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

项目摘要/摘要 上皮和内皮形成屏障，将内外环境隔开并保持隔离 生物体内的隔间。这些屏障由组装在一起的细胞间紧密连接密封。 通过克拉丁蛋白聚合物网络。除了形成屏障外，笨拙还会产生大小和电荷-- 选择性的细胞旁通道，可容纳离子和水。由于这些不同的功能， 个别的克拉丁被归类为封闭型或造孔型。对老鼠和人类的研究表明， 封闭或形成孔洞的克拉丁突变是遗传性疾病的原因。即使没有突变， Claudin异构体表达的调节变化参与了疾病的发病机制。例如，肠道 上皮claudin-2在结肠炎中表达上调，我们已经证明claudin-2通道失活 通过遗传或药理学方法显着减轻实验性免疫介导性结肠炎。 直到最近，克拉丁通道还被认为是允许持续的细胞旁流量的固定管道。我们的 跨紧连接膜片钳的发展使得claudin-2改变了范式的发现 通道动态地打开和关闭以创建量子细胞旁电导事件(Weber等人，eLife， 2015年)。这一观察产生了许多新的问题，包括基础性、翻译性和治疗性 冲击力。然而，使用跨紧连接贴片还不可能解决这些问题 钳位法，仅测量单个、非常小的连接点面积，已被证明过于劳动密集型 除了我们的原则证明分析之外，在技术上也很难应用。例如，它还没有， 可以确定是否所有的claudin通道都是动态的；如果不同的claudin使用 不同的生物物理特性，例如，打开概率或电导事件大小；或者这些特性如何 可受细胞调节过程和药理作用的调节。 这项探索性拨款提案旨在通过以下方式将纳米技术应用于克拉丁通道功能的分析 制造由可单独寻址的、安装在纳米柱上的电极阵列填充的纳米芯片。之后 在这些芯片上培养上皮细胞单层，电极在横向细胞间隙内，就在下面 紧结可以用来评估沟道电导。这种方法省去了困难的过程 在相邻细胞之间的细胞旁间隙修补GΩ密封层。通过取消贴片移液管 并使单个单层内的多个结和通道的并发分析成为可能， 紧密结传感纳米芯片将克服跨紧结膜片钳的主要局限性。 这种新颖的使能技术将导致新的、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 治疗不同部位的上皮屏障功能障碍，包括肠道、肾脏和肺。

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.