Abstract
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.
Highlights
Microbial rhodopsins are photosensitive retinylidene proteins that are utilized by fungi, algae, and prokaryotes to sense and adapt to light or to harness light energy[1,2]
In-vitro measurements of KR2 reconstituted into black lipid membranes (BLMs) support the assumption of an active, light-induced Na+ transport by KR2 and show that transport activity prevails when Na+ is removed, which could be explained by alternative H+ pumping[9,16]
We demonstrated that the enhanced expression of enhanced KR2 (eKR2) is preserved in cultured hippocampal mouse neurons and enabled the inhibition of action potential (AP) firing upon moderate green-light illumination, rendering eKR2 a potent optogenetic tool for efficiently silencing neurons
Summary
Microbial rhodopsins are photosensitive retinylidene proteins that are utilized by fungi, algae, and prokaryotes to sense and adapt to light or to harness light energy[1,2]. KR2 pumps Na+ out of the cell, as determined from pH measurements in suspensions of KR2-expressing E. coli cells[17]. Based on this assay, it was suggested that KR2 actively transports H+ in the absence of extracellular Na+ 9. Voltage-clamp experiments in mammalian cells aimed at investigating light-induced ion transport under defined intra- and extracellular ionic conditions and under the control of the membrane voltage are still lacking. We correlated spectroscopic flash-photolysis data recorded at different [Na+] and pH values with laser-induced single turnover measurements of eKR2 photocurrents generated in mammalian cells under similar buffer conditions. In contrast to other inhibitory tools, Na+-based silencing with eKR2 would neither influence the distribution of H+ or Cl− nor rely on the anion gradient
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