WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells

<jats:p> The electric excitability of muscle, heart, and brain tissue relies on the precise interplay of Na <jats:sup>+</jats:sup> - and K <jats:sup>+</jats:sup> -selective ion channels. The involved ion fluxes are controlled in optogenetic studies using light-gated channelrhodopsins (ChRs). While non-selective cation-conducting ChRs are well established for excitation, K <jats:sup>+</jats:sup> -selective ChRs (KCRs) for efficient inhibition have only recently come into reach. Here, we report the molecular analysis of recently discovered KCRs from the stramenopile <jats:italic>Hyphochytrium catenoides</jats:italic> and identification of a novel type of hydrophobic K <jats:sup>+</jats:sup> selectivity filter. Next, we demonstrate that the KCR signature motif is conserved in related stramenopile ChRs. Among them, WiChR from <jats:italic>Wobblia lunata</jats:italic> features a so far unmatched preference for K <jats:sup>+</jats:sup> over Na <jats:sup>+</jats:sup> , stable photocurrents under continuous illumination, and a prolonged open-state lifetime. Showing high expression levels in cardiac myocytes and neurons, WiChR allows single- and two-photon inhibition at low irradiance and reduced tissue heating. Therefore, we recommend WiChR as the long-awaited efficient and versatile optogenetic inhibitor. </jats:p>