Abstract
The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.
Highlights
Anion channelrhodopsins (ACRs) are natural light-gated anion channels first discovered in the cryptophyte alga Guillardia theta (GtACR1 and GtACR2) (Govorunova et al, 2015)
Of the 35 ACR homologs found in cryptophyte algae (Govorunova et al, 2018; Govorunova et al, 2016; Wietek et al, 2016), GtACR1 is the best characterized in terms of its gating mechanism and photochemical reaction cycle (Sineshchekov et al, 2015; Sineshchekov et al, 2016), and is the only ACR for which light-gated anion conductance has been proven to be maintained in vitro in a purified state (Li et al, 2016) further recommending it as the preferred ACR for crystallization
The GtACR1 protein was expressed in insect (Sf9) cells and purified as a disulfide-crosslinked homodimer (Figure 1—figure supplement 1)
Summary
Anion channelrhodopsins (ACRs) are natural light-gated anion channels first discovered in the cryptophyte alga Guillardia theta (GtACR1 and GtACR2) (Govorunova et al, 2015). Their large Cl- conductance makes GtACRs and other ACRs later found in various cryptophyte species (Govorunova et al, 2018; Govorunova et al, 2017b) the most potent neuron-silencing optogenetic tools available. We report the atomic structure of the dark (closed) state of GtACR1, which is essential for elucidating the mechanism of its unique natural function of light-gated anion conductance through biological membranes. Understanding ACR mechanisms at the atomic scale would enable rational engineering to tailor their use as optogenetic tools
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