Structural and functional analyses of E-face-associated tight junction strands formed by expression of claudin-1 mutant in the second extracellular loop in MDCKII cells

当通过冷冻断裂电子显微镜观察上皮细胞时，分别在原生质（P）面和外质（E）面观察到由膜内颗粒和无颗粒的互补凹槽组成的连续紧密连接链（P型紧密连接）。相反，周围小静脉内皮细胞的紧密连接颗粒位于E面的凹槽上（E型紧密连接）。紧密连接蛋白是紧密连接处的完整膜蛋白，由至少24个成员组成，并且在没有闭合蛋白的情况下在成纤维细胞中重建紧密连接链。紧密连接蛋白由四个跨膜结构域组成，其中NH 2-和COOH-末端均位于细胞质中，两个细胞外环（ECL 1和ECL 2）和一个细胞质环。据认为，紧密连接的结构和功能可能由紧密连接中表达的紧密连接蛋白种类的组合和混合比例决定。我们构建了含有claudin-1的表达载体， ...更多信息 在ECL 2中缺失两个氨基酸（151 P152 L）的1CLmyc（1CLAPLmyc），和在COOH-末端具有myc-表位的claudin-10（10 CLmyc），以及claudin-10本身。然后我们在MDCK I或II细胞中表达它们。当1CLmyc表达细胞的冷冻断裂电子显微镜观察，P型紧密连接检测到侧质膜。10 CLmyc在侧膜形成E型紧密连接。而1CLAPLmyc形成中间型紧密连接，其膜内颗粒与P面和E面的结合相等。表达1CLmyc和1CLAPLmyc的细胞的跨上皮电阻显著增加，但claudin-10的跨上皮电阻显著降低。这些结果表明claudin-1的ECL 2参与决定紧密连接颗粒是否与P面或E面相关，并且P型和中间型紧密连接增加跨上皮电阻，但E型紧密连接降低跨上皮电阻。少

When epithelial cells are observed by freeze-fracture electron microscopy, continuous tight junction strands consisted of intramembranous particles and complementary grooves without particles are observed in the protoplasmic (P)-face and exoplasmic (E)-face, respectively (P-type tight junction). In contrast, tight junction particles of endothelial cells of peripheral venules are located on the grooves in the E-face (E-type tight junction). Claudins are integral membrane proteins at tight junctions, consist of at least 24 members, and reconstitute tight junction strands in fibroblasts without occludin. Claudins consist of four transmembrane domains with both NH2-and COOH-termini located in the cytoplasm, two extracellular loops (ECL1 and ECL2), and one cytoplasmic loop. It is thought that structure and function of tight junctions may be determined by combination and mixing ratio of claudin species expressed in the tight junction. We constructed expression vectors containing claudin-1 wi … More th a myc-epitope at the COOH-terminus (1CLmyc), 1CLmyc deleted two amino acids (151P152L) in the ECL2(1CLAPLmyc), and claudin-10 with a myc-epitope at the COOH-terminus (10CLmyc), and claudin-10 itself. We then expressed them in MDCK I or II cells. When 1CLmyc-expressing cells were observed by freeze-fracture electron microscopy, P-type tight junctions were detected in lateral plasma membranes. 10CLmyc formed E-type tight junction in lateral membranes. However, 1CLAPLmyc formed intermediate type of tight junction whose intramembranous particles were associated equally with the P- and E-face. Transepithelial electrical resistances of 1CLmyc-and 1CLAPLmyc-expressing cells were significantly increased, but that of claudin-10 was significantly decreased. These results suggest that the ECL2 of claudin-1 is involved in determining whether tight junction particles associate with the P- or E-face, and that P-type and intermediate-type tight junctions increased transepithelial electrical resistance but E-type tight junction decreased transepithelial electrical resistance. Less