Yan Xiaojie, Zhou Hao, Zhang Jinxiu, Shi Chaowei, Xie Xingqiao, Wu Yinan, Tian Changlin, Shen Yuequan, Long Jiafu
Tianjin Key Laboratory of Protein Science, College of Life Science, Nankai University, Tianjin 300071, China.
J Mol Biol. 2009 Oct 2;392(4):967-76. doi: 10.1016/j.jmb.2009.07.060. Epub 2009 Jul 25.
Inwardly rectifying potassium channel 2.3 (Kir2.3) is specifically targeted on the basolateral membranes of epithelial and neuronal cells, and it thus plays an important role in maintaining potassium homeostasis. Tax-interacting protein-1 (TIP-1), an atypical PDZ-domain-containing protein, binds to Kir2.3 with a high affinity, causing the intracellular accumulation of Kir2.3 in cultured epithelial cells. However, the molecular basis of the TIP-1/Kir2.3 interaction is still poorly understood. Here, we present the crystal structure of TIP-1 in complex with the C-terminal Kir2.3-peptide (residues 436-445) to reveal the molecular details of the interaction between them. Moreover, isothermal titration calorimetry experiments show that the C-terminal Kir2.3-peptide binds much more strongly to TIP-1 than to mammalian Lin-7, indicating that TIP-1 can compete with mammalian Lin-7 to uncouple Kir2.3 from its basolateral membrane anchoring complex. We further show that the phosphorylation/dephosphorylation of Ser443 within the C-terminal Kir2.3 PDZ-binding motif RRESAI dynamically regulates the Kir2.3/TIP-1 association in heterologous HEK293T cells. These data suggest that TIP-1 may act as an important regulator for the endocytic pathway of Kir2.3.
内向整流钾通道2.3（Kir2.3）特异性定位于上皮细胞和神经元细胞的基底外侧膜上，因此在维持钾离子稳态中发挥重要作用。Tax相互作用蛋白1（TIP-1）是一种含非典型PDZ结构域的蛋白，它以高亲和力与Kir2.3结合，导致Kir2.3在培养的上皮细胞内积累。然而，TIP-1/Kir2.3相互作用的分子基础仍知之甚少。在此，我们展示了TIP-1与Kir2.3 C末端肽（残基436-445）复合物的晶体结构，以揭示它们之间相互作用的分子细节。此外，等温滴定量热法实验表明，Kir2.3 C末端肽与TIP-1的结合比与哺乳动物Lin-7的结合更强，这表明TIP-1可以与哺乳动物Lin-7竞争，使Kir2.3与其基底外侧膜锚定复合物解偶联。我们进一步表明，Kir2.3 C末端PDZ结合基序RRESAI内Ser443的磷酸化/去磷酸化动态调节异源HEK293T细胞中Kir2.3/TIP-1的结合。这些数据表明，TIP-1可能是Kir2.3内吞途径的重要调节因子。
