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bioRxiv · 10.1101/2022.09.26.509509

Structural foundations of potassium selectivity in channelrhodopsins

Govorunova, E. G.·Sineshchekov, O. A.·Brown, L.·Bondar, A.-N.·Spudich, J. L.
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Abstract

Kalium channelrhodopsins (KCRs) are light-gated K+ channels recently found in the stramenopile protist Hyphochytrium catenoides. When expressed in neurons, KCRs enable high-precision optical inhibition of spiking (optogenetic silencing). KCRs are capable of discriminating K+ from Na+ without the conventional K+-selectivity filter found in classical K+ channels. The genome of H. catenoides also encodes a third paralog that is more permeable for Na+ than for K+. To identify structural motifs responsible for the unusual K+ selectivity of KCRs, we systematically analyzed a series of chimeras and mutants of this protein. We found that mutations of three critical residues in the paralog convert its Na+ selective channel into a K+ selective one. Our characterization of homologous proteins from other protists (Colponema vietnamica, Cafeteria burkhardae and Chromera velia) and metagenomic samples confirmed the importance of these residues for K+ selectivity. We also show that Trp102 and Asp116, conserved in all three H. catenoides orthologs, are necessary, although not sufficient, for K+ selectivity. Our results provide the foundation for further engineering of KCRs for optogenetic needs. IMPORTANCERecently discovered microbial light-gated ion channels (channelrhodopsins) with a higher permeability for K+ than for Na+ (kalium channelrhodopsins, or KCRs) demonstrate an alternative K+ selectivity mechanism, unrelated to well-characterized "selectivity filters" of voltage- and ligand-gated K+ channels. KCRs can be used for optogenetic inhibition of neuronal firing, and potentially for the development of gene therapies to treat neurological and cardiovascular disorders. In this study we identify structural motifs that determine the K+ selectivity of KCRs that provide the foundation for that provide the foundation for elucidating their K+ selectivity mechanism and for their further engineering as optogenetic tools.

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Govorunova, E. G., Sineshchekov, O. A., Brown, L., Bondar, A.-N., Spudich, J. L.. 2022-09-27. Structural foundations of potassium selectivity in channelrhodopsins. https://doi.org/10.1101/2022.09.26.509509

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