Molecular architecture of claudin-based tight junction strands and paracellular ion channels

紧密连接(TJ)调节细胞旁跨越上皮和内皮组织屏障的溶质和水的通透性。这一功能的关键结构决定因素是Claudin家族的四螺旋跨膜蛋白(CLDN)。它们构成膜内TJ链的主干，并根据亚型的不同，形成小阳离子、阴离子或水的屏障或细胞旁通道。Claudins在结晶、结构预测、MD模拟和功能分析等方面的进展有力地促进了对TJ结构和功能的理解。然而，TJ链和细胞旁通道的分子结构仍在争论中。虽然已经确定了影响组装和功能的claudin序列决定因素，但导致TJ(阳离子、阳离子和水或阴离子或离子屏障的通道)功能多样性的三维结构差异尚不清楚。该项目的主要目标是阐明TJ链和细胞旁通道的详细分子结构，包括Claudin亚型依赖的多样性。在此之前，我们建立并成功地应用了以下方法：(A)剖析TJ链组装的多步骤过程，包括Claudins之间的同亲和异亲、顺式和反式相互作用；(B)研究TJ的运输生理；(C)在膜环境中模拟和模拟Claudin低聚体的分子动力学。对于TJ领域至关重要的是，TJ链上的细胞生理、纳米和结构/模拟数据的汇聚仍然在很大程度上缺失。这一资助期的总体目标是实现在胃肠道和/或肾脏中表达的克拉丁蛋白的这种融合。其具体目的是阐明(I)类CLDN10b/CLDN15类阳离子通道以及CLDN10a-和CLDN17阴离子通道的选择性，(Ii)屏障(CLDN1，-3，-5)与通道形成的结构和机制基础，(Iii)Claudin在TJ链上混合和分离成功能不同的片段，以及(Iv)致病突变对Claudin通道或屏障功能的干扰。该提案将细胞生理学和结构生物信息学专家捆绑在一起，研究经典的克拉丁蛋白。由于TJ功能对所有器官都是必不可少的，而且在许多影响肠道、肾脏、腺体或大脑的屏障和运输相关疾病中受到干扰，该项目将在组织屏障的分子生理学和病理生理学方面提供关键的见解。

Tight junctions (TJ) regulate the paracellular permeability for solutes and water across epithelial and endothelial tissue barriers. The key structural determinants for this function are the tetra-helical transmembrane proteins of the claudin (CLDN) family. They form the backbone of intramembrane TJ strands and depending on the subtype either barriers or paracellular channels for small cations, anions or water. Progress in crystallization, structure prediction, MD simulations and functional analysis of claudins strongly advanced the understanding of TJ structure and function. However, the molecular architecture of TJ strands and paracellular channels is still under debate. Although claudin sequence determinants influencing assembly and function have been identified, the 3D-structural differences leading to the functional diversity of TJs (channels for cation, cation & water or anions or ion barriers) are unclear. Main goal of the project is to elucidate the detailed molecular architecture of TJ strands and paracellular channels including claudin subtype-dependent diversity. Previously, we established and successfully applied methods (a) to dissect the multistep process of TJ strand assembly that includes homo- and heterophilic, cis- and trans-interactions between claudins (b) to investigate the transport physiology of TJs and (c) to model and simulate the molecular dynamics of claudin oligomers in a membrane environment. Crucially for the TJ field, convergence of cell-physiological, nanoscopic and structural/simulation data on TJ strands is still largely missing. The general aim of this funding period is to achieve this convergence for claudins expressed in the gastrointestinal tract and/or the kidney. The specific aims are elucidation of the structural and mechanistic basis for (i) selectivity of CLDN10b/CLDN15-like cation channels and that of CLDN10a- and CLDN17 anion channels, (ii) barrier (CLDN1, -3, -5) versus channel formation, (iii) claudin intermixing and segregation into functionally distinct segments along TJ strands and (iv) disturbance of channel or barrier function of claudins by pathogenic mutations. The proposal bundles cell-physiological and structural bioinformatics expertises on classic claudins. Since TJ function is essential for all organs and disturbed in a multitude of barrier- and transport-related diseases affecting the intestine, kidney, glands or the brain, the project will provide key insights in the molecular physiology and pathophysiology of tissues barriers in general.