The femtosecond-to-second photochemistry of red-shifted fast-closing anion channelrhodopsin PsACR1

Abstract

Anion channelrhodopsins (ACRs) are of great interest due to their ability to inhibit electrical signaling in optogenetic experiments. The photochemistry of ACRs is currently poorly understood and an improved understanding would be beneficial for rational design of ACRs with modified properties. Activation/deactivation of ACRs involves a series of photoreactions ranging from femtoseconds to seconds, thus real-time observation is essential to comprehend the full complexity of the photochemical processes. Here we investigate the photocycle of an ACR from Proteomonas sulcata (PsACR1), which is valuable for optogenetic applications due to the red-shifted absorption and action spectra compared to the prototype ACRs from Guillardia theta: GtACR1 and GtACR2, and the fast channel closing properties. From femto-to-submillisecond transient absorption spectroscopy, flash photolysis, and point mutations of acidic residues near the retinal Schiff base (RSB), E64, and D230, we found that the photoisomerization occurs in ∼500 fs independent of the protonation state of E64. Notably, E64 is involved in the rearrangement of the hydrogen-bond network near the RSB after photoisomerization. Furthermore, we suggest that E64 works as a primary proton acceptor during deprotonation of the RSB as has been proposed for GtACR1. Our findings allow for a deeper understanding of the photochemistry on the activation/deactivation of ACRs.

Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl− concentrations

“…During this transition, the absorption at 610 nm transiently increased together with the decrease in the absorption at 500 nm. In a previous study, these transient absorption changes corresponded to the re-establishment of the K/L equilibrium in favor of K 12 . However, based on the conventional photocycle scheme, it is more straight-forward to assign the transient increase in 610 nm as the generation of a new photo-intermediate rather than the re-establishment of the K/L equilibrium.…”

“…Here we explain the photocycle overview in the presence of 100 mM Cl − as an example. Figure 2A illustrates the flash-induced light-minus-dark difference absorption spectra from 10 μs to 1.4 s. After the flash excitation, the absorption for the initial state at 540 nm disappeared together with the concomitant appearance of three photo-intermediates with absorptions at 610 nm, 450 nm and 400 nm, tentatively assigned as K-, P 450 - and M-intermediates (abbreviated as K, P 450 and M), respectively 6 , 12 . Over time, these photo-intermediates increased and then decreased together with the recovery of the initial state and therefore the photocycle was completed.…”

“…P 0 represents the pure retinal spectrum of the initial state Psu ACR1. From the spectra, we assigned two additional photo-intermediates with absorption peaks at 500 nm and 540 nm as the L- and ACR’-intermediates (abbreviated as L and ACR’), respectively, by reference to previous reports 6 , 12 , 23 – 25 .…”

“…Several in vivo and in vitro investigations have revealed their optogenetic availability 3 , their channel gating mechanism during the photocycle 5 – 7 , the roles of positively charged residues for anion conductance 8 , and their structure and structural changes around the chromophore 9 , 10 . On the other hand, another homologous ACR from a marine cryptophyte alga Proteomonas sulcata (abbreviated as Psu ACR1 or Ps ACR1) has also been investigated regarding its electrophysiological 4 , 11 and spectroscopic properties 12 . Those studies have shown that Gt ACRs have the ability to work under weak light intensity and that Psu ACR1 has rapid channel closing kinetics, rapid dark recovery of the peak photocurrent, and the most red-shifted absorption wavelength among the known ACRs.…”

“…Previous investigations of the channel gating mechanism of Gt ACR1 and Psu ACR1 have revealed the relationships between the photo-intermediates in the photocycle and the open and closed states of the channel 4 – 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.…”