最近发表的SLC22家族的人类有机阳离子转运蛋白的cryo-EM结构揭示了七个,顺序排列的谷氨酸和天冬氨酸残基,这可能与带正电荷的底物的相互作用有关。我们分析了通过创建D155N去除这些负电荷的功能后果,E232Q,D382N,E390Q,E451Q,E459Q,和OCT3的D478N突变体。E232Q,E459Q,和D478N导致在外细胞膜中缺乏定位并且没有相关的摄取活性。然而,D155N和E451Q显示出底物特异性的转运活性损失,尽管膜定位正确,但E390Q没有剩余活性。相比之下,D382N显示几乎野生型样摄取。D155位于底物结合袋的入口处,可以,因此参与引导阳离子底物朝向结合袋的内部。对于E390,我们确认了其对转运功能的关键功能,正如最近在OCT1中相应位置显示的那样。有趣的是,E451似乎位于运输机的向外开放确认中的装订袋的底部。E451Q变体的转运活性的底物特异性丧失表明在作为机会结合位点的一部分的特定物质的转运循环中的重要作用。总的来说,我们的研究强调了cryo-EM结构在指导诱变研究中的影响,以了解转运蛋白-配体相互作用的分子水平,它也证实了在多特异性OCTs的诱变研究中测试多种底物的重要性。
Recently published cryo-EM structures of human organic cation transporters of the SLC22 family revealed seven, sequentially arranged glutamic and aspartic acid residues, which may be relevant for interactions with positively charged substrates. We analyzed the functional consequences of removing those negative charges by creating D155N, E232Q, D382N, E390Q, E451Q, E459Q, and D478N mutants of OCT3. E232Q, E459Q, and D478N resulted in a lack of localization in the outer cell membrane and no relevant uptake activity. However, D155N and E451Q showed a substrate-specific loss of transport activity, whereas E390Q had no remaining activity despite correct membrane localization. In contrast, D382N showed almost wild-type-like uptake. D155 is located at the entrance to the substrate binding pocket and could, therefore be involved in guiding cationic substrates towards the inside of the binding pocket. For E390, we confirm its critical function for transporter function as it was recently shown for the corresponding position in OCT1. Interestingly, E451 seems to be located at the bottom of the binding pocket in the outward-open confirmation of the transporter. Substrate-specific loss of transport activity of the E451Q variant suggests an essential role in the transport cycle of specific substances as part of an opportunistic binding site. In general, our study highlights the impact of the cryo-EM structures in guiding mutagenesis studies to understand the molecular level of transporter-ligand interactions, and it also confirms the importance of testing multiple substrates in mutagenesis studies of polyspecific OCTs.