The Experts below are selected from a list of 5337 Experts worldwide ranked by ideXlab platform
Petrine Wellendorph - One of the best experts on this subject based on the ideXlab platform.
-
exploring the molecular determinants for subtype selectivity of 2 amino 1 4 5 6 tetrahydropyrimidine 5 carboxylic acid analogs as betaine GABA Transporter 1 bgt1 substrate inhibitors
Scientific Reports, 2020Co-Authors: Stefanie Kickinger, Maria E K Lie, Gerhard F Ecker, Bente Frolund, Anas Alkhawaja, Anne S Haugaard, Rebekka Loffler, Maria Damgaard, Francesco Bavo, Petrine WellendorphAbstract:We have previously identified 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) as the most potent substrate-inhibitor of the betaine/GABA Transporter 1 (BGT1) (IC50 2.5 µM) reported to date. Herein, we characterize the binding mode of 20 novel analogs and propose the molecular determinants driving BGT1-selectivity. A series of N1-, exocyclic-N-, and C4-substituted analogs was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA Transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogs retained subtype-selectivity for hBGT1, though with lower inhibitory activities (mid to high micromolar IC50 values) compared to ATPCA. Further characterization of five of these BGT1-active analogs in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting they interact with the orthosteric site of the Transporter. In silico-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 as well as the conformational flexibility of the compounds potentially contribute to the subtype-selectivity of ATPCA and its analogs. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors. The findings may guide the rational design of BGT1-selective pharmacological tool compounds for future drug discovery.
-
pharmacological characterization of a betaine GABA Transporter 1 bgt1 inhibitor displaying an unusual biphasic inhibition profile and anti seizure effects
Neurochemical Research, 2020Co-Authors: Maria E K Lie, Arne Schousboe, Gerhard F Ecker, Stefanie Kickinger, Rasmus P. Clausen, Jonas Skovgaardpetersen, Steve H White, Petrine WellendorphAbstract:Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA Transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA Transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1R,2S)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 values (4.7 and 556 μM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.
-
structural and molecular aspects of betaine GABA Transporter 1 bgt1 and its relation to brain function
Neuropharmacology, 2019Co-Authors: Stefanie Kickinger, Arne Schousboe, Gerhard F Ecker, Eva Hellsberg, Bente Frolund, Petrine WellendorphAbstract:ɣ-aminobutyric-acid (GABA) functions as the principal inhibitory neurotransmitter in the central nervous system. Imbalances in GABAergic neurotransmission are involved in the pathophysiology of various neurological diseases such as epilepsy, Alzheimer's disease and stroke. GABA Transporters (GATs) facilitate the termination of GABAergic signaling by transporting GABA together with sodium and chloride from the synaptic cleft into presynaptic neurons and surrounding glial cells. Four different GATs have been identified that all belong to the solute carrier 6 (SLC6) Transporter family: GAT1-3 (SLC6A1, SLC6A13, SLC6A11) and betaine/GABA Transporter 1 (BGT1, SLC6A12). BGT1 has emerged as an interesting target for treating epilepsy due to animal studies that reported anticonvulsant effects for the GAT1/BGT1 selective inhibitor EF1502 and the BGT1 selective inhibitor RPC-425. However, the precise involvement of BGT1 in epilepsy remains elusive because of its controversial expression levels in the brain and the lack of highly selective and potent tool compounds. This review gathers the current structural and functional knowledge on BGT1 with emphasis on brain relevance, discusses all available compounds, and tries to shed light on the molecular determinants driving BGT1 selectivity. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
-
pharmacological characterization of 3h atpca as a substrate for studying the functional role of the betaine GABA Transporter 1 and the creatine Transporter
ACS Chemical Neuroscience, 2017Co-Authors: Anas Alkhawaja, Bente Frolund, Anne S Haugaard, Ales Marek, Rebekka Loffler, Louise Thiesen, Monica Santiveri, Maria Damgaard, Christoffer Bundgaard, Petrine WellendorphAbstract:The betaine/γ-aminobutyric acid (GABA) Transporter 1 (BGT1) is one of the four GABA Transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABAA receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [3H]ATPCA. Using traditional uptake assays at recombinant Transporters expressed in cell lines, [3H]ATPCA displayed a striking selectivity for BGT1 among the four GATs (Km and Vmax values of 21 μM and 3.6 nmol ATPCA/(min × mg protein), respectivel...
