Sonderforschungsbreich 35

Transmembrane Transporters
in Health and Disease

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Projects

Former SFB 35 projects:


Interaction of transmembrane transporters with low molecular weight ligands and their potential role as pharmacological chaperones

Peter Chiba

Work of the first funding period defined a novel concept for interaction of solute-type ligands with the model ABC transporter P-glycoprotein. It demonstrated: (i) a dual interaction mode caused by rotational pseudosymmetry of the transporter and (ii) the ability of novel chemical entities to rescue trafficking deficient ABCB1 mutants. Based on these findings we should like to (i) define interaction sites of ABC transporters as a basis for pharmacological interference, (ii) define the interaction with ligands on a single molecule level and (iii) study the interaction of the hepatic ABC-transporter ABCB11 and trafficking deficient variants with chemical entities and their effect on intrahepatic cholestasis. We will continue the productive collaboration with P02/Ecker on modelling and data driven docking of ABC transporters and P13/Müller on design and synthesis of PET tracers for imaging of ABC-transporters.


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Molecular basis of drug-transporter interaction

Gerhard F. Ecker

Within the first funding period experimental data guided ligand docking into protein homology models of ABCB1 and SERT followed by experimental validation allowed to propose a binding mode of propafenone-type inhibitors of ABCB1 and to identify the binding mode of imipramine and analogous tricyclic antidepressants in SERT. The methods developed during the first funding period lay the ground for the proposed research programme for the next 3 years. Specifically, we aim at (i) experimental data guided docking into protein homology models of transporters expressed in the liver and at the blood-brain barrier (ABCB4, ABCB11 and ABCG2) and experimental validation of the binding hypotheses, (ii) Identification of structure-based hypotheses to design subtype selective GAT ligands, and (iii) pharmacophore-based screening of vendor libraries to identify new ligands and pharmacochaperones and experimental validation of hit compounds retrieved.


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ER export of neurotransmitter transporters

Michael Freissmuth

The work of the first funding period confirmed that: (i) SERT differs from other SLC6-family members in its specificity for SEC24C, (ii) ER export via the COPII-dependent pathway is required for correct axonal targeting, (iii) the C-terminus of SERT is required for correct folding of the protein. Based on these findings, we should like to (i) determine the molecular basis for the interaction of SERT/DAT/NET with SEC24C/D proteins by creating chimerae and explore the effects of human coding variants of SEC24C on ER export; (ii) generate a mouse with a variant SEC24C that does not assist ER export of SERT: this should preclude axonal delivery of SERT but still allow for SERT to accumulate in the somatodendritic compartment; (iii) identify the proteins which assist folding of the C-terminus to verify our chaperone/COPII-exchange model. In addition, we intend to pursue the fruitful collaboration with P04/Ecker on modelling the drug- and substrate binding site of SERT and DAT.

 


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Single molecular forces involved in recognition and transport of trans¬membrane transporters

Peter Hinterdorfer

Our studies will use the potential of single molecule force spectroscopy and recognition imaging to investigate membrane proteins on the surface of a living cell. Thereby it is aimed to study the dynamics of monoamine and ABC transporters with various antidepressants for a better understanding of structure, function, and kinetics of such transporters in native conformational states and environments. More specifically, we will explore (i) the interaction energy landscape, (ii) the stoichiometry and cooperativity, and (iii) the lateral distribution of ligand binding to transmembrane transporters. Our studies will be closely correlated with (i) macroscopic binding and transport assays, (ii) single molecule fluorescence microscopy and (iii) molecular modelling and steered molecular dynamics simulation (Stockner, MUV).


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Molecular structure-function analysis and inhibition of the human ABCG2 ABC transporter

Karl Kuchler

We have pursued a detailed structure-function and modeling analysis of certain yeast PDR transporters, which will continue in the second SFB phase. Fungal PDR transporters are orthologues of mammalian ABCG transporters, some of which are implicated in hepatobiliary liver diseases, antitumor resistance and gout. The key hypothesis is that the principle drug transport mechanism is conserved among PDRs and ABCGs. Thus, we will study drug transport and substrate interference in multiple-transfected MDCK cells expressing BCRP and ABCB11/BSEP, and in primary hepatocytes. The analysis will include disease-relevant BCRP mutations and structure predictions with molecular dynamics simulations and computational docking. Further, we will study the intracellular targeting mechanisms of BCRP, and the role of BCRP in hepatobiliary detoxification. Finally, we shall delineate the substrate interference of BCRP and BSEP with ABCB1/P-gp, as well as a potential function of BCRP in the chemoprotection in the blood-brain barrier .


