title: Transmembrane domain three contributes to the ion conductance pathway of channelrhodopsin-2.
abstract: Channelrhodopsin-2 (ChR2) is a light-activated nonselective cation channel that is found in the eyespot of the unicellular green alga Chlamydomonas reinhardtii. Despite the wide employment of this protein to control the membrane potential of excitable membranes, the molecular determinants that define the unique ion conductance properties of this protein are not well understood. To elucidate the cation permeability pathway of ion conductance, we performed cysteine scanning mutagenesis of transmembrane domain three followed by labeling with methanethiosulfonate derivatives. An analysis of our experimental results as modeled onto the crystal structure of the C1C2 chimera demonstrate that the ion permeation pathway includes residues on one face of transmembrane domain three at the extracellular side of the channel that face the center of ChR2. Furthermore, we examined the role of a residue at the extracellular side of transmembrane domain three in ion conductance. We show that ion conductance is mediated, in part, by hydrogen bonding at the extracellular side of transmembrane domain three. These results provide a starting point for examining the cation permeability pathway for ChR2.
authors.affiliation: Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, MA, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Gaiko
authors.forename: Robert E
authors.initials: RE
authors.lastname: Dempski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D007477:Ions / Q000378:metabolism; D008698:Mesylates / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010539:Permeability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D007477:Ions; D008698:Mesylates; C093171:methanethiosulfonate ethylammonium; C109153:(2-sulfonatoethyl)methanethiosulfonate; D012243:Rhodopsin; D005020:Ethyl Methanesulfonate