title: Photoinactivation of glutamate receptors by genetically encoded unnatural amino acids.
abstract: Ionotropic glutamate receptors (iGluRs) are ubiquitous in the mammalian brain, and the AMPA-subtype is essential for fast, glutamate-activated postsynaptic currents. We incorporated photoactive crosslinkers into AMPA receptors using genetically encoded unnatural amino acid mutagenesis in a mammalian cell line. Receptors rescued by incorporation of unnatural amino acids, including p-benzoyl-l-phenylalanine (BzF, also known as Bpa), had largely similar properties to wild-type channels and were expressed at similar levels. BzF incorporation at subunit interfaces afforded photocrosslinking of subunits, as assessed by biochemical experiments. In electrophysiological recordings, BzF incorporation allowed selective and potent UV-driven photoinactivation of both homomeric (GluA2) and heteromeric (GluA2:GluA1) AMPA receptors. State dependence of trapping at two sites in the lower lobe of the ligand binding domain is consistent with deformation of these domains as well as intersubunit rearrangements during AMPA receptor desensitization.
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie, Berlin 13125, Germany, and Cluster of Excellence NeuroCure, Charité, Berlin 10117, Germany.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Klippenstein
authors.forename: Valentina
authors.initials: V
authors.lastname: Ghisi
authors.forename: Marcus
authors.initials: M
authors.lastname: Wietstruk
authors.forename: Andrew J R
authors.initials: AJ
authors.lastname: Plested
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics*; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D055668:Photochemical Processes; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017470:Receptors, Glutamate / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D017470:Receptors, Glutamate
keywords: UV light; benzophenone; crosslinking; mammalian cells; orthogonal tRNA; synthetase