Abstract 1074 Structural insights into Potassium-selectivity in Kalium Channelrhodopsins (KCRs)

Recently we discovered two channelrhodopsins (ChRs) in the protist Hyphochytrium catenoides that show greater permeability for K+ than Na+, for which we named them "kalium channelrhodopsins" (KCRs) [1]. HcKCR1 from H. catenoides, is in use for optogenetic silencing of mammalian neurons. The K+ selectivity was a mystery since the KCRs do not contain the canonical tetrameric K+ selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a cation channelrhodopsin paralog we named HcCCR, a paralog of HcKCR1 that selects Na+ over K+ [2]. In collaboration with Oliver P. Ernst's laboratory (Toronto, Canada), we expressed and purified HcKCR1 and HcCCR and used cryo-electron microscopy to determine their atomic structures embedded in peptidiscs to elucidate the structural basis of their different cation selectivity. Together with electrophysiology patch-clamping and structure-guided mutagenesis [2,3], we found that K+ versus Na+ selectivity is determined at two distinct sites on the putative ion conduction pathway: in a patch of critical residues in the intracellular segment (Leu69/Phe69, Ile73/Ser73 and Asp116) and within a cluster of aromatic residues in the extracellular segment (primarily, Trp102 and Tyr222). The two filters are on the opposite sides of the photoactive site involved in channel gating. A possible need for the precise location of Tyr222 is its potential dehydration of K+ by π-electron cloud interaction, and Asp116 is involved in partial dehydration of K+ entering from the cytoplasmic side, hence, the dehydration of K+ is one of the primary mechanisms for its selectivity. These findings provide more information to guide mutagenesis to provide novel optogenetics tools. REFERENCES [1] Govorunova EG, Gou Y, Sineshchekov OA, Li H, Lu X, Wang Y, Brown LS, St-Pierre F, Xue M, and Spudich JL. (2022) Kalium channelrhodopsins are natural light-gated potassium channels that mediate optogenetic inhibition. Nature Neurosci. 25(7):967-974. [2] Govorunova EG, Sineshchekov OA, Brown LS, Bondar A-N, and Spudich JL (2022) Structural Foundations of Potassium Selectivity in Channelrhodopsins. mBio.13(6):e0303922. [3] Morizumi T, Kim K, Li H, Govorunova EG, Sineshchekov OA, Wang Y, Zheng L, Bertalan É, Bondar AN, Askari A, Brown LS, Spudich JL, Ernst OP (2023) Structures of channelrhodopsin paralogs in peptidiscs explain their contrasting K+ and Na+ selectivities. Nature Commun. 14(1):4365. https://doi.org/10.1038/s41467-023-40041-2 This work was supported by the National Institutes of Health grants R35GM140838 and U01NS118288 (J.L.S.); Robert A. Welch Foundation Endowed Chair AU-0009 (J.L.S.)