Exploring Protonation State, Ion Binding, and Photoactivated Channel Opening of an Anion Channelrhodopsin by Molecular Simulations

Channelrhodopsins are light-gated ion channels with a retinal chromophore found in microbes and are widely used in optogenetics, a field of neuroscience that utilizes light to regulate neuronal activity. <i>Gt</i>ACR1, an anion conducting channelrhodopsin derived from <i>Guillardia theta</i>, has attracted attention for its application as a neuronal silencer in optogenetics because of its high conductivity and selectivity. However, atomistic mechanisms of channel photoactivation and ion conduction have not yet been elucidated. In the present study, we investigated the molecular characteristics of <i>Gt</i>ACR1 and its photoactivation processes by molecular simulations. The QM/MM RWFE-SCF method which combines highly accurate quantum chemistry calculations with long-time molecular dynamics (MD) simulations were used to model protein structures of the wild-type and mutants with different protonation states of key groups and to calculate absorption energies for verification of the models. The QM/MM modeling together with MD simulations of free-energy calculations favors protonation of a key counterion carboxyl group of Asp234 with a strong binding of a chloride ion in the extracellular pocket in the dark state. A channel open state was also successfully modeled by the QM/MM RWFE-SCF free-energy optimizations, providing atomistic insights into the channel activation mechanism.