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The molecular mechanism of drug transport mediated by fungal ABC transporters

Karl Kuchler

ATP-binding cassette (ABC) proteins form one of the largest protein families with more than 5000 members operating in all living cells from bacteria to yeast as well as man. ABC proteins often act at crossroads of vital cellular processes, because most carry intrinsic transport activities. Eukaryotic ABC transporters mediate multidrug resistance in cancer therapy and infectious diseases, and several human ABCs are intimately connected to prominent genetic diseases. Although the domain organization of ABC proteins is conserved in evolution, the molecular basis for their broad yet distinct drug substrate specificities has remained an unsolved mystery. At present we even have a limited molecular understanding of their transport mechanisms, which is, however, a mandatory prerequisite to combat or cure, ABC-mediated diseases and drug resistance phenomena.


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PET tracers for the in vivo imaging of multidrug transporters in the CNS.

Markus Müller, Oliver Langer

Drug resistance in epilepsy may be caused, among other factors, by active efflux transport of antiepileptic drugs by P-glycoprotein (Pgp) at the blood-brain barrier (BBB), which hampers the built-up of therapeutically effective drug concentration levels in brain tissue. A diagnostic method which can non-invasively assess cerebral Pgp function/expression could find clinical application in preselecting epilepsy patients with increased transporter activity for treatment with Pgp modulating drugs in a personalized medicine approach. In the first funding period we have developed a new positron emission tomography (PET) imaging protocol based on paired scans with the 11C-labeled third-generation Pgp inhibitor [11C]tariquidar and the Pgp substrate (R)-[11C]verapamil, which allows for independent non-invasive assessment of cerebral Pgp expression and function in rodents. In the upcoming funding period we will apply this PET imaging protocol in healthy volunteers and therapy refractory and responsive temporal lobe epilepsy patients to investigate regional differences in cerebral Pgp expression/function. In parallel, we will continue, in collaboration with Chiba and Ecker, to develop improved PET tracers for mapping of cerebral Pgp expression, which have higher signal-to-noise ratios than [11C]tariquidar. In addition, we will support Trauner in the PET visualization of breast cancer resistance protein (BCRP) function in the liver and Pollak in the imaging of SERT density in brain.


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The Large Neutral Amino Acids Transporter LAT1 in Autism

Gaia Novarino

Recently I have identified several patients affected by autism spectrum disorder (ASD) carrying mutations in the gene BCKDK. Patients carrying mutations in BCKDK hyper-metabolize serum branched chain amino acids (BCAAs). In our first manuscript, we showed that Bckdk-/- mice have neurobehavioral phenotypes resembling our patients. In addition, metabolic studies showed decreased levels of BCAAs and increased levels of other large neutral amino acids (LNAAs) in the brain of Bckdk-/- mice due to abnormal flux through the facilitative transporter, LAT1, present at the blood brain barrier (BBB). These findings suggested that the phenotype in the patients with BCKDK mutations may result from defective transport of BCAAs and LNAAs (several of which are important precursors for neurotransmitters) across the BBB. Using the Slc7a5 conditional knockout mouse, currently available in my laboratory, we would like to
i) measure steady state brain LNAA levels and LAT-1 mediated LNAA flux at the blood brain barrier (with Langer)
ii) investigate how the LAT1 transporter influences neurogenesis
iii) study how LAT1-mediated LNAAs flux influences mouse behavior (with Pollak).


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Neurotransmitter transporters in mouse models of psychiatric disorders

Daniela Pollak

The mode of action of the most commonly used pharmacological agents for the treatment of mood disorders is based upon their inhibitory effect on the serotonin transporter (SERT). Variations in the promoter region of SERT constitute a genetic predisposition for the development of mood disorders. Environmental conditions, primarily life stress are also known to be causally involved in the pathogenesis of mood disorders and interact with genetic factors. However, the biological basis mediating the effect of the environmentally acquired vulnerability to disease is not well understood. Here we to examine whether chronic mild stress, a behavioral model of depression in mice, alters SERT functional activity at the cellular and molecular level in brain tissue and in-vivo and to study the underlying molecular signaling pathways level. In a translational approach, candidate molecular key players will be further studied at the genetic level and related to brain activity in human patients with depression. This study may for the first time link environmental depressiogenic conditions to alterations in brain monoamine transporters and define some of the molecular elements involved. Identification of functionally relevant genetic contributors in the human brain may provide the biological basis for the nature-nurture interaction believed to form the grounds for the multifaceted network of factors contributing to the development of mood disorders.


