Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behavior studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. We used molecular dynamics simulations along with spectroscopic and electrophysiological analyses to provide unexpected insights into substrate recognition, channel gating, and ion selectivity of both dACRs and nACRs. We found that many sets of residues that appear to play a similar role in dACRs and nACRs based on crystal structures displayed vastly different dynamics and, accordingly, mutagenesis of these regions gave distinct results. These results enabled us to create—the first optogenetics tool able to reliably block single action potentials—an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

Elucidating molecular mechanisms of anion channelrhodopsins to design new optogenetics tools

2019 Fall National Meeting

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