Sonderforschungsbreich 35

Transmembrane Transporters
in Health and Disease
Britisch Flag English

email office@sfb35.at
Tel.: +43 1 4277 64106
(Mo-Do: 09:00 - 12:00)

Colloquia in Transmembrane Transport:

Please see also the seminar series in "Computational Life Sciences" at the Pharmacy Center, University of Vienna

- You can find the lecture series here

 

"ABC transporters regulate stem and progenitor cell proliferation, leukocytosis, thrombocytosis and atherosclerosis"

Leukocytosis is a risk factor for athero-thrombotic disease in humans, and develops in animal models of atherosclerosis in response to feeding high fat, high cholesterol diets. The ATP binding cassette transporters ABCA1 and ABCG1 promote cholesterol efflux to apoA-1 and HDL, respectively, and are targets of LXR transcription factors. Mice lacking ABCA1/G1 develop a dramatic myeloproliferative phenotype with monocytosis and neutrophilia, associated with expansion and proliferation of hematopoietic stem and myeloid progenitor populations (HSPCs). The transporters are highly expressed in HSPCs where they act to control proliferative responses to growth factors (IL-3, GM-CSF) by regulating plasma membrane lipid rafts and cell surface expression of the common beta subunit of the IL-3/GM-CSF receptor. ApoE is highly expressed on hematopoietic stem and progenitor cells where it interacts with ABCA1/G1 to control stem cell proliferation, monocytosis and accumulation of monocytes in atherosclerotic lesions. ABCG4 is the closest relative of ABCG1 and also promotes cholesterol efflux to HDL. ABCG4 has a restricted distribution in the bone marrow megakaryocyte progenitor (MkP) population, where it acts to control the cell surface levels of the thrombopoeitin (TPO) receptor, megakaryocyte formation, platelet counts and atherogenesis. The findings suggest a common theme wherein ABC transporters mediating cellular cholesterol efflux act to suppress cell surface expression of growth factor receptors and thus match cellular proliferative responses to cholesterol availability. When cholesterol efflux pathways are suppressed, excessive cellular proliferation may contribute to atherosclerosis and neoplasia.

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"Optochemical Genetics"

Transmembrane receptors allow a cell to communicate with its environment in response to a variety of input signals. These can be changes in the concentration of ligands (e.g. hormones or neurotransmitters), temperature, pressure (e.g. via acoustic waves or touch), transmembrane potential, or light intensity. Many important receptors have now been characterized in atomic detail and our understanding of their functional properties has markedly increased in recent years. As a consequence, these sophisticated molecular machines can be reprogrammed to respond to unnatural input signals. Arguably, the most useful of these signals is light. I will show how ligand-gated ion channels, G-protein cou-pled receptors, as well as voltage-gated ion channels, can be manipulated with synthetic photoswitches to become light-sensitive. The resulting hybrid photoreceptors can be used to optically control neurons with very high precision. They have been used to dissect neu-ral networks and have already found applications in the restoration of vision and the con-trol of other sensations (such as pain). This combination of synthetic photoswitches and receptor proteins augments the field of Optogenetics and adds a new functional dimension to Chemical Genetics. As such, we propose to call it “Optochemical Genetics”.

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"Metal Assisted Synthetic Approaches to Small Molecules Modifying Cell Differen-tiation"

Regenerative Medicine is understood as one of the most promising therapeutic approaches to alleviate a multitude of diseases. While there is significant progress in the experimental development of this ap-proach related to gene therapeutic intervention often in combination with employing embryonic stem cells, the regulatory implications of such therapies are yet unclear and ethical aspects represent a ma-jor obstacle in the further development of such strategies. Hence, small molecules capable to affect cell differentiation towards particular tissues have received significant attention, as the prospect of employ-ing such agents in regenerative medicine seem highly attractive. Within the past years we have been developing several compounds capable to trigger differentiation of certain progenitor cells towards particular tissues otherwise difficult to regenerate. Case studies will be presented in the area of small molecule induced cardiomyogenesis and neurogenesis, as well as in differentiation acceleration towards skeletal muscle cells. Within a very recent project we have identi-fied lead compounds capable to efficiently induce cardiomyogenesis starting from embryonic or pro-genitor cells, ultimately leading to independently beating heart cells. Considering the fact, that cardiac infarctation represents one of the major death causes in the developed world, the prospect of tissue regeneration of damaged heart muscle tissue offers a highly innovative perspective of regaining heart function, as this organ in not capable to functional repair on its own. Synthetic approaches towards the individual target compounds exploited modular strategies based on metal assisted catalysis, in particular sequential coupling strategies (C-C, C-N, and C-H activation). Optimization of functional decorations of the particular heterocyclic scaffolds will be discussed in detail.

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"Dopamine Transporter Mutant Mice in Experimental Neuropharmacology"

The dopamine transporter tightly regulates the extracellular dynamics of dopamine by recapturing released neurotransmitter into the presynaptic terminals, and genetic deletion of this protein results in profound alterations in both the presynaptic homeostasis and the extracellular dynamics of dopamine. By using this model of severe dopaminergic dysregulation, significant progress has been made in defining the major target of psychotropic drugs, understanding the mechanisms of their action, unraveling novel signaling events relevant for dopaminergic transmission, and mapping neuronal pathways involved in dopamine-related behaviors. Furthermore, DAT mutant mice provided an opportunity to model in vivo conditions of extreme dopaminergic dysfunction that could be relevant for human disorders such as ADHD, schizophrenia, and Parkinson's disease and, thus, could serve as test systems for developing novel treatments for these and related disorders.

