Effect of Simultaneously Replacing Putative TM6 and TM12 of Human NBCe1-A with Those from NBCn1 on Surface Abundance in Xenopus Oocytes
Effect of Simultaneously Replacing Putative TM6 and TM12 of Human NBCe1-A with Those from NBCn1 on Surface Abundance in Xenopus Oocytes
复制标题
同时用 NBCn1 替换假定的人 NBCe1-A TM6 和 TM12 对非洲爪蟾卵母细胞表面丰度的影响
DOI：
10.1007/s00232-012-9421-y
复制
发表时间：
2012-03-01
影响因子：
2.4
通讯作者：
Boron, Walter F.
中科院分区：
文献类型：
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作者：
Chen, Li-Ming;Qin, Xue;Boron, Walter F.
通过产电Na/HCO 3-共转运蛋白NBCe 1的HCO 3-跨质膜转运在细胞内pH调节和跨上皮HCO 3-转运中起重要作用。然而，转运蛋白功能的结构决定因素在很大程度上仍然未知。先前的研究表明，推定的第四胞外环（EL 4）在确定NBCe 1的产电性中起着至关重要的作用。在本研究中，我们产生了8个新的嵌合体的人NBCe 1-A和NBCn 1-A。所有具有推定的NBCe 1 EL 4和产电。当细胞外液中加入CO2/HCO 3-时，嵌合体O产生的超极化最小（1-2 mV），其中NBCe 1的第六跨膜片段（TM 6）和C末端（Ct）的EL 5被相应的NBCn 1序列取代。生物素化实验表明，O在质膜上的丰度非常低。然而，嵌合体中，我们同时取代了推定的TM 6和较小的NBCn 1序列的EL 5-Ct区域的亚结构域是强烈的产电除了嵌合体T，其中我们取代了TM 6和TM 12的NBCn 1与相应的区域。与野生型NBCe 1-A相比，T表现出大大降低的转运蛋白表面表达，同时保留至少一些产电特征。我们假设假定TM 6和TM 12是功能单元的一部分，并且如果这两个TM被相同转运蛋白类型的TM所取代，则高表面表达将需要周围的TM也来自相同的转运蛋白类型。
HCO3- translocation across the plasma membrane via the electrogenic Na/HCO3- cotransporter NBCe1 plays an important role in intracellular pH regulation and transepithelial HCO3- transport. However, the structural determinants of transporter function remain largely unknown. A previous study showed that the putative fourth extracellular loop (EL4) plays an essential role in determining the electrogenicity of NBCe1. In the present study, we generated eight new chimeras of human NBCe1-A and NBCn1-A. All possess the putative NBCe1 EL4 and are electrogenic. Chimera O, in which the putative sixth transmembrane segment (TM6) and EL5 through the C terminus (Ct) of NBCe1 was replaced by corresponding NBCn1 sequence, produces the smallest hyperpolarization (1-2 mV) when CO2/HCO3- is added to the extracellular solution. Biotinylation experiments show that O has a very low abundance at the plasma membrane. However, chimeras in which we simultaneously replaced the putative TM6 and smaller subdomains of the EL5-Ct region for the NBCn1 sequence were strongly electrogenic except for chimera T, in which we replaced TM6 and TM12 of NBCe1 with the corresponding regions of NBCn1. T exhibited greatly reduced transporter surface expression compared to wild-type NBCe1-A, while retaining at least some electrogenic character. We hypothesize that putative TM6 and TM12 are part of a functional unit and that if the two TMs are replaced by those of the same transporter type, high surface expression would require that the surrounding TMs are also from the same transporter type.