Role of PDZK1 in membrane expression of renal brush border ion exchangers.
Role of PDZK1 in membrane expression of renal brush border ion exchangers.
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PDZK1 在肾刷状缘离子交换器膜表达中的作用。
DOI：
10.1073/pnas.0506578102
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发表时间：
2005
影响因子：
11.1
通讯作者：
Aronson,PeterS
中科院分区：
文献类型：
--
作者：
Thomson,RBrent;Wang,Tong;Thomson,BenjaminR;Tarrats,Luis;Girardi,Adriana;Mentone,SueAnn;Soleimani,Manoocher;Kocher,Olivier;Aronson,PeterS
钠-氢交换体NHE 3和氯-阴离子交换体CFEX（SLC 26 A6，PAT 1）在哺乳动物肾脏近端小管重吸收钠和氯中起主要作用。NHE 3和CFEX定位于近端小管刷状缘并维持在其中的机制在很大程度上是未知的。为了研究NHE 3和CFEX与含PDZ结构域的支架蛋白PDZK 1的可能相互作用，我们用GST融合蛋白和天然刷状缘膜蛋白进行了一系列体外相互作用测定。这些研究表明，NHE 3和CFEX不仅能够直接与PDZK 1相互作用，而且这种相互作用是通过其C-末端PDZ相互作用位点介导的。为了确定PDZK 1相互作用是否是NHE 3和CFEX在体内刷状缘定位所必需的，我们通过Western分析和免疫细胞化学检测了野生型和PDZK 1缺失突变小鼠肾脏中NHE 3和CFEX的表达。这些研究表明，尽管NHE 3的刷状缘表达不受PDZK 1缺失的影响，但CFEX的表达显著降低。最后，我们测定了CFEX功能活性，如刷状缘膜囊泡中的Cl-草酸盐交换和微灌注近端小管中的草酸盐刺激的体积吸收。与观察到的CFEX蛋白表达降低一致，CFEX功能活性的两种测量在PDZK 1缺失动物中均显著降低。总之，支架蛋白PDZK 1是必不可少的正常表达和功能的Cl-阴离子交换CFEX在近端小管的哺乳动物肾脏。
Na-H exchanger NHE3 and Cl-anion exchanger CFEX (SLC26A6, PAT1) play principal roles in the reabsorption of Na and Cl in the proximal tubule of the mammalian kidney. The mechanisms by which NHE3 and CFEX are localized to and maintained in the brush border of the proximal tubule are largely unknown. To investigate the possible interaction of NHE3 and CFEX with the PDZ-domain-containing scaffolding protein PDZK1, we performed a series ofin vitrointeraction assays with GST-fusion proteins and native brush border membrane proteins. These studies demonstrated that, not only were NHE3 and CFEX capable of directly interacting with PDZK1, but that this interaction was mediated through their C-terminal PDZ-interaction sites. To determine whether PDZK1 interaction is essential for brush border localization of NHE3 and CFEXin vivo, we examined the expression of NHE3 and CFEX in kidneys of wild-type and PDZK1-null mutant mice by both Western analysis and immunocytochemistry. These studies indicated that, although brush border expression of NHE3 was unaffected by the loss of PDZK1, the expression of CFEX was markedly reduced. Finally, we assayed CFEX functional activity as Cl–oxalate exchange in brush border membrane vesicles and oxalate-stimulated volume absorption in microperfused proximal tubules. Consistent with the observed decrease in CFEX protein expression, both measures of CFEX functional activity were dramatically reduced in PDZK1-null animals. In conclusion, the scaffolding protein PDZK1 is essential for the normal expression and function of Cl-anion exchanger CFEX in the proximal tubule of the mammalian kidney.