封件。通过消除补丁移液器 并同时分析单个单层内的多个连接和通道， 紧密的连接感应纳米芯片将克服跨紧密连接贴片夹的主要局限性。 这项小说，实现技术将导致新的基本，改变范式的发现，并最终，最终 为了调节细胞紧密障碍物的代理所需的知识和工具，以便 治疗在肠道，肾脏和肺在内的潜在地点的上皮屏障功能的疾病。