-
Pharmacological Characterization of [3H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter
2017Co-Authors: Anas Al-khawaja, Bente Frolund, Anne S Haugaard, Ales Marek, Louise Thiesen, Monica Santiveri, Maria Damgaard, Christoffer Bundgaard, Rebekka Löffler, Petrine WellendorphAbstract:The betaine/γ-aminobutyric acid (GABA) Transporter 1 (BGT1) is one of the four GABA Transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABAA receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [3H]ATPCA. Using traditional uptake assays at recombinant Transporters expressed in cell lines, [3H]ATPCA displayed a striking selectivity for BGT1 among the four GATs (Km and Vmax values of 21 μM and 3.6 nmol ATPCA/(min × mg protein), respectively), but was also found to be a substrate for the creatine Transporter (CreaT). In experiments with mouse cortical cell cultures, we observed a Na+-dependent [3H]ATPCA uptake in neurons, but not in astrocytes. The neuronal uptake could be inhibited by GABA, ATPCA, and a noncompetitive BGT1-selective inhibitor, indicating functional BGT1 in neurons. In conclusion, we report [3H]ATPCA as a novel radioactive substrate for both BGT1 and CreaT. The dual activity of the radioligand makes it most suitable for use in recombinant studies
Michael Freissmuth - One of the best experts on this subject based on the ideXlab platform.
-
switching the clientele a lysine residing in the c terminus of the serotonin Transporter specifies its preference for the coat protein complex ii component sec24c
Journal of Biological Chemistry, 2013Co-Authors: Sonja Sucic, Harald H Sitte, Oliver Kudlacek, Ali Elkasaby, Florian Koban, Thomas Stockner, Michael FreissmuthAbstract:The serotonin Transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the coat protein complex II component SEC24C for endoplasmic reticulum (ER) export. The closely related Transporters for noradrenaline and dopamine depend on SEC24D. Here, we show that discrimination between SEC24C and SEC24D is specified by the residue at position +2 downstream from the ER export motif ((607)RI(608) in SERT). Substituting Lys(610) with tyrosine, the corresponding residue found in the noradrenaline and dopamine Transporters, switched the SEC24 isoform preference: SERT-K610Y relied solely on SEC24D to reach the cell surface. This analysis was extended to other SLC6 (solute carrier 6) Transporter family members: siRNA-dependent depletion of SEC24C, but not of SEC24D, reduced surface levels of the glycine Transporter-1a, the betaine/GABA Transporter and the GABA Transporter-4. Experiments with dominant negative versions of SEC24C and SEC24D recapitulated these findings. We also verified that the presence of two ER export motifs (in concatemers of SERT and GABA Transporter-1) supported recruitment of both SEC24C and SEC24D. To the best of our knowledge, this is the first report to document a change in SEC24 specificity by mutation of a single residue in the client protein. Our observations allowed for deducing a rule for SLC6 family members: a hydrophobic residue (Tyr or Val) in the +2 position specifies interaction with SEC24D, and a hydrophilic residue (Lys, Asn, or Gln) recruits SEC24C. Variations in SEC24C are linked to neuropsychiatric disorders. The present findings provide a mechanistic explanation. Variations in SEC24C may translate into distinct surface levels of neurotransmitter Transporters.
-
the serotonin Transporter is an exclusive client of the coat protein complex ii copii component sec24c
Journal of Biological Chemistry, 2011Co-Authors: Sonja Sucic, Harald H Sitte, Oliver Kudlacek, Ali Elkasaby, Subhodeep Sarker, Philippe Marin, Michael FreissmuthAbstract:Abstract The Transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif (607RI608) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine Transporters and the more distantly related GABA Transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related Transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders.
