1) Name – Title of the research project in CCHD
Werner Sieghart – GABAA receptor-associated proteins
2) Coordinates of the Faculty Member
Center for Brain Research, Department of Biochemistry and Molecular Biology, Medical University Vienna, Spitalgasse 4, A-1090 Vienna; Tel +43 1 4277 62950; Fax +43 1 4277 62960; email: Werner.Sieghart@meduniwien.ac.at
3) Keywords
Ligand-gated ion channels, G protein-coupled receptors, GABAA receptor associated proteins, interaction, trafficking, function
4) Research interest of the Faculty Member
Dr. Sieghart is interested in the structure, pharmacology and function of GABAA receptor subtypes. Using highly selective antibodies against GABAA receptor subunits, he investigated their regional, cellular and subcellular distribution as well as the subunit composition and abundance of receptor subtypes in different brain tissues. Using recombinant GABAA receptor subunits, he studied the structure and assembly of GABAA receptors, the location of their different drug binding sites and their pharmacology. Finally, in an international collaboration he established a strategy for the investigation of the function of individual cell types and GABAA receptor subtypes in the brain, based on transgenic mice expressing pharmacologically impaired GABAA receptors throughout the brain, that are then cell-specifically replaced by wild-type receptors, allowing the electrical activity of these neurons to be selectively modulated by GABAergic drugs. In the recent past, he additionally started to investigate the interaction of GABAA receptors with so far unknown proteins as well as other ionotropic or metabotropic receptors.
5) Collaborations within CCHD
Together with S. Boehm direct interactions between GABAA receptors and other ionotropic receptors are being investigated. With M. Freissmuth the co-trafficking of GABAA receptors with ionotropic and metabotropic receptors is studied, and the group of G. Superti-Furga will be invaluable for the identification of GABAA receptor-associated proteins using mass-spectrometric techniques. Students of H. Lassmann are performing SDS-PAGE and Western blot analyses in the lab of W. Sieghart and H. Lassmann provides support in immunohistochemistry of GABAA receptor subunits. Several attempts to isolate recombinant antibodies against GABAA receptor subunits were performed in collaboration with D. Kerjaschki. With H. Stockinger, single dye tracking of GABAA receptors and its associated proteins and with E. Jensen-Jarolim epitope studies by mimotopes will be performed. With T. Klausberger, GABAA receptor subunits will be localized in various interneurons and the in vivo pharmacology of GABAA receptor subtypes will be investigated.
6) Collaborating research groups where PhD Students can perform their research stay
Dr. Antoine Triller, Inserm UR497, Ecole Normale Supérieure, Biologie Cellulaire de la Synapse N&P, 46 Rue d'Ulm, 75005 Paris, France.
Dr. Peter Somogyi, MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, University of Oxford, Oxford OX1 3TH, United Kingdom.
7) Industrial partners (if applicable)
There are 3 industrial partners (SME´s) within the integrated project “Neurocypres”, in which Dr. Sieghart participates, financed by the EC since February 2008.
8) Know-how and infrastructure of the research group
Dr. Sieghart was the first to demonstrate a molecular heterogeneity of GABAA receptors using [3H]flunitrazepam as a photoaffinity label (Sieghart and Karobath, 1980). He then confirmed the heterogeneity of GABAA receptors using a variety of biochemical and pharmacological techniques (Sieghart 1995). During the last 18 years his group generated highly selective antibodies against 13 different GABAA receptor subunits that were then used to determine the subunit composition of GABAA receptors in the brain (Jechlinger et al., 1998; Ogris et al., 2006). In addition, these antibodies were used for the investigation of the regional, cellular and subcellular distribution of these subunits in a variety of collaborative studies. In a series of studies his group identified various amino acid residues in GABAA receptor alpha1, beta3 or gamma2 subunits that seem to be important for the assembly of these pentameric receptors (Sarto-Jackson et al., 2006; 2007).
Using homology modeling techniques his group generated comparative models of the GABAA receptor extracellular and transmembrane domain using the structures of the acetylcholine binding protein and of the nicotinic acetylcholine receptor as a template (Ernst et al., 2005). These models not only allow to visualize the receptor in its three dimensional structure, but also can be used as a guide for further experiments aiming to identify intersubunit contact sites important for assembly, drug binding sites, or sites for the transduction of drug effects. Recently, he established a large scale expression and purification of recombinant GABAA receptor subtypes in insect cells (Kang et al., 2008) that is now used for attempts to crystallize GABAA receptors within the EC-supported integrated project “Neurocypres”.
In long-term collaborative studies with Robert Dodd (CNRS, Gif-sur-Yvette, France) and Jim Cook (Univ. Wisconsin, USA) he is developing new GABAA receptor subtype selective drugs with a possible clinical application (Sigel et al., 2001;; Savic et al., 2008) using receptor binding studies and electrophysiological investigations of recombinant receptors expressed in Xenopus oocytes. In another long-term international collaboration with Bill Wisden (Univ. Heidelberg, Germany, now Univ. Aberdeen, UK), Peter Somogyi (Univ. Oxford, UK), and Esa Korpi (Univ. Helsinki, Finland) he established a novel transgenic mouse model that allows to study the function of individual neuronal cell types in behaviour, learning and memory by using drugs interacting with GABAA receptors (Ogris et al., 2004; Wulff et al., 2007). A variation of this technique is now used within a National Research Network supported by the Austrian Science Fund to identify neurons within the amygdala that are involved in fear and anxiety. Finally, in a collaboration with Harald Aschauer (University Clinic for Psychiatry, Vienna) and Martha Feucht (University Clinic for Pediatrics, Vienna) his group is investigating possible causes of psychiatric and neurologic diseases by applying molecular genetic techniques. In the course of this CCHD program, his group established cell biological and immunohistochemical techniques using primary neurons in culture and is investigating the interaction of GABAA receptors with other proteins as well as with ionotropic and metabotropic receptors.
Dr. Sieghart is Head of the Department of Biochemistry and Molecular Biology, Center for Brain Research, that contains four cell culture labs with a total of 8 laminar flows and incubators, biochemistry labs equipped for protein chemistry, molecular biology, receptor binding assays, culture facilities for E. coli, yeast, neuronal cells, insect SF9 cell (baculovirus), primary cell culture, Western blots, immunoprecipitation, antibody purification and characterization, 4 electrophysiology sets for oocytes (two electrode voltage clamp) and transfected cells (patch clamp) in cell culture. Additional infrastructure includes in house animal rooms. The Department contains 4 research groups with a total of 40 researchers. Dr. Sieghart´s research group currently comprises 3 research assistants, 3 postdocs, 8 Ph.D. students, 1 diploma student, and 3 technicians.
