Parvin Most Nahid, Gerelsaikhan Tudevdagva, Turner R James
Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, DHHS, Bethesda, Maryland 20892-1190, USA.
Biochemistry. 2007 Aug 21;46(33):9630-7. doi: 10.1021/bi700881a. Epub 2007 Jul 27.
The "secretory" Na+-K+-2Cl- cotransporter, NKCC1, is a member of a small gene family of electroneutral cation-chloride cotransporters (CCCs) with 9 homologues in vertebrates. A number of these transporters, including NKCC1 itself, have been shown to exist as homodimers in the membrane, suggesting that this may be a common feature of the CCCs. Here we employ chemical cross-linking studies, a novel co-immunoprecipition assay, and NKCC1/CCC chimeras to further explore the basis and significance of NKCC1 dimerization. An N-terminally truncated NKCC1 (nttNKCC1), in which the first 20 kDa of the 28 kDa cytosolic N-terminus are deleted, forms homodimers as well as heterodimers with full-length NKCC1, indicating that this region of N-terminus is not required for dimerization. On the other hand, replacing the 50 kDa NKCC1 C-terminus with that of several other non-NKCC1 homologues results in chimeric proteins that form homodimers but show little or no heterodimerization with NKCC1, demonstrating that the C-terminus of NKCC1 plays an essential role in dimerization and that NKCC1 dimerization exhibits definite homologue-specificity. Using additional chimeras we find that the residues required for dimer formation lie between amino acids 751 and 998 of (rat) NKCC1. We also show that dramatically overexpressing the nonfunctional truncated protein nttNKCC1 relative to the endogenous NKCC1 in the HEK293 cells results in a modest inhibition of fluxes via the endogenous transporter and a change in its sensitivity to the specific inhibitor bumetanide. These latter results indicate that there is a functional interaction between dimer subunits but that nonfunctional subunits do not necessarily have a dominant negative effect as has been previously proposed.
“分泌型”钠 - 钾 - 2氯协同转运蛋白NKCC1是电中性阳离子 - 氯离子协同转运蛋白（CCC）小基因家族的成员，在脊椎动物中有9个同源物。已证明其中一些转运蛋白，包括NKCC1自身，在膜中以同二聚体形式存在，这表明这可能是CCC的共同特征。在这里，我们采用化学交联研究、一种新型的共免疫沉淀测定法以及NKCC1/CCC嵌合体，以进一步探索NKCC1二聚化的基础和意义。一种N端截短的NKCC1（nttNKCC1），其28 kDa胞质N端的前20 kDa被删除，能与全长NKCC1形成同二聚体和异二聚体，这表明N端的该区域对于二聚化不是必需的。另一方面，用其他几种非NKCC1同源物的50 kDa C端替换NKCC1的C端，会产生形成同二聚体但与NKCC1几乎没有或没有异二聚化的嵌合蛋白，这表明NKCC1的C端在二聚化中起关键作用，并且NKCC1二聚化表现出明确的同源物特异性。使用额外的嵌合体，我们发现二聚体形成所需的残基位于（大鼠）NKCC1的751至998位氨基酸之间。我们还表明，相对于HEK293细胞中的内源性NKCC1，显著过表达无功能的截短蛋白nttNKCC1会导致通过内源性转运蛋白的通量适度受到抑制，并改变其对特异性抑制剂布美他尼的敏感性。后一个结果表明二聚体亚基之间存在功能相互作用，但无功能亚基不一定像先前提出的那样具有显性负效应。