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"Pendrin, a transporter involved in hearing loss and Asthma bronchiale"

The sequencing of the human genome was a milestone in science and marked the beginning of the post-genomic era. Genetic make-up, proteomic interaction and epigenetic regulation of gene expression are important for n ormal cellular physiology and play key roles in both disease progression and responses to therapeutic intervention. The knowledge of the genomic make-up, however, does not by itself give insight into the function of single genes; nor does it give conclusive answers on the effect of single genes or gene products on regulatory circuits established in living cells. The challenge now and in the immediate future is to combine the genomic and epigenetic information with the functional information already known regarding gene products in their native environment. The research in our laboratory is focused on the genomics, epigenetics and functional-proteomics of pendrin. The SLC26A4 gene codes for the pendrin protein, which exchanges bicarbonate, iodide and other anions for chloride. Pendrin is mainly expressed in the thyroid gland, inner ear and kidney. The identification of the pendrin coding gene led to investigations regarding the origin of some types of hereditary deafness. 170 mutations located both in exons and introns of the pendrin gene have been linked to various human diseases. Individuals with disease-causing mutations can present distinct phenotypes, collectively named Pendred syndrome (PS, OMIM n° 274600), DFNB4 (OMIM n° 600792) or LVAS (OMIM n° 603545). PS is a rare disease causing 5-13% of childhood deafness. It is an autosomal recessive disorder characterized by sensorineural.

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"Integrins modulate the serotonin system through a three-fold interaction with the serotonin transporter"

Integrins are differentially expressed in the brain, where they modulate the assembly of synapses through the recruitment of scaffolding proteins that influence receptor localization and synaptic vesicle function. The integrin b3 gene (ITGB3) has been associated with autism, with a strong synergistic association with the serotonin transporter gene (SLC6A4, SERT). While SERTs have been extensively studied in the central nervous system, little is known on integrin b3 function in the brain. In the brain, integrin b3 binds to the integrin av subunit to form the vitronectin receptor (avb3). Here we utilize mouse models to determine the role of integrin avb3 on the modulation of the serotonergic system. Integrin b3 and SERT haploinsufficiency leads to several behaviors associated to altered serotonergic signaling. These behavioral modifications are correlated with reductions in av expression and integrin-linked signaling pathways due to the synergistic genetic interaction between these two genes. The genetic interaction also points to a functional interaction, where the activation of integrin avb3 signaling pathways are associated with increases in SERT reuptake activity. Increased adhesive properties in cells expressing avb3 also increase SERT uptake of serotonin in heterologous cells. Finally, some, but not all of the changes associated with avb3 activity may result from a direct interaction with the carboxyl-terminus of SERT. While we are still in the process of deciphering the multiple levels of interaction between integrin b3 and SERTs, we have discovered a complex mechanism by which cell adhesion may influence serotonin synaptic function and plasticity in vivo.

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"Reading The Labels On Your Medication Targets: A Walk-Through From Serotonin Transporter Cloning To Brain Disorders"

Presynaptic biogenic amine transporters mediate reuptake of released amines from the synapse, thus regulating serotonin, dopamine and norepinephrine neurotransmission. Medications utilized in the treatment of depression, attention deficit-hyperactivity disorder and other psychiatric disorders possess high affinity for amine transporters. In addition, amine transporters are targets for psychostimulants. Altered expression of biogenic amine transporters has long been implicated in several psychiatric and neurodegenerative disorders. Therefore, appropriate regulation and maintenance of biogenic amine transporter activity is critical for normal amine homoeostasis. I will present our efforts to understand (1) how cellular protein kinases and phosphatases regulate serotonin transporter expression, activity, trafficking and degradation, (2) how transporter phosphorylation regulates amine clearance, (3) how transporter phosphorylation is dysregulated in human disease and (4) our translational approach from in vitro, ex vivo to in vivo animal model to understand the role of transporter phosphorylation in aminergic neurotransmission and in human disease. We believe that understanding the molecular mechanisms by which phosphorylation events affect amine transporter activity is essential for understanding the contribution of transporter phosphorylation to the regulation of monoamine neurotransmission and for identifying potential new targets for the treatment of various brain diseases.

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"Ion coupling and dynamics of amino acid transporters"

Amino acids, which are important for cellular function and metabolism, are transported across cell membranes by specific transporter proteins. If the transport is uphill, against a transmem-brane concentration gradient of the amino acid, it is often coupled to the co- or counter-transport of a driving inorganic ion, which provides free energy by flowing down its own trans-membrane concentration gradient. In mammalian cells, active amino acid transport is often driven by the co-transport of Na+ ion(s). The transporters are thought to catalyze transport by simultaneously binding extracellular amino acid and Na+ ion(s) in their transmembrane do-main, followed by a structural rearrangement of the transporter-amino-acid-Na+ complex to allow dissociation of the substrates into the intracellular space. Therefore, transport is a multistep process that is composed of a number of sequential, individual reaction steps. Here, I describe ongoing efforts in our laboratory to dissect the mechanism of this multistep transport reaction, by applying rapid chemical kinetic techniques and measuring kinetic and thermody-namic parameters of amino acid interaction with the transport protein in two different model systems, the glutamate transporter EAAC1, and the neutral amino acid transporter ASCT2. Using structure-function analysis, we have also identified novel sites of interaction with the cation in these transporters.

