College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Int J Mol Sci. 2023 Sep 22;24(19):14414. doi: 10.3390/ijms241914414.
Engineering light-controlled K pumps from Na-pumping rhodopsins (NaR) greatly expands the scope of optogenetic applications. However, the limited knowledge regarding the kinetic and selective mechanism of K uptake has significantly impeded the modification and design of light-controlled K pumps, as well as their practical applications in various fields, including neuroscience. In this study, we presented K-dependent photocycle kinetics and photocurrent of a light-driven Na pump called rhodopsin 2 (NdR2). As the concentration of K increased, we observed the accelerated decay of M intermediate in the wild type (WT) through flash photolysis. In 100 mM KCl, the lifetime of the M decay was approximately 1.0 s, which shortened to around 0.6 s in 1 M KCl. Additionally, the K-dependent M decay kinetics were also observed in the G263W/N61P mutant, which transports K. In 100 mM KCl, the lifetime of the M decay was approximately 2.5 s, which shortened to around 0.2 s in 1 M KCl. According to the competitive model, in high KCl, K may be taken up from the cytoplasmic surface, competing with Na or H during M decay. This was further confirmed by the K-dependent photocurrent of WT liposome. As the concentration of K increased to 500 mM, the amplitude of peak current significantly dropped to approximately ~60%. Titration experiments revealed that the ratio of the rate constant of H uptake (k) to that of K uptake (k) is >10. Compared to the WT, the G263W/N61P mutant exhibited a decrease of approximately 40-fold in k/k. Previous studies focused on transforming NaR into K pumps have primarily targeted the intracellular ion uptake region of rhodopsin 2 (KR2) to enhance K uptake. However, our results demonstrate that the naturally occurring WT NdR2 is capable of intracellular K uptake without requiring structural modifications on the intracellular region. This discovery provides diverse options for future K pump designs. Furthermore, we propose a novel photocurrent-based approach to evaluate K uptake, which can serve as a reference for similar studies on other ion pumps. In conclusion, our research not only provides new insights into the mechanism of K uptake but also offers a valuable point of reference for the development of optogenetic tools and other applications in this field.
从 Na 泵浦视紫红质（NaR）工程光控 K 泵极大地扩展了光遗传学应用的范围。然而，对于 K 摄取的动力学和选择性机制的有限认识，极大地阻碍了光控 K 泵的修饰和设计，以及它们在神经科学等各个领域的实际应用。在这项研究中，我们介绍了一种称为视紫红质 2（NdR2）的光驱动 Na 泵的依赖 K 的光循环动力学和光电流。随着 K 浓度的增加，我们通过闪光光解观察到野生型（WT）中 M 中间体的加速衰减。在 100mM KCl 中，M 衰减的寿命约为 1.0s，在 1MKCl 中缩短至约 0.6s。此外，在 G263W/N61P 突变体中也观察到依赖 K 的 M 衰减动力学，该突变体转运 K。在 100mM KCl 中，M 衰减的寿命约为 2.5s，在 1MKCl 中缩短至约 0.2s。根据竞争模型，在高 KCl 中，K 可能从细胞质表面摄取，与 M 衰减过程中的 Na 或 H 竞争。这进一步通过 WT 脂质体的依赖 K 的光电流得到证实。随着 K 浓度增加到 500mM，峰电流的幅度显著下降至约~60%。滴定实验表明，H 摄取速率常数（k）与 K 摄取速率常数（k）的比值>10。与 WT 相比，G263W/N61P 突变体的 k/k 降低了约 40 倍。以前的研究主要集中在将 NaR 转化为 K 泵上，针对的是 2（KR2）的细胞内离子摄取区域，以增强 K 摄取。然而，我们的结果表明，天然存在的 WT NdR2 能够在不要求对细胞内区域进行结构修饰的情况下进行细胞内 K 摄取。这一发现为未来的 K 泵设计提供了多种选择。此外，我们提出了一种新的基于光电流的评估 K 摄取的方法，可为其他离子泵的类似研究提供参考。总之，我们的研究不仅为 K 摄取机制提供了新的见解，也为光遗传学工具的发展和该领域的其他应用提供了有价值的参考。
