Electrical properties, substrate specificity and optogenetic potential of the engineered light-driven sodium pump eKR2

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.