AbstractsBiology & Animal Science

Photoinactivation of glutamate receptors by genetically encoded unnatural amino acids

by Viktoria Klippenstein

Institution: Freie Universität Berlin
Degree: PhD
Year: 2015
Record ID: 1102391
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000099210


Ionotropic glutamate receptors (iGluRs) are ubiquitous in the mammalian brain and the AMPA subtype is essential for fast, glutamate-activated postsynaptic currents. In the framework of this study, photoactive unnatural amino acid (UAA) crosslinkers as p-Benzoyl-L-phenylalanine (BzF, also known as Bpa) or p-Azido-L-phenylalanine (AzF) were incorporated site-specifically into AMPA receptors expressed in mammalian cells. Incorporation was performed via genetically encoded UAA mutagenesis using specific orthogonal tRNA / tRNA-synthetase pairs. This approach has been shown to be a powerful extension of site-directed mutagenesis, which has allowed systematic structure-function studies of a range of ion channels, however, its application to ligand-gated ion channels as the glutamate receptors has been sparse. The aim of this study was the extension of the use of UAAs in photoinactivation of AMPA receptors. The establishment and thorough characterization of UAA photocrosslinking in AMPA receptors has been a fundamental part of the study. Methodologically, UV-driven photocrosslinking was detected by means of rapid perfusion outside-out patch-clamp electrophysiology, and assessed by biochemical experiments. Electrophysiological measurements revealed, that receptors rescued by incorporation of AzF or BzF within the lower ligand-binding domain (LBD) interface had nearly invariably similar properties to wild-type (WT) channels and were expressed at similar levels. BzF incorporation at these subunit interfaces within the LBD allowed photocrosslinking of subunits, which was induced by UV irradiation at 365 nm from a mercury lamp. Photoinactivation was characterized by peak current reduction of ~90% within ~10 seconds of cumulative UV exposure. BzF incorporation led to 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 LBD is consistent with deformation of the LBDs as well as intersubunit rearrangements during AMPA receptor desensitization. These experiments demonstrate, that UAAs are a useful tool for exploring structural rearrangements of the AMPA receptor and suggest relevant applications of photoinactivation of glutamate receptor ion channels in native systems. Ionotrope Glutamatrezeptoren (iGluRs) gehören zu einer der häufigsten Rezeptorfamilien in unserem Gehirn, wobei der AMPA-Subtyp mit seiner Erzeugung von glutamat-aktivierten elektrischen Strömen in der Postsynapse eine zentrale Rolle bei der Signalübertragung zwischen Nervenzellen spielt. Im Rahmen dieser Arbeit wurden photoaktive unnatürliche Aminosäuren wie p-Benzoyl-L-phenylalanin (BzF, auch bekannt als Bpa) oder p-Azido-L-phenylalanin (AzF) positions-spezifisch innerhalb von AMPA-Rezeptoren eingebaut, die in Säugetierzellen (HEK-293) exprimiert wurden. Der Einbau erfolgte über die so genannte genetisch-kodierte unnatürliche Aminosäure-Mutagenese, die auf die Verwendung spezifischer orthogonaler tRNA /…