Caron L, Rousseau F, Gagnon E, Isenring P
Groupe de Recherche en Néphrologie, Department of Medicine, and the Unité de Recherche en Génétique Humaine et Moléculaire, Laval University, Québec G1R 2J6, Canada.
J Biol Chem. 2000 Oct 13;275(41):32027-36. doi: 10.1074/jbc.M000108200.
To date, the cation-Cl(-) cotransporter (CCC) family comprises two branches of homologous membrane proteins. One branch includes the Na(+)-K(+)-Cl(-) cotransporters (NKCCs) and the Na(+)-Cl(-) cotransporter, and the other branch includes the K(+)-Cl(-) cotransporters. Here, we have isolated the first member of a third CCC family branch. This member shares approximately 25% identity in amino acid sequence with each of the other known mammalian CCCs. The corresponding cDNA, obtained from a human heart library and initially termed WO(3.3), encodes a 914-residue polypeptide of 96.2 kDa (calculated mass). Sequence analyses predict a 12-transmembrane domain (tm) region, two N-linked glycosylation sites between tm(5) and tm(6), and a large intracellular carboxyl terminus containing protein kinase C phosphorylation sites. Northern blot analysis uncovers an approximately 3.7-kilobase pair transcript present in muscle, placenta, brain, and kidney. With regard to function, WO(3. 3) expressed either in HEK-293 cells or Xenopus laevis oocytes does not increase Rb(+)-, Na(+)-, and Cl(-)-coupled transport during 5- or 6-h fluxes, respectively. In the oocyte, however, WO(3.3) specifically inhibits human NKCC1-mediated (86)Rb(+) flux. In addition, coimmunoprecipitation studies using lysates from WO(3. 3)-transfected HEK-293 cells suggest a direct interaction of WO(3.3) with endogenous NKCC. Thus, we have cloned and characterized the first putative heterologous CCC-interacting protein (CIP) known at present. CIP1 may be part of a novel family of proteins that modifies the activity or kinetics of CCCs through heterodimer formation.
迄今为止，阳离子 - 氯（Cl⁻）共转运蛋白（CCC）家族由同源膜蛋白的两个分支组成。一个分支包括钠 - 钾 - 氯（Na⁺-K⁺-Cl⁻）共转运蛋白（NKCCs）和钠 - 氯（Na⁺-Cl⁻）共转运蛋白，另一个分支包括钾 - 氯（K⁺-Cl⁻）共转运蛋白。在此，我们分离出了CCC家族第三个分支的首个成员。该成员与其他已知的哺乳动物CCC在氨基酸序列上具有约25%的同一性。从人心脏文库获得并最初命名为WO(3.3)的相应cDNA，编码一个914个残基、96.2 kDa（计算质量）的多肽。序列分析预测有一个12跨膜结构域（tm）区域，在tm(5)和tm(6)之间有两个N - 连接糖基化位点，以及一个含有蛋白激酶C磷酸化位点的大的细胞内羧基末端。Northern印迹分析揭示在肌肉、胎盘、脑和肾中存在一个约3.7千碱基对的转录本。关于功能，在HEK - 293细胞或非洲爪蟾卵母细胞中表达的WO(3.3)在5小时或6小时通量期间分别不会增加铷（Rb⁺）、钠（Na⁺）和氯（Cl⁻）偶联的转运。然而，在卵母细胞中，WO(3.3)特异性抑制人NKCC1介导的⁸⁶Rb⁺通量。此外，使用WO(3.3)转染的HEK - 293细胞裂解物进行的免疫共沉淀研究表明WO(3.3)与内源性NKCC存在直接相互作用。因此，我们已经克隆并鉴定了目前已知的首个假定的异源CCC相互作用蛋白（CIP）。CIP1可能是通过异二聚体形成修饰CCC活性或动力学的新型蛋白质家族的一部分。
