Structure, function, and modulation of claudin cation channels in the GI tract

摘要 肠道疾病，包括炎症性肠病（IBD）、乳糜泻和感染性结肠炎， 与上皮屏障功能障碍相关，导致腹泻和营养吸收不良。紧 连接密封上皮细胞之间的空间，并通过控制细胞旁流量来维持屏障功能。的 紧密连接蛋白的claudin家族在定义对离子和小分子的紧密连接屏障中是关键的 分子。在胃肠道中，claudin-2和claudin-15在它们的调节作用中特别重要。 细胞旁钠流及其改变的表达可促进肠道疾病的发展。 利用一种新的膜片钳技术，我们证明了claudin-2和claudin-15形成门控Na+选择性离子， 细胞旁间隙的通道。这一点很重要，因为它证明了claudin具有 类似于跨膜离子通道。然而，我们对这些阳离子如何选择性地 claudins有助于电荷和大小选择性的细胞旁孔，这两个claudins如何单独或 共同影响肠道功能，以及它们如何影响疾病进程。为了解决这些问题，我们 建立了claudin-2和claudin-15的全原子计算机模型，使我们能够模拟claudin结构和孔 功能这些模型还帮助我们鉴定了几种一流的claudin通道阻滞剂， 孔在低微摩尔浓度。 我们建议使用这些独特和新颖的计算和小分子抑制剂工具来研究 claudin-2和-15通道如何控制通过紧密连接的单价阳离子通量，以及它们如何 差异调节健康和疾病中的阳离子转运。在目标1中，我们将使用claudin-2和claudin-15 计算机模型和通道阻断剂，以确定决定大小和 电荷选择性在目标2中，我们将使用现有的和新的claudin通道阻滞剂来定义个体 以及紧密连接蛋白-2和紧密连接蛋白-15对正常肠生理学和疾病表现的组合贡献， 发展我们将确定claudin-2和claudin-15通道在Na+耦合营养共转运中的作用。 介导的屏障调节、结肠炎小鼠模型和人IBD中的作用。完成这项研究是 通过提供这些通道如何介导的关键见解，有望为人类健康做出积极贡献 营养吸收，导致结肠炎背景下的腹泻，并可能有助于发展 新疗法

ABSTRACT Intestinal diseases, including inflammatory bowel disease (IBD), celiac disease, and infectious colitis are associated with epithelial barrier dysfunction that contribute to diarrhea and nutrient malabsorption. Tight junctions seal spaces between epithelial cells and maintain barrier function by controlling paracellular flux. The claudin family of tight junction proteins is critical in defining the tight junction barrier to ions and small molecules. In the gastrointestinal tract, claudin-2 and -15 are particularly important in their role regulating paracellular sodium flux and their altered expression can contribute to intestinal disease development. Using a novel patch clamp technique, we demonstrated that claudin-2 and -15 form gated Na+ selective ion channels in the paracellular space. This is important because it demonstrates that claudins have properties similar to transmembrane ion channels. However, we have limited understanding of how these cation selective claudins contribute to charge and size selective paracellular pores, how these two claudins function singly or together to impact intestinal function, and how they impact disease processes. To address these questions, we built all-atom computer models for claudin-2 and -15 which allowed us to model both claudin structure and pore function. These models also helped us to identify several first-in-class claudin channel blockers that block the pore at low micromolar concentrations. We propose to use these unique and novel computational and small molecule inhibitor tools to investigate how claudin-2 and -15 channels control monovalent cation flux across the tight junction, and how they may differentially regulate cation transport in health and disease. In Aim 1, we will use our claudin-2 and -15 computer models and channel blockers to determine key molecular and structural features that dictate size and charge selectivity. In Aim 2, we will use our existing and new claudin channel blockers to define the individual and combined contributions of claudin-2 and -15 to normal intestinal physiology and disease presentation and development. We will determine the role of claudin-2 and -15 channels in Na+-coupled nutrient co-transport- mediated barrier regulation, mouse models of colitis, and in human IBD. Completion of this line of study is expected to contribute positively to human health by providing key insight into how these channels mediate nutrient absorption, contribute to diarrhea in the setting of colitis, and potentially aid in the development of novel therapies.