-
sec24 and arfgap1 dependent trafficking of GABA Transporter 1 is a prerequisite for correct axonal targeting
The Journal of Neuroscience, 2008Co-Authors: Veronika Reiterer, Susanne Maier, Harald H Sitte, Alexander Kriz, Markus A Ruegg, Hanspeter Hauri, Michael Freissmuth, Hesso FarhanAbstract:The GABA Transporter-1 (GAT1) is a prototypical protein of the synaptic specialization. Export of GAT1 from the endoplasmic reticulum (ER) is contingent on its interaction with the COPII (coatomer protein-II) coat subunit Sec24D. Here we show that silencing all four Sec24 isoforms strongly inhibits transport of GAT1 to the cell surface. In contrast, transport of GAT1-RL/AS, a mutant that is deficient in Sec24D recruitment, was not inhibited, suggesting a nonconventional, COPII-independent pathway. However, ARFGAP1 bound directly to the C terminus of both GAT1-RL/AS and wild-type GAT1. Surface expression of GAT1-RL/AS involved ARFGAP1. GAT1-RL/AS appeared to bypass the ER-Golgi-intermediate compartment, but its pathway to the plasma membrane still involved passage through the Golgi. Thus, the GAT1-RL/AS mutant allowed to test whether COPII-dependent ER-export is required for correct sorting of GAT1 to the axon terminal in neuronal cells. In contrast to wild-type GAT1, GAT1-RL/AS failed to be specifically enriched at the tip of neurite extensions of CAD.a cells (a neuroblastoma cell line that can be differentiated into a neuron-like phenotype) and in the axon terminals of hippocampal neurons. These findings indicate that correct sorting to the axon is contingent on ER export via the COPII machinery and passage through the ER-Golgi-intermediate compartment.
-
signal dependent export of GABA Transporter 1 from the er golgi intermediate compartment is specified by a c terminal motif
Journal of Cell Science, 2008Co-Authors: Hesso Farhan, Veronika Reiterer, Harald H Sitte, Alexander Kriz, Hanspeter Hauri, Margit Pavelka, Michael FreissmuthAbstract:The C-terminus of GABA Transporter 1 (GAT1, SLC6A1) is required for trafficking of the protein through the secretory pathway to reach its final destination, i.e. the rim of the synaptic specialization. We identified a motif of three hydrophobic residues (569VMI571) that was required for export of GAT1 from the ER-Golgi intermediate compartment (ERGIC). This conclusion was based on the following observations: (i) GAT1-SSS, the mutant in which 569VMI571 was replaced by serine residues, was exported from the ER in a COPII-dependent manner but accumulated in punctate structures and failed to reach the Golgi; (ii) under appropriate conditions (imposing a block at 15°C, disruption of COPI), these structures also contained ERGIC53; (iii) the punctae were part of a dynamic compartment, because it was accessible to a second anterograde cargo [the temperature-sensitive variant of vesicular stomatitis virus G protein (VSV-G)] and because GAT1-SSS could be retrieved from the punctate structures by addition of a KKxx-based retrieval motif, which supported retrograde transport to the ER. To the best of our knowledge, the VMI-motif of GAT1 provides the first example of a cargo-based motif that specifies export from the ERGIC.
-
mutations within an intramembrane leucine heptad repeat disrupt oligomer formation of the rat GABA Transporter 1
Journal of Biological Chemistry, 2002Co-Authors: Petra Scholze, Michael Freissmuth, Harald H SitteAbstract:Abstract Na+/Cl−-dependent neurotransmitter Transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat γ-aminobutyric acid Transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of Forster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane.
Martin E Keck - One of the best experts on this subject based on the ideXlab platform.
-
long term anxiolytic and antidepressant like behavioural effects of tiagabine a selective GABA Transporter 1 gat 1 inhibitor coincide with a decrease in hpa system activity in c57bl 6 mice
Journal of Psychopharmacology, 2010Co-Authors: Christoph K Thoeringer, Florian Holsboer, Angelika Erhardt, Inge Sillaber, M. Mueller, Martin E KeckAbstract:Gamma-aminobutyric acid (GABA) system plays a pivotal role in the pathophysiology of anxiety and mood disorders. This study was aimed to assess the anxiolytic and antidepressant-like properties of tiagabine, an inhibitor of the GABA Transporter-1 (GAT-1), after acute and chronic administration in C57BL/6JOlaHsD mice with paroxetine as a positive control. In first experiments, the acute administration of tiagabine (7.5 mg/kg, orally [PO]) and paroxetine (10 mg/kg PO) induced anxiolytic effects in the elevated plus maze test and the modified hole board test and an antidepressant-like effect in the forced swim test. Chronic application of tiagabine (7.5 mg/kg PO) and paroxetine (10 mg/kg PO) for 22 days revealed an anxiolytic and antidepressant-like efficacy of tiagabine only. In a further experiment, we analysed the impact of chronic tiagabine versus paroxetine treatment on the hypothalamic-pituitary-adrenocortical (HPA) system regulation. GAT-1 blockade induced a setpoint-shift of the stress hormone system...
