Structural Foundations of Potassium Selectivity in Channelrhodopsins

Potassium-selective 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 paralogs, are necessary, although not sufficient, for K(+) selectivity. Our results provide the foundation for further engineering of KCRs for optogenetic needs.