title: Bioinformatic and mutational analysis of channelrhodopsin-2 protein cation-conducting pathway.
abstract: 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. Although several ChR2 variants with modified properties have been generated, the structural determinants of the protein function are largely unresolved. We used bioinformatic modeling of the 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, Gln-56 is important for ion conductance, whereas Ser-63, Thr-250, and Asn-258 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.
authors.affiliation: Department of Biomedical Sciences, University of Padova, viale Colombo 3, 35100 Padova, Italy.
authors.forename: Anna Pia
authors.initials: AP
authors.lastname: Plazzo
authors.forename: Nicola
authors.initials: N
authors.lastname: De Franceschi
authors.forename: Francesca
authors.initials: F
authors.lastname: Da Broi
authors.forename: Francesco
authors.initials: F
authors.lastname: Zonta
authors.forename: Maria Federica
authors.initials: MF
authors.lastname: Sanasi
authors.forename: Francesco
authors.initials: F
authors.lastname: Filippini
authors.forename: Marco
authors.initials: M
authors.lastname: Mongillo
mesh_terms: D000075402:Channelrhodopsins; D019295:Computational Biology / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008958:Models, Molecular; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D000075402:Channelrhodopsins; D009419:Nerve Tissue Proteins