-
long term anxiolytic and antidepressant like behavioural effects of tiagabine a selective GABA Transporter 1 gat 1 inhibitor coincide with a decrease in hpa system activity in c57bl 6 mice
Journal of Psychopharmacology, 2010Co-Authors: Christoph K Thoeringer, Florian Holsboer, Angelika Erhardt, Inge Sillaber, M. Mueller, Frauke Ohl, Martin E KeckAbstract:Gamma-aminobutyric acid (GABA) system plays a pivotal role in the pathophysiology of anxiety and mood disorders. This study was aimed to assess the anxiolytic and antidepressant-like properties of tiagabine, an inhibitor of the GABA Transporter-1 (GAT-1), after acute and chronic administration in C57BL/6JOlaHsD mice with paroxetine as a positive control. In first experiments, the acute administration of tiagabine (7.5 mg/kg, orally [PO]) and paroxetine (10 mg/kg PO) induced anxiolytic effects in the elevated plus maze test and the modified hole board test and an antidepressant-like effect in the forced swim test. Chronic application of tiagabine (7.5 mg/kg PO) and paroxetine (10 mg/kg PO) for 22 days revealed an anxiolytic and antidepressant-like efficacy of tiagabine only. In a further experiment, we analysed the impact of chronic tiagabine versus paroxetine treatment on the hypothalamic-pituitary-adrenocortical (HPA) system regulation. GAT-1 blockade induced a setpoint-shift of the stress hormone system toward lower levels as indicated by decreased plasma corticosterone concentrations and attenuated gene expression levels of corticotropin-releasing factor in the paraventricular nucleus of the hypothalamus and of hippocampal steroid receptors. This data indicate that both acute and long-term anxiolytic and antidepressant-like properties of brain GAT-1 inhibition coincide with a reduction in HPA system activity in mice.
Stefanie Kickinger - One of the best experts on this subject based on the ideXlab platform.
-
exploring the molecular determinants for subtype selectivity of 2 amino 1 4 5 6 tetrahydropyrimidine 5 carboxylic acid analogs as betaine GABA Transporter 1 bgt1 substrate inhibitors
Scientific Reports, 2020Co-Authors: Stefanie Kickinger, Maria E K Lie, Gerhard F Ecker, Bente Frolund, Anas Alkhawaja, Anne S Haugaard, Rebekka Loffler, Maria Damgaard, Francesco Bavo, Petrine WellendorphAbstract:We have previously identified 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) as the most potent substrate-inhibitor of the betaine/GABA Transporter 1 (BGT1) (IC50 2.5 µM) reported to date. Herein, we characterize the binding mode of 20 novel analogs and propose the molecular determinants driving BGT1-selectivity. A series of N1-, exocyclic-N-, and C4-substituted analogs was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA Transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogs retained subtype-selectivity for hBGT1, though with lower inhibitory activities (mid to high micromolar IC50 values) compared to ATPCA. Further characterization of five of these BGT1-active analogs in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting they interact with the orthosteric site of the Transporter. In silico-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 as well as the conformational flexibility of the compounds potentially contribute to the subtype-selectivity of ATPCA and its analogs. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors. The findings may guide the rational design of BGT1-selective pharmacological tool compounds for future drug discovery.
-
pharmacological characterization of a betaine GABA Transporter 1 bgt1 inhibitor displaying an unusual biphasic inhibition profile and anti seizure effects
Neurochemical Research, 2020Co-Authors: Maria E K Lie, Arne Schousboe, Gerhard F Ecker, Stefanie Kickinger, Rasmus P. Clausen, Jonas Skovgaardpetersen, Steve H White, Petrine WellendorphAbstract:Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA Transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA Transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1R,2S)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 values (4.7 and 556 μM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.
