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

当用冷冻断裂电镜观察上皮细胞时，在原生质(P)面和外质(E)面分别观察到由膜内颗粒组成的连续紧密连接链和没有颗粒的互补凹槽（P型紧密连接）。外周小静脉内皮细胞紧密连接颗粒位于e面凹槽上（e型紧密连接）。claudin是紧密连接的完整膜蛋白，由至少24个成员组成，在无occludin的情况下重建成纤维细胞的紧密连接链。claudin由四个跨膜结构域（nh2和cooh末端均位于细胞质中）、两个细胞外环（ECL1和ECL2）和一个细胞质环组成。我们认为紧密连接的结构和功能可能是由紧密连接中表达的claudin物种的组合和混合比例决定的。我们构建了含有claudin-1的表达载体，claudin-1在cooh末端有一个myc表位（1CLmyc）， 1CLmyc在ECL2中删除了两个氨基酸（151P152L） （1clplmyc）， claudin-10在cooh末端有一个myc表位（10CLmyc）， claudin-10本身。然后我们在MDCK I或II细胞中表达它们。冷冻断裂电镜观察表达1clmyc的细胞，在侧质膜上检测到p型紧密连接。clmyc在侧膜形成e型紧密连接。然而，1CLAPLmyc形成了中间型紧密连接，其膜内颗粒与P面和e面相等。表达1clmyc和1clplmyc的细胞上皮电阻值显著升高，而表达claudin-10的细胞上皮电阻值显著降低。这些结果表明，cludin -1的ECL2参与决定紧密连接颗粒是否与P面或e面结合，并且P型和中间型紧密连接增加了穿过上皮的电阻，而e型紧密连接降低了穿过上皮的电阻。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