Structural basis for ion selectivity in potassium-selective channelrhodopsins
Seiya Tajima, Yoon Seok Kim, Masahiro Fukuda, YoungJu Jo, Peter Y. Wang, Joseph M. Paggi, Masatoshi Inoue, Eamon F.X. Byrne, Koichiro Kishi, Seiwa Nakamura, Charu Ramakrishnan, Shunki Takaramoto, Takashi Nagata, Masae Konno, Masahiro Sugiura, Kota Katayama, Toshiki E. Matsui, Keitaro Yamashita, Suhyang Kim, Hisako Ikeda, Jaeah Kim, Hideki Kandori, Ron O. Dror, Keiichi Inoue, Karl Deisseroth, Hideaki Kato
Abstract
KCR channelrhodopsins (K + -selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K + selectivity is achieved. Here, we present 2.5–2.7 Å cryo-electron microscopy structures of Hc KCR1 and Hc KCR2 and of a structure-guided mutant with enhanced K + selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K + selectivity; rather than forming the symmetrical filter of canonical K + channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for Hc KCR1/ Hc KCR2 spectral differences), and design of corresponding KCR variants with increased K + selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo . Thus, discovery of a mechanism for ion-channel K + selectivity also provides a framework for next-generation optogenetics.