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Subunit stoichiometry and supermolecular organization of transmembrane transporters

Gerhard Schütz

We will employ state-of-the-art single molecule fluorescence microscopy for obtaining insights into the oligomeric state, the interaction kinetics, the mobility, and the nanoscopic organization of transmembrane transporters. The single molecule approach allows us to go beyond conventional ensemble measurements: with brightness analysis and two-color colocalization studies, we will precisely quantify molecular associations; the high temporal resolution of one millisecond will be employed to capture transient phenomena such as weak interactions with immobile sites in the membrane; finally, we will exploit the high localization precision of ~20nm for unraveling structures influencing the diffusional paths and for obtaining insights into the nanoscopic arrangements of the molecules. In particular, we will address the organization of the monoamine transporters SERT and DAT in live cells; this study will be performed in close collaboration with Sitte and Freissmuth, who will provide and test GFP fusion constructs, fluorescent ligands, cell lines, and ex vivo cultured neurons. The developed techniques shall be further applied for studying the organization of ABC and PDR transporters. We will coordinate all experiments with Hinterdorfer, who performs complimentary force spectroscopy measurements to determine the interaction energies between transmembrane transporters and their ligands and substrates.


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EGFR-mediated regulation of transmembrane transporters

Maria Sibilia

Mice lacking the epidermal growth factor receptor (EGFR) develop a neurodegeneration shortly after birth affecting the frontal cortex and olfactory bulbs. We found that EGFR signaling controls the survival of cortical but not of midbrain astrocytes which might provide a mechanism for the region-specific neurodegeneration in EGFR-/- mice. Furthermore, our preliminary studies demonstrate that mice lacking the EGFR in astrocytes are more sensitive to seizures induced by kainic acid. In an attempt to identify the molecular basis of this phenotype, we found that 2 members of the family of glutamate transporters, GLAST and GLT1, are down-regulated in EGFR deficient cortical astrocytes. Based on our findings we hypothesize that impaired glutamate transporter expression and glutamate uptake in EGFR deficient astrocytes might promote the development of neurodegenerative diseases, stroke and epilepsy. We would therefore like to understand the mechanism how EGFR signaling affects expression and function of glutamate transporters in different astrocyte populations of the mouse brain and if the impaired glutamate turnover is causally involved in the development of neurological diseases. In collaboration with Trauner we also found that the ABC transporters Mdr1a/b/ABCB1 und Bcrp/ABCG2, mainly involved in conferring multidrug resistance, are expressed at reduced levels in EGFR deficient livers. We will therefore also investigate how EGFR signaling influences the expression of these ABC transporters and if their reduced expression in EGFR deficient livers affects the development of cholestatic liver diseases and the resistance to anti-cancer treatments in mice.


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Structure/function relationships of bacterial and human transporter orthologues

Harald H. Sitte

We plan to explore the structural determinants of function of SERT and GAT1 by a comparative approach using bacterial orthologues. The crystallization of LeuTAa ushered the advent of a new era: the structure of LeuTAa provides a starting point for comparative studies to assess which residues are involved in ligand binding to SERT and how conformational changes in SERT account for the transport cycle. We assume that inferences from in-silico modelling, pharmacoinformatics and proteomics can be verified by the creation of site-specific mutants and testing in biochemical and electrophysiological assays. We further plan to model LeuTAa, GAT1 and SERT and dock leads into the structural context. We will then compare purified and reconstituted LeuTAa to SERT- or GAT1 expressed in Xenopus laevis oocytes.


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Investigating the pathophysiological role of ABCB6 and the mechanism of action of ABCB1

Gergely Szakacs

Recently, we have identified that the putative mitochondrial transporter ABCB6 is a glycoprotein present in the membrane of mature erythrocytes and in exosomes released from reticulocytes during the final steps of erythroid maturation. Preliminary data supporting the proposal suggest that ABCB6 is expressed in the endolysosomal compartment of HeLa cells and in the melanosomes of MNT-1 cells. Our goal is to establish structure-function relationships by analyzing the consequences of mutations and polymorphic variations on the intracellular trafficking and the function of ABCB6. We will express ABCB6 variants in yeast, insect cells and several human cell lines to analyze function, targeting and intracellular trafficking (with Freissmuth, Chiba & Kuchler). Another aim is to establish the link between the conformational switch of the transmembrane domains and the ATPase cycle of ABCB1 by following the binding of the conformation sensitive anti-Pgp antibody UIC2, ATPase measurements, azido-ATP labeling and transport studies. We will characterize the UIC2-reactivity, nucleotide and substrate affinity and the rates of the formation and dissociation of the pre- and post-hydrolytic catalytic intermediates of Pgp variants carrying mutations at the drug-binding and the ATPase sites (with Ecker & Stockner).


