Bioinformatic and mutational analysis of channelrhodopsin-2 cation conducting pathway.

Channelrhodopsin-2 (ChR2) is a light-gated cation channel widely used as a biotechnological tool to control membrane depolarization in various cell types and tissues. Even though several ChR2 variants with modified properties have been generated, the structural determinants of the protein function are largely unresolved. We used bioinformatic modeling of ChR2 structure to identify the putative cationic pathway within the channel which is formed by a system of inner cavities that are uniquely present in this microbial rhodopsin. Site-directed mutagenesis combined with patch clamp analysis in HeLa cells was used to determine key residues involved in ChR2 conductance and selectivity. Among them, Q56 is important for ion conductance, whereas S63, T250 and N258 are previously unrecognized residues involved in ion selectivity and photocurrent kinetics. This study widens the current structural information on ChR2 and can assist in the design of new improved variants for specific biological applications.