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"Single Molecule Biology - Studying Movements and Meetings within the Plasma Membrane"

Current scientific research throughout the natural sciences aims at the exploration of the Nanocosm, the collectivity of structures with dimensions between 1 and 100nm. In the life sciences, the diversity of this Nanocosm attracts more and more researchers to the emerging field of Nanobiotechnology. In my lecture, I will show examples how to obtain insights into the organization of the cellular Nanocosm by single molecule experiments. Our primary goal is to understand the molecular organization of the plasma membrane and its impact on signaling processes. For this, we apply single molecule tracking to resolve the plasma membrane structure at sub-diffraction-limited length-scales by employing the high precision for localizing biomolecules of ~15nm. Brightness and single molecule colocalization analysis allows for studying stable or transient molecular associations in vivo. In particular, we developed a technique to detect molecular cluster formation directly in the live cell plasma membrane. With this methodology, individual aggregates can be selectively imaged, and the load of each cluster can be determined. We applied this technique to investigate the association of a fluorescent lipid analogues and lipid anchored proteins (1). Aggregates containing up to 4 probe lipids were observed to diffuse freely as stable platforms, shedding new light on the current debate concerning the existence of “lipid rafts”. Using the same technique we could further quantify the subunit stoichiometry of ion channels (2). Next, single molecule tools enable the direct observation of the random transitions a biomolecule experiences during its movements through the plasma membrane. For example, transient proximity due to interactions with other proteins can be directly monitored and quantified. I will present results on the interaction between antigen-loaded MHC and the T cell receptor directly in the interface region of a T cell with a mimicry of an antigen-presenting cell, using single molecule FRET (3). Finally, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions (4). This technology brings together our interest in immune signaling, and the capability for ultra-sensitive readout of large biochip surfaces. Cells transfected with a fluorescent fusion protein (“prey”) are grown on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of a membrane protein (“bait”); the fluorescence copatterning is used as readout for the bait-prey interaction. We applied this technology for the study of the interaction between CD4 – the major coreceptor for T cell activation – and Lck, an important tyrosine kinase in early T cell signaling. In addition to the well-known zinc-clasp structure, we found strong contributions of Lck membrane anchorage to the binding of the two proteins.

1. M. Brameshuber et al., J Biol Chem 285, 41765 (2010).

2. J. Madl et al., J Biol Chem 285, 41135 (2010).

3. J. B. Huppa et al., Nature 463, 963 (2010).

4. M. Schwarzenbacher et al., Nat Methods 5, 1053 (2008)

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"Mitochondrial K+/H+ exchange in Health and Disease: From Ion and Volume Homeostasis to Seizures in the Wolf Hirschhorn Syndrome"

Mitochondrial K+/H+ exchange in Health and Disease: From Ion and Volume Homeostasis to Seizures in the Wolf Hirschhorn Syndrome The protein family Mdm38/LETM1 is essential for the mitochondrial electro-neutral K+/H+ exchange (KHE), driven by the inside-directed H+ gradient. To qualify the proteins as a component of the mitochondrial KHE and measure the K+ fluxes, we have developed a novel method involving H+ and K+ sensitive fluorescent dyes entrapped in submitochondrial particles (SMPs). Our data showed that the KHE activity was nearly abolished in mutant SMPs. LETM1 is a part of a protein complex whose components are presently under study. In the absence of LETM1, yeast mitochondria swell due to an overload of K+, the most abundant cation in the organelle, and are subjected to autophagic degradation (´mitophagy´). Up- or downregulation of the human LETM1 expression results in early cell death. Importantly, nigericin, an ionophore catalyzing KHE, fully reverted all LETM1 deletion phenotypes. Moreover, transcriptional profiling associated to compromised mitochondrial K+ homeostasis in yeast revealed a significant induction of intracellular Zn2+ transporters and changes in ergosterol biosyn-thetic pathways. Human Wolf-Hirschhorn Syndrome (WHS) is a complex disease resulting from the hemizygous termi-nal deletion on chromosome 4. A critical region as been defined (WHSCR1) which correlates with the hallmark of WHS including growth and mental retardation and facial dysmorphismus. LETM1 is 80 kb distal to this region and is always deleted in WHS patients with seizures while it is preserved in patients without seizures. These findings suggest that LETM1 is the candidate gene for seizures events associated with WHS. Using Drosophila as a model organism for understanding the molecular defects in Wolf-Hirschhorn Syndrome we demonstrated the function of LETM1 as a mitochondrial osmoregulator through its KHE activity. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues showed that depletion of LETM1 resulted in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of LETM1 resulted in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. These results will prompt us to study in more details presynaptic mitochondria in absence of LETM1 .

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"Structural Basis for Protein Translocation in Molecular Machines"

Abstract - following soon.

