Stabilized Ion Selectivity Corrects Activation Drift in Kalium Channelrhodopsins.

Effective optogenetic inhibition of neuronal activity requires tools that reliably silence neurons across cell types and conditions. K+-selective channelrhodopsins (KCRs) have emerged as attractive alternatives to chloride-conducting channels for optogenetic inhibition of cellular excitability, but many KCR variants exhibit an ion selectivity shift toward Na+ under prolonged illumination. Through behavioral and electrophysiological analyses in Drosophila, and Caenorhabditis elegans, it is found that both the absolute K+ to Na+ permeability ratio and its stability over time determine the inhibition to activation transition, which limits their utility for silencing neural circuits. Among tested variants, the KCR1-C29D mutant shows a relatively high and the most stable K+ to Na+ permeability ratio during illumination. While other KCR variants often evoke excitatory responses, KCR1-C29D consistently provides robust in vivo inhibition across cell types, illumination conditions, and species. This work addresses a key limitation of KCR optogenetics and establishes KCR1-C29D as a superior and reliable inhibitory tool. These findings highlight the stability of ion selectivity as a design criterion and provide guidance for the design of next generation optogenetic tools.