Department of Biological Sciences, Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science & Technology, Wuhan 430074, Hubei Province, China.
J Membr Biol. 2012 Mar;245(3):131-40. doi: 10.1007/s00232-012-9421-y. Epub 2012 Mar 2.
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.
HCO₃⁻ 通过电中性 Na/HCO₃⁻ 共转运蛋白 NBCe1 跨质膜转运在细胞内 pH 调节和跨上皮 HCO₃⁻ 转运中发挥重要作用。然而，转运体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，假定的第四细胞外环（EL4）在决定 NBCe1 的电中性方面起着至关重要的作用。在本研究中，我们生成了人 NBCe1-A 和 NBCn1-A 的八个新嵌合体。所有这些嵌合体都具有假定的 NBCe1 EL4，并且是电中性的。嵌合体 O，其中假定的第六跨膜片段（TM6）和 EL5 以及 NBCe1 的 C 末端（Ct）被相应的 NBCn1 序列取代，当 CO₂/HCO₃⁻ 被添加到细胞外溶液中时，产生的超极化最小（1-2 mV）。生物素化实验表明，O 在质膜上的丰度非常低。然而，当我们同时将假定的 TM6 和 EL5-Ct 区域的较小亚结构替换为 NBCn1 序列时，除了嵌合体 T 之外，所有嵌合体都具有很强的电中性，在嵌合体 T 中，我们用 NBCn1 的相应区域替换了 NBCe1 的 TM6 和 TM12。与野生型 NBCe1-A 相比，T 表现出转运体表面表达大大降低，而保留了至少一些电中性特征。我们假设假定的 TM6 和 TM12 是一个功能单元的一部分，如果这两个 TM 被同一转运体类型的 TM 取代，那么高表面表达需要周围的 TM 也来自同一转运体类型。