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"Investigation of the consequences of nucleotide binding to a multidrug re-sistance ABC transporter with magnetic resonance techniques"

The multidrug ATP Binding Cassette (ABC) transporter LmrA is an integral membrane protein and a functional homologue of human P-glycoprotein involved in resistance against anti-cancer drugs in chemotherapy. LmrA forms a homodimer comprising the typical ABC transporter architecture of two nucleotide binding domains (NBDs) and two transmembrane do-mains (TMDs). It utilizes ATP binding or hydrolysis at its NBDs to drive the extrusion of toxic hydrophobic compounds via its TMDs. Combined, NMR and EPR present excellent tools to investigate all aspects associated with membrane transporter functionality, such as dynamics (e.g. side-chain or domain mobility) or structural (e.g. domain movements, substrate binding) effects. With a tri-fold magnetic resonance approach including solid-state NMR on the recon-stituted full-length protein, solution NMR on the isolated NBD as well as pulsed EPR on cysteine mutants of the full-length protein, a comprehensive picture on the dynamics of LmrA throughout its catalytic cycle could be developed. In order to investigate the ATPase activity of reconstituted LmrA, a 31P solid-state NMR based assay was developed that allows the simultaneous observation of all phosphor resonances in the ATP hydrolysis reaction. Because solid-state NMR is not limited by phase separation effects, this assay can be utilized to investigate simultaneous reactions in the aqueous as well as the lipid phase.

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"Patho-Genetics of Anxiety"

Twin studies propose a strong genetic contribution to the pathogenesis of anxiety disorders and panic disorder in particular with a heritability of about 50%. The present talk will give an overview of linkage and cytogenetic studies in panic disorder as well as association studies yielding support for several candidate genes contributing to the genetic risk for panic disorder such as the catechol-O-methyltransferase (COMT), the serotonin receptor 1A (5-HT1A), the serotonin transporter (5-HTT) and neuropeptide Y (NPY) genes. Additionally, evidence for a gene-environment interaction between 5-HTT gene variation and life events, respectively, will be reported. On a system level, fMRI activation in brain regions critical for emotional and learning processes has been proposed as a promising intermediate phenotype for genetic studies in psychiatric disorders. Thus, recent imaging genetic findings with respect to the influence of COMT, 5-HT1A and neuropeptide Y gene variants on neuronal activation correlates of emotional processing will be presented. Finally, pharmacogenetic studies in the field of anxiety suggest 5-HTT and NPY gene variants to drive inter-individual differences in treatment response to antidepressants in panic disorder as well as in anxious depression. These studies will be discussed with respect to their potential benefit in future efforts to develop an individu-ally tailored therapy for patients with anxiety disorders.

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"Molecular modelling: connecting protein structure to function using in silico methods"

Molecular modelling is gaining importance in studying protein function as its application spectrum in-creases. More importantly, improved predictive capability now allows for thorough experimental scru-tiny and validation of obtained results. An overview of methods (homology modelling and simulations) will be given and their application will be discussed. Analyses of the function of proteins by experimental techniques provide us with a large body of data that highlight e.g. the importance of amino acids, their accessibility and changes in distances. A full understanding of the function of proteins at the molecular level requires knowledge about relevant con-formations and structural changes. Although the number of protein crystal structures increases expo-nentially (presently above 50.000), only about 200 unique membrane protein structures have been sol-ved. Structures of bacterial homologs of the ABC and the NSS transporter subfamilies have become available, while the structures of the human counterparts remain elusive. Molecular modelling techniques can be used to predict the structure of these human transporters if a suitable template is available. The models created by homology modelling allow to project biochemical information onto the transporter structure. Hypotheses about the molecular details of protein function can be elaborated. Validated models can serve as a catalyst for the design of biochemical experi-ments. This is highlighted by the example of its application to the ABC transporter P-glycoprotein. Even if the structure of the human membrane transporter will become available, the crystal structure represents a single snapshot that still might not reveal the functionally relevant conformational changes. Molecular dynamics simulations are an in silico method that allows to explore the accessible phase space (accessible conformations) while fulfilling the laws of physics. Dynamics and involved conformational transitions of membrane inserted proteins can be studied at atomic resolution. Examples of applications to ABC transporters will be discussed.

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"Symmetry and the art of transport: a role for inverted-topology structural repeats in neurotransmitter transport"

Transport of solutes such as neurotransmitters across cell membranes is carried out by specific trans-port proteins that, for example, couple solute flux to the movement of ions along their concentration gradients. This has been proposed to occur via alternate exposure of a substrate binding-site to either side of the membrane, requiring at least two distinct conformations of the protein. X-ray crystallographic data for several secondary transporters from diverse families support this hypothesis and provide key insights into specific transport mechanisms. However, to date, most of these structures have been solved for only one state in the transport cycle. An intriguing feature of these structures is the presence of internal repeats with inverted topologies with respect to the membrane. We have carried out computational modelling using these structural repeats in order to model the opposite conformations of two families of neurotransmitter transporters. Experimental support for these models is provided by biochemical accessibility measurements and cross-linking studies by our collaborators, and by very recent X-ray structural data. The implications for these results will be discussed in terms of a potentially general role of inverted-topology repeats – and of symmetry – in the pseudo-symmetric alternating-access mechanism of secondary transport.

