Abstract
Natural anion channelrhodopsins (ACRs) have recently received increased attention because of their effectiveness in optogenetic manipulation for neuronal silencing. In this study, we focused on Proteomonas sulcata ACR1 (PsuACR1), which has rapid channel closing kinetics and a rapid recovery to the initial state of its anion channel function that is useful for rapid optogenetic control. To reveal the anion concentration dependency of the channel function, we investigated the photochemical properties of PsuACR1 using spectroscopic techniques. Recombinant PsuACR1 exhibited a Cl− dependent spectral red-shift from 531 nm at 0.1 mM to 535 nm at 1000 mM, suggesting that it binds Cl− in the initial state with a Kd of 5.5 mM. Flash-photolysis experiments revealed that the photocycle was significantly changed at high Cl− concentrations, which led not only to suppression of the accumulation of the M-intermediate involved in the Cl− non-conducting state but also to a drastic change in the equilibrium state of the other photo-intermediates. Because of this, the Cl− conducting state is protracted by one order of magnitude, which implies an impairment of the rapid channel closing of PsuACR1 in the presence of high concentrations of Cl−.
Highlights
Microbial rhodopsins are photoreceptor proteins produced in diverse microbes, such as archaea, bacteria and eukaryotes
We investigated the Cl− dependent changes in the photochemical properties of Proteomonas sulcata ACR1 (PsuACR1) using static and time-resolved spectroscopic techniques that revealed that the photocycle, which is directly connected to the anion channel function, is strongly affected by Cl− concentration
We found that PsuACR1 is able to bind Cl− in the initial state at a Kd of 5.5 mM, which was estimated by the Cl− dependent spectral red-shift
Summary
Microbial rhodopsins are photoreceptor proteins produced in diverse microbes, such as archaea, bacteria and eukaryotes. Previous investigations of the channel gating mechanism of GtACR1 and PsuACR1 have revealed the relationships between the photo-intermediates in the photocycle and the open and closed states of the channel[4,5,6,11,12] In these cases, the anion transport starts together with the formation of the L-intermediate, which is observed in the early stage of the photocycle, whereas it stops together with the formation of the M-intermediate. PsuACR1 was expressed in and extracted from methylotrophic yeast Pichia pastoris cells as a recombinant protein in the presence of the detergent dodecyl-β-D-maltoside (DDM) These spectroscopic measurements revealed information about the Cl− binding ability in the initial state and the Cl− concentration-dependent changes of the photocycle that are directly connected to its anion channel function
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