-
structural and molecular aspects of betaine GABA Transporter 1 bgt1 and its relation to brain function
Neuropharmacology, 2019Co-Authors: Stefanie Kickinger, Arne Schousboe, Gerhard F Ecker, Eva Hellsberg, Bente Frolund, Petrine WellendorphAbstract:ɣ-aminobutyric-acid (GABA) functions as the principal inhibitory neurotransmitter in the central nervous system. Imbalances in GABAergic neurotransmission are involved in the pathophysiology of various neurological diseases such as epilepsy, Alzheimer's disease and stroke. GABA Transporters (GATs) facilitate the termination of GABAergic signaling by transporting GABA together with sodium and chloride from the synaptic cleft into presynaptic neurons and surrounding glial cells. Four different GATs have been identified that all belong to the solute carrier 6 (SLC6) Transporter family: GAT1-3 (SLC6A1, SLC6A13, SLC6A11) and betaine/GABA Transporter 1 (BGT1, SLC6A12). BGT1 has emerged as an interesting target for treating epilepsy due to animal studies that reported anticonvulsant effects for the GAT1/BGT1 selective inhibitor EF1502 and the BGT1 selective inhibitor RPC-425. However, the precise involvement of BGT1 in epilepsy remains elusive because of its controversial expression levels in the brain and the lack of highly selective and potent tool compounds. This review gathers the current structural and functional knowledge on BGT1 with emphasis on brain relevance, discusses all available compounds, and tries to shed light on the molecular determinants driving BGT1 selectivity. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
-
structure activity relationship pharmacological characterization and molecular modeling of noncompetitive inhibitors of the betaine γ aminobutyric acid Transporter 1 bgt1
Journal of Medicinal Chemistry, 2017Co-Authors: Lars N Jorgensen, Stefanie Kickinger, Jonas Skovgaardpetersen, Anas Alkhawaja, Rebekka Loffler, Karsten K Madsen, Stine B Vogensen, Emil Rosenthal, Nrupa Borkar, Hans BraunerosborneAbstract:N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a noncompetitive inhibitor of the betaine/GABA Transporter 1 (BGT1). We here report the synthesis and structure–activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated Transporters, and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin Transporter (hSERT). On the basis of these experiments, we propose a binding mode involving residues within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel noncom...
-
Structure–Activity Relationship, Pharmacological Characterization, and Molecular Modeling of Noncompetitive Inhibitors of the Betaine/γ-Aminobutyric Acid Transporter 1 (BGT1)
2017Co-Authors: Lars Jørgensen, Stefanie Kickinger, Karsten K Madsen, Stine B Vogensen, Anas Al-khawaja, Rebekka Löffler, Jonas Skovgaard-petersen, Emil Rosenthal, Nrupa Borkar, Hans Bräuner-osborneAbstract:N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a noncompetitive inhibitor of the betaine/GABA Transporter 1 (BGT1). We here report the synthesis and structure–activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated Transporters, and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin Transporter (hSERT). On the basis of these experiments, we propose a binding mode involving residues within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel noncompetitive inhibitors
Harald H Sitte - One of the best experts on this subject based on the ideXlab platform.
-
switching the clientele a lysine residing in the c terminus of the serotonin Transporter specifies its preference for the coat protein complex ii component sec24c
Journal of Biological Chemistry, 2013Co-Authors: Sonja Sucic, Harald H Sitte, Oliver Kudlacek, Ali Elkasaby, Florian Koban, Thomas Stockner, Michael FreissmuthAbstract:The serotonin Transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the coat protein complex II component SEC24C for endoplasmic reticulum (ER) export. The closely related Transporters for noradrenaline and dopamine depend on SEC24D. Here, we show that discrimination between SEC24C and SEC24D is specified by the residue at position +2 downstream from the ER export motif ((607)RI(608) in SERT). Substituting Lys(610) with tyrosine, the corresponding residue found in the noradrenaline and dopamine Transporters, switched the SEC24 isoform preference: SERT-K610Y relied solely on SEC24D to reach the cell surface. This analysis was extended to other SLC6 (solute carrier 6) Transporter family members: siRNA-dependent depletion of SEC24C, but not of SEC24D, reduced surface levels of the glycine Transporter-1a, the betaine/GABA Transporter and the GABA Transporter-4. Experiments with dominant negative versions of SEC24C and SEC24D recapitulated these findings. We also verified that the presence of two ER export motifs (in concatemers of SERT and GABA Transporter-1) supported recruitment of both SEC24C and SEC24D. To the best of our knowledge, this is the first report to document a change in SEC24 specificity by mutation of a single residue in the client protein. Our observations allowed for deducing a rule for SLC6 family members: a hydrophobic residue (Tyr or Val) in the +2 position specifies interaction with SEC24D, and a hydrophilic residue (Lys, Asn, or Gln) recruits SEC24C. Variations in SEC24C are linked to neuropsychiatric disorders. The present findings provide a mechanistic explanation. Variations in SEC24C may translate into distinct surface levels of neurotransmitter Transporters.