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"Role of canalicular phospholipid export pump ABCB4 for pathogenesis and treatment of cholestatic liver diseases"

Bile is primarily formed by canalicular excretion of bile acids and other biliary constituents including cholesterol, phospholipids, and bilirubin conjugates via specific ATP-binding cassette (ABC) transport-ers. The canalicular phospholipid flippase ABCB4 mediates biliary excretion of phosphatidylcholine which forms mixed micelles with bile acids and cholesterol to protect the bile duct epithelium from the detergent properties of bile acids. Mice with targeted disruption of the Abcb4 gene (Abcb4-/- mice) spontaneously develop sclerosing cholangitis with macroscopic and microscopic features of human primary sclerosing cholangitis. Bile duct injury in these mice is linked to defective biliary phospholipid secretion resulting in an increased concentration of free non-micellar bile acids which subsequently cause bile duct epithelial cell (cholan-giocyte) injury with development of sclerosing cholangitis, severe liver fibrosis and liver tumors (hepa-tocellular carcinomas). In analogy to the Abcb4-/- mouse model of sclerosing cholangitis, hereditary and acquired ABCB4 defects may play a role in the pathogenesis of a broad spectrum of hepatobiliary diseases in humans ranging from progressive familial intrahepatic cholestasis in neonates to intra-hepatic cholestasis of pregnancy, drug-induced cholestasis, intrahepatic cholelithiasis, non-anastomotic biliary strictures after liver transplantation, sclerosing cholangitis and biliary cirrhosis in adults. Therapeutic strategies for these disorders may target bile composition/toxicity via key nuclear receptors (e.g., FXR, PPAR) regulating biliary phospholipid and bile acid excretion. The therapeutically used bile acid ursodeoxycholic acid (UDCA) shows some of these properties, but has limited efficacy in the treatment of sclerosing cholangitis and other cholestatic disorders in humans. In contrast to UDCA, its side chain-shortened homologue norUDCA undergoes cholehepatic shunting between cholangiocytes and hepatocytes leading to a bicarbonate-rich hypercholeresis counteracting bile acid toxicity. Moreover, norUDCA has direct has anti-inflammatory, anti-fibrotic and anti-proliferative effects in hepatocytes, cholangiocytes and myofibroblasts. norUDCA is able to reverse the disease phenotype in the Abcb4-/- mouse model of sclerosing cholangitis. Upcoming clinical trials will have to demonstrate whether norUDCA or other side chain-modified bile acids are also clinically effec-tive in humans.

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"Neurogenetic mechanisms and distributed neural processing in schizophrenia"

Disturbed connectivity has been hypothesized to be central for the pathogenesis of schizo-phrenia since Wernicke. Neuroimaging has confirmed and specified this hypothesis by dem-onstrating aspects of decreased, but also increased connectivity, with a focus on prefrontal cortex, the extended limbic system, and the connections between the two. A dysregulation of prefrontal control, impacting on dopaminergic neurotransmission and salience attribution, may be a final common pathway to psychosis, leading to positive symptoms of stimulation of pre-frontal cortex, and abnormal connectivity between prefrontal cortex and hippocampus has been found to be a regionally specific abnormality in schizophrenia. New methods to quantify the entire connectome, derived from graph theory, have extended these regional findings by showing a generalized abnormality in the hierarchical wiring of neocortex and limbic system in schizophrenia. Since the disease is highly heritable, the study of the neural effects of genetic variants implicated in risk for schizophrenia offers a research strategy to dissect the neuroge-netic risk architecture of the disorder leading to, and potentially preceding, first episode psy-chosis. In this talk we review results from the study of risk genes related to dopaminergic stimulation (COMT, RGS4), canonical (DARPP-32) and non-canonical, beta-arrestin mediated signal transduction (AKT1), as well as results from the study of newly discovered variants ge-nome-wide significantly associated with risk for schizophrenia (ZNF804A, CACNA1C) identify-ing systems of disturbed connectivity mediating genetic risk for the disease.

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"Correcting the Trafficking Defect of Mutant CFTR"

The main cause of Cystic Fibrosis (CF), one of the most common life-shortening recessive genetic diseases, is the F508del mutation, a deletion of 3 nucleotides in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. CFTR (also ABCC7) is a cAMP-activated chloride (Cl-) channel expressed at the apical mem-brane of most surface epithelial cells, like the airways, the gastrointestinal tract, and in cells lining the ducts of several glands, like the sweat gland, the pancreas and the submucosal glands. Due to the absence/ impairment of functional CFTR in epithelia of CF patients very little or no CFTR-mediated Cl- transport is generally observed. The molecular and cellular defects associated with the most prevalent mutation (F508del), is at the level of CFTR intracellular localization, since the mutant protein fails to traffic to the plasma membrane, being mostly retained and degraded at the level of the endoplasmic reticu-lum (ER) [1,2,3]. The focus of this talk will be on the mechanisms of the ER quality control which are responsi-ble for the intrecellular retention of F508el-CFTR as well as on the pharmacological strategies to rescue this mutant by promising therapeutic small molecules which have started to emerge from the research to the clinical setting [4].
Grants: TargetScreen2 EU-FP6-2005-LH-7-037365; FCT-POCTI/MGI/47382/2002 (Portugal/ EU).
References:
1. Farinha CM & Amaral MD (2005) Most F508del-CFTR is targeted to degradation at an early folding check-point and independently of calnexin. Mol Cell Biol 25, 5242-5252.
2. Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, Sheppard DN, Amaral MD (2006) Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by dif-ferent mechanisms. Proc Natl Acad Sci USA 103, 17891-17896.
3. Scott-Ward TS, Amaral MD (2009) Deletion of F508 in the first nucleotide binding domain of CFTR increases its affinity to bind the Hsc70 chaperone. FEBS J 276, 7097-7109.
4. Amaral MD, Kunzelmann K (2007) Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis. Trends Pharmacol Sci 28: 334-341.

