Van Itallie Christina M, Rogan Sarah, Yu Alan, Vidal Lucia Seminario, Holmes Jennifer, Anderson James M
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA.
Am J Physiol Renal Physiol. 2006 Dec;291(6):F1288-99. doi: 10.1152/ajprenal.00138.2006. Epub 2006 Jun 27.
Members of the large claudin family of tight junction (TJ) proteins create the differences in paracellular conductance and charge selectivity observed among different epithelia. Previous studies demonstrated that ionic charge selectivity is influenced by acidic or basic amino acids on the first extracellular domain of claudins. We noted two alternatively spliced variants of claudin-10 in the database, 10a and 10b, which are predicted to encode two different first extracellular domains and asked whether this might be a novel mechanism to generate two different permselectivities from a single gene. Using quantitative PCR, we found that claudin-10b is widely expressed among tissues including the kidney; however, claudin-10a is unique to the kidney. Using a nondiscriminating antibody, we found that claudin-10 (a plus b) is expressed in most segments of the nephron. In situ hybridization, however, showed that mRNA for 10a is concentrated in the cortex, and mRNA for 10b is more highly expressed in the medulla. Expression in Madin-Darby canine kidney (MDCK) II and LLC-PK1 cells reveals that both variants form low-resistance pores, and that claudin-10b is more selective for cations than claudin-10a. Charge-reversing mutations of cationic residues on 10a reveal positions that contribute to its anion selectivity. We conclude that alternative splicing of claudin-10 generates unique permselectivities and might contribute to the variable paracellular transport observed along the nephron.
紧密连接（TJ）蛋白的大claudin家族成员造成了不同上皮细胞间观察到的细胞旁电导和电荷选择性差异。先前的研究表明，离子电荷选择性受claudin蛋白第一个细胞外结构域上酸性或碱性氨基酸的影响。我们在数据库中注意到claudin-10的两个选择性剪接变体，10a和10b，预计它们编码两个不同的第一个细胞外结构域，并询问这是否可能是从单个基因产生两种不同通透选择性的新机制。通过定量PCR，我们发现claudin-10b在包括肾脏在内的多种组织中广泛表达；然而，claudin-10a仅在肾脏中表达。使用非特异性抗体，我们发现claudin-10（a加b）在肾单位的大多数节段中表达。然而，原位杂交显示，10a的mRNA集中在皮质，10b的mRNA在髓质中表达更高。在Madin-Darby犬肾（MDCK）II细胞和LLC-PK1细胞中的表达表明，这两种变体均形成低电阻孔，并且claudin-10b对阳离子的选择性高于claudin-10a。10a上阳离子残基的电荷反转突变揭示了对其阴离子选择性有贡献的位置。我们得出结论，claudin-10的选择性剪接产生独特的通透选择性，并可能导致沿肾单位观察到的可变细胞旁转运。