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Molecular regulation of hepatobiliary ABC transporters in cholestatic liver diseases

Michael Trauner

Hepatobiliary ATP-binding cassette (ABC) transporters are responsible for excretion of various endo- and xenobiotics including bile salts, other biliary lipids, drugs and carcinogens into bile. Based on our central hypothesis that alterations of hepatobiliary ABC transporters critically determine cholestatic liver injury and bile duct diseases (cholangiopathies) we first aim to explore role of the canalicular bile salt export pump (ABCB11) for downstream bile duct injury using genetic and pharmacological animal models with increased and reduced ABCB11 function. In our second aim we will unravel the so far neglected role of breast cancer-related protein (ABCG2) and multidrug resistance protein (ABCB1) as potential compensatory canalicular efflux system for bile salts and carcinogens in genetic mouse models and acquired (mechanically-obstructive, lithocholic acid and drug-induced) cholestasis using functional imaging studies with PET tracers in corresponding mouse models and human disease. This project is expected to provide novel mechanistic insights into the pathogenesis and progression of bile duct injury, which represents an key clinical problem and leading indication for liver transplantation. Finally, the results of this project are expected to result in novel diagnostic and therapuetic strategies targeting hepatobiliary ABC transporters in cholestatic liver diseases.


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Membrane transporters in drug refractory epilepsy

Christoph Baumgartner

The goal of this project part is to systematically investigate the role of multidrug transporters in medically refractory focal epilepsy affecting 30-40% of all epilepsy patients. Specifically, we will assess the relative contribution of a genetically determined inherited overexpression of multidrug efflux transporters versus an acquired and transient overexpression as a consequence of uncontrolled seizures. We will establish a data base of patients with medically refractory epilepsy undergoing presurgical evaluation at our center and perform genetic testing for polymorphisms on the expression of specific drug transporters. Patients will undergo PET scans for in vivo imaging of multidrug transporters (see project by Markus Müller). Furthermore, we will perform intraoperative electrophysiological studies to assess local epileptogenicity as well as intraoperative microdialysis to measure local drug concentrations in the resected brain tissue. The local expression of multidrug transporter proteins will be studied by subsequent histochemical analysis of the resected brain tissue. Finally, we will provide specimens of human epileptic brain tissue for further analysis by the other research groups within the SFB.


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Multimodal Imaging of human Brain Monoamine Transporters

Lukas Pezawas

Introduction: Serotonin (5-HT) and dopamine (DA) are critically involved in the development and function of brain circuitries of emotion and have clinically been related to depression and temperamental risk factors of depression such as anxiety and impulsivity. By the use of an Imaging Genetics and Gene Discovery approach, we will investigate effects of functional genetic variants and possible genetic candidates of SLC6A4 and SLC6A3, which are related to mood disorders and comorbid condition such as Attention Deficit and Hyperactivity Disorder (ADHD), in a large sample of healthy controls and remitted depressive patients. Furthermore, our study aims at elucidating the complex relationship between DAT functionality and D2-receptor binding and its correlates on a brain systems level, a relationship, which has not sufficiently been studied so far.
Objective: To study implications of genetic variation of the 5-HT and DA transporter genes on brain development and function on a brain systems level as well as the relationship between DAT functionality and D2-receptor density. Local anatomical and functional measures such as grey matter and subcortical volumes, cortical thickness, parcellelated cortical areas, and BOLD activity as well as brain system related measures such as functional, effective, and structural connectivity will be utilized to detect subtle genetic effects and will be related to D2-receptor binding. Behavioral significance will be evaluated by means of neuropsychological parameters, which have been identified as risk factors for mood disorders.


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Selection of Stable Transporters to Facilitate their Structural Characterisation

Peggy Stolt-Bergner

Structural and functional studies on transmembrane transporters are crucial to understanding mechanisms of substrate translocation. However, structural work on membrane proteins is difficult due to their hydrophobic nature and instability outside of the membrane. We will focus on efforts to produce and purify mammalian transporters of interest to the consortium, in large enough quantity for both structural and functional studies. We are also attempting to crystallize prokaryotic homologues of these transporters in parallel. In addition, we are interested in identifying stable transporter mutants that may aid structural and functional studies.


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