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"Regulation of D-glucose absorption in the kidney and small intestine"

According to text books luminal D-glucose is reabsorbed by sodium-coupled secondary active transporters (SGLT-family) located in the apical brush border membrane of epithe-lial cells in the kidney and small intestine. D-glucose is then released into the blood stream by facilitated diffusion via transporters of the GLUT-family located in the basal-lateral pole of the epithelial cell. However, recent results suggest that D-glucose absorp-tion is a much more dynamic process. There is intensive trafficking of the transporters involved between the plasma membrane and intracellular sites. Furthermore, there is short-term redistribution of transporters in epithelial cells in response to a changing nutritional state of the individual. The mecha-nisms of D-glucose transporter trafficking as well as their potential role as therapeutic targets for the treatment of obesity and type2 diabetes will be discussed in this presentation.

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"Ion channels as drug targets"

The present talk will be divided into 3 parts: 1) A brief introduction to the biotech company NeuroSearch specialized in performing research and development of compounds targeting membrane proteins. This introduction will include drugs that targets transporters. 2) A general introduction to ion channels and 3) A detailed description of a research project aiming at developing new principles for the treatment of cardiac arrhythmias. A more elaborated introduction to point 3) is as follows: The cardiac action potential is the result of an orchestrated function of a number of different ion channels. Action potential repolarisation in humans relies on three current components named IKr, IKs and IK1 with party overlapping functions. The ion channel α-subunits conducting these currents are hERG1 (Kv11.1), KCNQ1 (Kv7.1) and Kir2.1 (KCNJ2). Loss of function in any of these currents can result in long QT syndrome. Long QT is a pro-arrhythmic disease with increased risk of developing lethal ventricular arrhythmias such as Torsade de Pointes and ventricular fibrillation. In addition to congenital long QT, acquired long QT can also constitute a safety risk. Especially unintended inhibition of the hERG1 channel constitutes a major concern in the development of new drugs. Based on this knowledge is has been speculated whether activation of the hERG1 channel could be anti-arrhythmic and thereby constitute a new a principle in treatment of cardiac arrhythmogenic disorders. The first hERG1 channel agonist was reported 4 years ago and a limit number of this new compound class is now available. The present talk will illustrate how a research program aiming at developing a new principle for anti-arrhythmic treatment can be accomplished. Results will include data from in vitro to in vivo. The biophysical mode of action for hERG1 channel activators will be revealed by different electrophysiological experiments after heterologous expression in both Xenopus laevis oocytes and mammalian cells. Patch clamp experiments will further be included in characterization of hERG1 channel activators using native cardiomyocytes. Finally, the physiological and anti-arrhythmic properties of hERG1 channel activators will be demonstrated in isolated hearts and in different in vivo models.

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"Understanding Dopamine Transmission through Protein-Protein Interactions"

Our research group focuses on how neurons in the brain regulate dopamine homeostasis and the role of neurotransmitter transporters. Transmitter re-uptake through plasma membrane transporters is crucial in terminating synaptic transmission and ensuring that vesicular pools of transmitter are available for subsequent release. The dopamine transporter (DAT) is the main target site for major psychostimulants and for drugs used to treat mental illnesses. We have revealed the critical role of protein-protein interactions in the cell biology of DAT. These findings included the role of the PDZ synaptic protein PICK1 in targeting DAT to presynaptic terminals; the role of the adaptor protein Hic-5 in DAT trafficking; and the identification of a domain involved in DAT oligomerization. Recently, we have discovered a physical and functional link between DAT and synaptic vesicles through an interaction with the synaptic vesicle protein synaptogyrin-3. Our findings suggest that this novel interaction facilitates synaptic vesicle docking at the plasma membrane near DAT to provide efficient vesicle loading of extracellular dopamine during the reuptake process. These findings may represent a novel target for the actions of psychostimulants.

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"Amino acid transport across epithelia and blood brain barrier"

Small intestine and kidney proximal tubule epithelia are important cellular barriers where transcellular (re)absorption of amino acid takes place. The defect of the luminal sodium-dependent amino acid transporter B0AT1 (SLC6A19) causes Hartnup disorder. This transporter and other members of the SLC6 family require for their efficient surface expression in kidney and intestine association with collectrin (Tmem27) or ACE2, respectively. Since some mutations of B0AT1 differentially interact with collectrin and ACE2, a new mechanism leading the variable phenotype of Hartnup disorder is proposed. The closely related and previously orphan transporter XT2 (SC6A18) is shown to function as a higher affinity neutral amino acid transporter and to localize to the late kidney proximal tubule. We thus propose to rename it B0AT3. The basolateral amino acid transport machinery of these epithelia is less well understood and involves the interplay of exchangers and facilitated diffusion pathways such that it can fulfil, next to the efflux function, a role for specialized metabolic tasks and cellular housekeeping. A similarly complex task is that of the brain microvascular endothelial cells of the blood brain barrier across which an amino acid concentration gradient is also maintained.

