Pore-Opening and Ion-Conduction Mechanism in Channelrhodopsins C1C2, ChR2, and iChloC by Computational Electrophysiology and Constant-pH Simulations

Channelrhodopsins (ChRs) are photoreceptors that function as light-gated ion channels. Over the last two decades, they have become essential tools in optogenetics, enabling precise manipulation of neurons, neural circuits, and animal behavior through light. Although structural studies have provided important mechanistic insights into channelrhodopsins, a detailed understanding of their ion conduction mechanism and selectivity was proven to be challenging due to difficulties in experimentally resolving open-state structures. Here, we employed molecular dynamics (MD)-based computational electrophysiology and constant-pH simulations to obtain the fully open states of three different ChRs. A significant number of spontaneous K + , Na + , and Ca 2+ permeation events were observed in the cation-conducting C1C2 and ChR2, as well as Cl – permeations in the anion-conducting iChloC. Analyses of the ion binding sites and hydration profiles provided key insights into ion selectivity. Our study presents a robust computational framework for establishing the fully open-state structures of channelrhodopsins, laying the groundwork for the rational engineering of ion selectivity and conductivity.