Experimental Biophysics, Institute for Biology, Humboldt-Universität zu Berlin, Invalidenstr. 42, 10115, Berlin, Germany.
Sci Rep. 2018 Jun 18;8(1):9316. doi: 10.1038/s41598-018-27690-w.
A new microbial rhodopsin class that actively transports sodium out of the cell upon illumination was described in 2013. However, poor membrane targeting of the first-identified sodium pump KR2 in mammalian cells has hindered the direct electrical investigation of its transport mechanism and optogenetic application to date. Accordingly, we designed enhanced KR2 (eKR2), which exhibits improved membrane targeting and higher photocurrents in mammalian cells to facilitate molecular characterization and future optogenetic applications. Our selectivity measurements revealed that stationary photocurrents are primarily carried by sodium, whereas protons only play a minor role, if any. Combining laser-induced photocurrent and absorption measurements, we found that spectral changes were not necessarily related to changes in transport activity. Finally, we showed that eKR2 can be expressed in cultured hippocampal mouse neurons and induce reversible inhibition of action potential firing with millisecond precision upon illumination with moderate green-light. Hence, the light-driven sodium pump eKR2 is a reliable inhibitory optogenetic tool applicable to situations in which the proton and chloride gradients should not be altered.
2013 年，人们描述了一个新的微生物视紫红质家族，该家族在光照时能主动将钠离子从细胞内运出。然而，在哺乳动物细胞中，第一个被鉴定的钠离子泵 KR2 的膜靶向性较差，这阻碍了对其运输机制的直接电研究和光遗传学应用。因此，我们设计了增强型 KR2（eKR2），它在哺乳动物细胞中表现出改善的膜靶向性和更高的光电流，以促进分子表征和未来的光遗传学应用。我们的选择性测量表明，固定光电流主要由钠离子携带，而质子即使有作用，也只起次要作用。结合激光诱导光电流和吸收测量，我们发现光谱变化不一定与运输活性的变化有关。最后，我们表明 eKR2 可以在培养的海马体小鼠神经元中表达，并在适度绿光照射下以毫秒级精度诱导动作电位发射的可逆抑制。因此，光驱动的钠离子泵 eKR2 是一种可靠的抑制性光遗传学工具，适用于不应改变质子和氯离子梯度的情况。