-
the serotonin Transporter is an exclusive client of the coat protein complex ii copii component sec24c
Journal of Biological Chemistry, 2011Co-Authors: Sonja Sucic, Harald H Sitte, Oliver Kudlacek, Ali Elkasaby, Subhodeep Sarker, Philippe Marin, Michael FreissmuthAbstract:Abstract The Transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif (607RI608) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine Transporters and the more distantly related GABA Transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related Transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders.
-
sec24 and arfgap1 dependent trafficking of GABA Transporter 1 is a prerequisite for correct axonal targeting
The Journal of Neuroscience, 2008Co-Authors: Veronika Reiterer, Susanne Maier, Harald H Sitte, Alexander Kriz, Markus A Ruegg, Hanspeter Hauri, Michael Freissmuth, Hesso FarhanAbstract:The GABA Transporter-1 (GAT1) is a prototypical protein of the synaptic specialization. Export of GAT1 from the endoplasmic reticulum (ER) is contingent on its interaction with the COPII (coatomer protein-II) coat subunit Sec24D. Here we show that silencing all four Sec24 isoforms strongly inhibits transport of GAT1 to the cell surface. In contrast, transport of GAT1-RL/AS, a mutant that is deficient in Sec24D recruitment, was not inhibited, suggesting a nonconventional, COPII-independent pathway. However, ARFGAP1 bound directly to the C terminus of both GAT1-RL/AS and wild-type GAT1. Surface expression of GAT1-RL/AS involved ARFGAP1. GAT1-RL/AS appeared to bypass the ER-Golgi-intermediate compartment, but its pathway to the plasma membrane still involved passage through the Golgi. Thus, the GAT1-RL/AS mutant allowed to test whether COPII-dependent ER-export is required for correct sorting of GAT1 to the axon terminal in neuronal cells. In contrast to wild-type GAT1, GAT1-RL/AS failed to be specifically enriched at the tip of neurite extensions of CAD.a cells (a neuroblastoma cell line that can be differentiated into a neuron-like phenotype) and in the axon terminals of hippocampal neurons. These findings indicate that correct sorting to the axon is contingent on ER export via the COPII machinery and passage through the ER-Golgi-intermediate compartment.
-
signal dependent export of GABA Transporter 1 from the er golgi intermediate compartment is specified by a c terminal motif
Journal of Cell Science, 2008Co-Authors: Hesso Farhan, Veronika Reiterer, Harald H Sitte, Alexander Kriz, Hanspeter Hauri, Margit Pavelka, Michael FreissmuthAbstract:The C-terminus of GABA Transporter 1 (GAT1, SLC6A1) is required for trafficking of the protein through the secretory pathway to reach its final destination, i.e. the rim of the synaptic specialization. We identified a motif of three hydrophobic residues (569VMI571) that was required for export of GAT1 from the ER-Golgi intermediate compartment (ERGIC). This conclusion was based on the following observations: (i) GAT1-SSS, the mutant in which 569VMI571 was replaced by serine residues, was exported from the ER in a COPII-dependent manner but accumulated in punctate structures and failed to reach the Golgi; (ii) under appropriate conditions (imposing a block at 15°C, disruption of COPI), these structures also contained ERGIC53; (iii) the punctae were part of a dynamic compartment, because it was accessible to a second anterograde cargo [the temperature-sensitive variant of vesicular stomatitis virus G protein (VSV-G)] and because GAT1-SSS could be retrieved from the punctate structures by addition of a KKxx-based retrieval motif, which supported retrograde transport to the ER. To the best of our knowledge, the VMI-motif of GAT1 provides the first example of a cargo-based motif that specifies export from the ERGIC.
-
mutations within an intramembrane leucine heptad repeat disrupt oligomer formation of the rat GABA Transporter 1
Journal of Biological Chemistry, 2002Co-Authors: Petra Scholze, Michael Freissmuth, Harald H SitteAbstract:Abstract Na+/Cl−-dependent neurotransmitter Transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat γ-aminobutyric acid Transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of Forster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane.