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"Molecular aspects of coupled, uncoupled and leak currents in glutamate and dopamine transporters"

L-glutamic acid (glutamate) is a ubiquitous metabolite from the mitochondria and a neu-rotransmitter in neurons that express vesicular glutamate transporters (vGLUTs). Dopa-mine on the other hand is a neurotransmitter in those neurons that contains tyrosine hy-droxylase and vesicular monoamine transporters (vMATs). vGLUT and vMAT accumulate their substrate into synaptic vesicles, which upon fusion with the pre-synaptic membrane release the neurotransmitter into the perisynaptic space. The released neurotransmitter elicits a postsynaptic response through its pre- and post-synaptic receptors. Excitatory amino acid transporters (EAATs) and the dopamine transporter (DAT) remove the re-leased glutamate and dopamine, respectively, whereby they modulate the postsynaptic response. The plasmamembrane localized EAATs utilize the electrochemical energy from the sodium and potassium gradients created by Na+-K+-ATPase. In each stoichiometric, i.e. coupled, cycle the EAATs translocate one glutamate together with one proton and three sodium ions in exchange for one potassium ion. In the presence of glutamate EAATs also mediate a chloride flux, that does not influence the coupled cycle, and is therefore termed uncou-pled. We investigated and characterized the coupled and uncoupled fluxes as part of structure-function investigations aimed at understanding the molecular basis of the cation dependence of EAATs. In contrast to glutamate transporters, DAT utilizes only the electrochemical energy from the sodium gradient created by the Na+-K+-ATPase. DATs translocate one dopamine to-gether with two sodium and one chloride ion and results in the coupled, i.e. stoichiomet-ric, current. Human DAT is the best characterized of the DATs and mediates a leak cur-rent in absence of dopamine and a poorly characterized uncoupled chloride current in presence of dopamine. I will present some recent unpublished electrophysiological data on the functional differences, and similarities, between species homologues of DAT with special focus on the leak and uncoupled currents.

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"Targeting of the peroxisomal ABC-transporter ALDP"

Peroxisomes are ubiquitous, single membrane bound organelles, which exert a variety of metabolic reactions such as the degradation of hydrogen peroxide and of various fatty acids, or the biosynthesis of plasmalogens, docosahexaenoic acid and bile acids. Perox-isomal membrane proteins are assumed to be imported from the cytosol and the respon-sible targeting signals are usually recognized by the protein PEX19. The peroxisomal membrane protein ALDP belongs to the family of ABC-transporter proteins and mutations in this protein are associated with the neurodegenerative disease X-linked adrenoleu-kodystrophy (X-ALD). ALDP harbours a peroxisomal targeting signal and a binding site for PEX19 was identified. However, we found that mutations that destroy this interaction do not block the transport of ALDP to peroxisomes. Further investigations of the targeting process of ALDP revealed another relevant sequence and improve the understanding of the transport of peroxisomal membrane proteins.

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"Sert gene variation and risk for emotional disorders - contributions from rodent studies"

Identifying biological mechanisms through which genes lead to individual differences in emotional behavior is paramount to our understanding of how such differences confer risk for neuropsychiatric illness. The emergence of techniques such as in vivo imaging of brain function in humans and genetic engineering in rodents has provided important new in-sights into the impact of serotonin (5-HT), a key modulator of emotional behavior, on neural systems subserving anxiety and depression. A major finding has been the discov-ery of genetic variation in a crucial regulatory molecule within the 5-HT system, the 5HT transporter (5-HTT), and its influence on emotional traits. The study of the 5-HTT pro-vides a new foundation for understanding the neurobiological and genetic basis of emo-tional regulation and affective illness.

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"ABC transporters in Cancer drug resistance and ADME-tox"

Chemotherapy resistance is a recurrent problem, and the mechanism of resistance should be clarified in order to help further drug development. The active extrusion of several therapeutic agents by the MDR-ABC transporters should be considered as a potential cause of clinical resistance. The three major types of multidrug resistance (MDR) transporter proteins in humans include members of the ABCB, the ABCC, and the ABCG subfamily. These pumps recognize a wide range of drug substrates, mostly hydrophobic compounds, but also a variety of amphipathic anions and cations. MDR-ABC proteins play an important role in cancer drug resistance, but also in the absorption, distribution, metabolism and toxicity (ADME-tox) of several pharmaceutical agents. This concept is summarized as a "chemoimmunity" network, protecting our body against hydrophobic toxic agents.
This talk will discuss the role of MDR-ABC transporters in ADME-Tox and in drug resistance phenomena, focusing on the potential role of the ABCG2 transporter. This protein provides tissue protection against numerous toxic compounds and accounts for multidrug-resistant phenotype in various cancer cells.
Selective tyrosine kinase inhibitors (TKI) are key agents in modern cancer chemotherapy, and combination of TKIs with classical chemotherapeutic drugs may help to overcome currently untreatable metastatic cancers. However, ABCG2 displays a high affinity interaction with several clinically effective small molecule tyrosine kinase inhibitors, including Iressa (Gefitinib), an inhibitor of EGF-receptor dependent signaling, or Imatinib (Glivec) that inhibits the Bcr-Abl fusion protein, the molecular basis of chronic myeloid leukemia (CML). Recently we investigated the interaction of ABCG2 with several second generation Bcr-Abl inhibitors, already in clinical trials. Our data suggest that TKI interactions with ABC transporters may modulate drug metabolism, including the fate of diverse, chemically or target-wise unrelated drugs. These effects are based on multiple forms of MDR-ABC transporter interactions with TKIs, as these compounds may be both substrates and/or inhibitors of an ABC transporter. These interactions should be carefully considered in clinical application. Also, a combined MDR-ABC transporter and TKI effect may bring an advantage in future drug development.

