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 与导致腹泻和营养物质吸收不良的上皮屏障功能障碍有关。紧绷 连接处封闭上皮细胞之间的间隙，并通过控制细胞旁流量来维持屏障功能。这个 紧密连接蛋白的claudin家族在确定对离子和小分子的紧密连接屏障中起着关键作用。 分子。在胃肠道中，claudin-2和-15在它们的调节作用中尤为重要 细胞旁钠流量及其表达的改变可能导致肠道疾病的发生。 利用新的膜片钳技术，我们证明了Claudin-2和-15形成门控Na选择离子 细胞旁间隙中的通道。这一点很重要，因为它证明了克劳迪斯具有 类似于跨膜离子通道。然而，我们对这些阳离子的选择性了解有限 Claudins有助于电荷和大小选择性的细胞旁毛孔，这两个Claudins如何单独发挥作用或 共同影响肠道功能，以及它们如何影响疾病过程。为了解决这些问题，我们 建立了claudin-2和-15的全原子计算机模型，使我们能够同时对claudin结构和孔洞进行建模 功能。这些模型还帮助我们确定了几种一流的克拉丁通道阻滞剂，它们可以阻止 微摩尔浓度较低的孔洞。 我们建议使用这些独特而新颖的计算和小分子抑制物工具来研究 Claudin-2和-15通道如何控制紧密连接上的单价阳离子通量，以及它们如何 对健康和疾病中的阳离子转运进行差异化调控。在目标1中，我们将使用claudin-2和-15 计算机模型和通道阻滞剂以确定决定大小和结构的关键分子和结构特征 电荷选择性。在目标2中，我们将使用我们现有的和新的Claudin通道阻滞剂来定义个体 Claudin-2和-15在正常肠道生理和疾病表现中的联合作用 发展。我们将确定claudin-2和-15通道在钠偶联营养共运输中的作用。 介导性屏障调节，小鼠结肠炎模型，以及人类IBD。完成这一研究方向是 希望通过提供对这些渠道如何调节的关键洞察力，为人类健康做出积极贡献 营养吸收，有助于结肠炎背景下的腹泻，并可能有助于 新奇的疗法。

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.