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"Monoamine transporters as the targets of psychostimulants"

Cocaine is a powerful psychostimulant and an addictive drug of abuse. There are three known high-affinity targets for cocaine: the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Decades of studies sup-port the dopamine (DA) hypothesis that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate cocaine reward and reinforcement. Contrary to ex-pectations, DAT knockout mice (DAT-KO), and SERT or NET knockout mice still display the rewarding property of cocaine. These studies indicate that none of these transporters are required for the cocaine effects and led to the re-evaluation of the DA hypothesis and the proposal of redundant reward pathways. However, the knockout mice have very sig-nificant adaptive changes during development to compensate for the complete absence of a critical protein, which might have altered how cocaine produces its effects in these mice. Therefore, we have engineered a functional but cocaine-insensitive mutant of DAT and generated a knock-in mouse line carrying this DAT mutant (DAT-CI mice). Normal doses of cocaine still block SERT and NET but have little effect on DAT in these mice which provide a unique tool to study the role of DAT in mediating cocaine effects. In DAT-CI mice, cocaine did not elevate extracellular DA in the nucleus accumbens (NAc), cocaine did not stimulate locomotor activity but suppressed it, and cocaine failed to pro-duce reward as measured by conditioned place preference and by drug self administra-tion. In contrast, amphetamine, another psychostimulant, was able to stimulate locomo-tor activity and produce reward, indicating that the reward system functioned well in these mice. In addition, re-introducing wild type DAT back into the brains of fully devel-oped DAT-CI mice restored the ability of cocaine to stimulate locomotor activity and to produce conditioned place preference. Our results indicate that cocaine blockade of DAT is required for the stimulating and rewarding effects of cocaine in mice with a functional DAT. Furthermore, we are now in the process of generating mouse lines carry a cocaine-resistant SERT or NET mutant to study the roles of these transporters in the complex co-caine effects.

 

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"The Binding Site for Dopamine and Cocaine on the Dopamine Transporter are Overlapping"

Cocaine is a widely abused substance with psychostimulant effects attributed to inhibition of the dopamine transporter. It has been debated whether or not the cocaine binding site overlaps with that of dopamine. If the binding sites were separate, it would be possible, at least in theory, to generate a cocaine antidote that did not influence the transport of dopamine. Here we present molecular docking models of the dopamine- and cocaine binding site showing an almost complete overlap. The models were validated experimentally using site-directed mutagenesis, Zn2+-site engineering, and chemical cross-linkers. These data argue against the possibility of creating such a cocaine antagonist. However, there might be hope for the cocaine addicts after all...

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"Regulation of the serotonin transporter by interacting proteins"

The neurotransmitter serotonin (5-hydroxytrytamine, 5-HT) exerts broad modulatory con-trol over many fundamental aspects of physiology and behavior. Alterations in whole blood 5-HT levels are associated with a variety of neuropsychiatric disorders, suggesting that conserved molecular mechanisms regulate 5-HT homeostasis in brain and blood. One such example is the serotonin transporter (SERT), responsible for the maintenance of 5-HT levels in both the brain and in platelets. Peripheral findings suggest that genetic varia-tion impacting both platelet and brain 5HT levels contributes to multiple brain disorders and may underlie comorbidity with peripheral symptoms. We recently characterized a number of regulatory partners that dictate SERT trafficking and function, including in-tegrins and focal adhesion proteins. Hic-5 is an adaptor protein responsible for the mo-lecular organization of signaling complexes downstream of integrin activation. Hic-5 bind-ing to the carboxy-terminal of SERT leads to inactivation and internalization of the trans-porter. The interaction between SERT and Hic-5 is regulated by intracellular signaling pathways and 5HT availability. Integrins are cell surface receptors that interact with ex-tracellular matrix proteins and mediate the signal transduction cascades necessary for cell motility and shape change. Activation and clustering of integrins activate SERT transport activity through previously characterized p38MAPK-dependent mechanisms. Importantly, integrin genetic variation associated with 5HT blood levels also influence SERT uptake ac-tivity. These are but two examples of SERT-interacting proteins that dynamically interact and regulate transport function that are conserved in platelets and in the brain. Our latest efforts aim to parallel changes in 5HT signaling in adult brain with altered 5HT blood ho-meostasis and map the molecular changes in SERT interactions in disease. These studies reveal a framework for understanding the comorbidity of depression and cardiovascular disease.

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