The Experts below are selected from a list of 192 Experts worldwide ranked by ideXlab platform
Swen Hülsmann - One of the best experts on this subject based on the ideXlab platform.
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the postnatal development of ultrasonic vocalization associated breathing is altered in Glycine Transporter 2 deficient mice
The Journal of Physiology, 2019Co-Authors: Swen Hülsmann, Michal G Fortuna, Johannes Hirrlinger, Tobias A Latal, Yoshihiko Oke, Guillaume Mesuret, Marcus Niebert, J E Fischer, Kurt HammerschmidtAbstract:KEY POINTS Newborn mice produce ultrasonic vocalization to communicate with their mother. The neuronal Glycine Transporter (GlyT2) is required for efficient loading of synaptic vesicles in Glycinergic neurons. Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and the mice die in the second postnatal week. In the present study, we show that GlyT2-knockout mice do not acquire adult ultrasonic vocalization-associated breathing patterns. Despite the strong impairment of Glycinergic inhibition, they can produce sufficient expiratory airflow to produce ultrasonic vocalization. Because mouse ultrasonic vocalization is a valuable read-out in translational research, these data are highly relevant for a broad range of research fields. ABSTRACT Mouse models are instrumental with respect to determining the genetic basis and neural foundations of breathing regulation. To test the hypothesis that Glycinergic synaptic inhibition is required for normal breathing and proper post-inspiratory activity, we analysed breathing and ultrasonic vocalization (USV) patterns in neonatal mice lacking the neuronal Glycine Transporter (GlyT2). GlyT2-knockout (KO) mice have a profound reduction of Glycinergic synaptic currents already at birth, develop a severe motor phenotype and survive only until the second postnatal week. At this stage, GlyT2-KO mice are smaller, have a reduced respiratory rate and still display a neonatal breathing pattern with active expiration for the production of USV. By contrast, wild-type mice acquire different USV-associated breathing patterns that depend on post-inspiratory control of air flow. Nonetheless, USVs per se remain largely indistinguishable between both genotypes. We conclude that GlyT2-KO mice, despite the strong impairment of Glycinergic inhibition, can produce sufficient expiratory airflow to produce ultrasonic vocalization.
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Genetic ablation of VIAAT in Glycinergic neurons causes a severe respiratory phenotype and perinatal death
Brain structure & function, 2014Co-Authors: Jamilur Rahman, Christian Schnell, Johannes Hirrlinger, Volker Eulenburg, Stefanie Besser, Sonja M. Wojcik, Swen HülsmannAbstract:Both Glycinergic and GABAergic neurons require the vesicular inhibitory amino acid Transporter (VIAAT) for synaptic vesicle filling. Presynaptic GABA concentrations are determined by the GABA-synthesizing enzymes glutamate decarboxylase (GAD)65 and GAD67, whereas the presynaptic Glycine content depends on the plasma membrane Glycine Transporter 2 (GlyT2). Although severely impaired, Glycinergic transmission is not completely absent in GlyT2-knockout mice, suggesting that other routes of Glycine uptake or de novo synthesis of Glycine exist in presynaptic terminals. To investigate the consequences of a complete loss of Glycinergic transmission, we generated a mouse line with a conditional ablation of VIAAT in Glycinergic neurons by crossing mice with loxP-flanked VIAAT alleles with a GlyT2-Cre transgenic mouse line. Interestingly, conditional VIAAT knockout (VIAAT cKO) mice were not viable at birth. In addition to the dominant respiratory failure, VIAAT cKO showed an umbilical hernia and a cleft palate. Immunohistochemistry revealed an almost complete depletion of VIAAT in the brainstem. Electrophysiology revealed the absence of both spontaneous Glycinergic and GABAergic inhibitory postsynaptic currents from hypoglossal motoneurons. Our results demonstrate that the deletion of VIAAT in GlyT2-Cre expressing neurons also strongly affects GABAergic transmission and suggest a large overlap of the Glycinergic and the GABAergic neuron population during early development in the caudal parts of the brain.
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mixed miniature postsynaptic currents resulting from co release of Glycine and gaba recorded from Glycinergic neurons in the neonatal respiratory network
European Journal of Neuroscience, 2013Co-Authors: Jamilur Rahman, Johannes Hirrlinger, Tobias A Latal, Stefanie Besser, Swen HülsmannAbstract:Inhibitory neurons are involved in the generation and patterning of the respiratory rhythm in the adult animal. However, the role of Glycinergic neurons in the respiratory rhythm in the developing network is still not understood. Although the complete loss of Glycinergic transmission in vivo is lethal, the blockade of Glycinergic transmission in slices of the medulla has little effect on pre-Botzinger complex network activity. As 50% of the respiratory rhythmic neurons in this slice preparation are Glycinergic, they have to be considered as integrated parts of the network. We aimed to investigate whether Glycinergic neurons receive mixed miniature inhibitory postsynaptic currents (mIPSCs) that result from co-release of GABA and Glycine. Quantification of mixed mIPSCs by the use of different objective detection methods resulted in a wide range of results. Therefore, we generated traces of mIPSCs with a known distribution of mixed mIPSCs and mono-transmitter-induced mIPSCs, and tested the detection methods on the simulated data. We found that analysis paradigms, which are based on fitting the sum of two mIPSC templates, to be most acceptable. On the basis of these protocols, 20-40% of all mIPSCs recorded from respiratory Glycinergic neurons are mixed mIPSCs that result from co-release of GABA and Glycine. Furthermore, single-cell reverse transcriptase polymerase chain reaction revealed that 46% of Glycinergic neurons co-express mRNA of Glycine Transporter 2 together with at least one marker protein of GABAergic neurons. Our data suggest that significant co-transmission occurs in the pre-Botzinger complex that might be involved in the shaping of synaptic inhibition of respiratory Glycinergic neurons.
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development of synaptic inhibition in Glycine Transporter 2 deficient mice
Molecular and Cellular Neuroscience, 2010Co-Authors: Tobias A Latal, Volker Eulenburg, Thomas Kremer, Jesus Gomeza, Swen HülsmannAbstract:Abstract Mice deficient for the neuronal Glycine Transporter subtype 2 (GlyT2) die during the second postnatal week after developing neuromotor deficiencies, which resembles severe forms of human hyperekplexia. This phenotype has been attributed to a dramatic reduction in Glycinergic neurotransmission. In the present study we analyzed the development of GABAergic and Glycinergic synaptic transmission in GlyT2-knockout mice during early postnatal life. Anti-Glycine immunohistochemistry in spinal cord and brainstem slices and whole-cell voltage-clamp recordings of Glycinergic inhibitory postsynaptic currents (IPSCs) from hypoglossal motoneurons revealed strikingly reduced levels of synaptic Glycine already at birth. Since GABA and Glycine use the same vesicular inhibitory amino acid Transporter (VIAAT or VGAT) we also analysed GABAergic neurotransmission. No increase of GABA immunoreactivity was observed in the spinal cord and brainstem of GlyT2−/− mice at any stage of postnatal development. Correspondingly no up-regulation of GABAergic IPSCs was detected in GlyT2−/− hypoglossal motoneurons. These data suggest that in the first postnatal week, loss of the Glycine Transporter 2 is neither compensated by Glycine de-novo synthesis nor by up-regulation of the GABAergic transmission in GlyT2−/− mice.
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deletion of the mouse Glycine Transporter 2 results in a hyperekplexia phenotype and postnatal lethality
Neuron, 2003Co-Authors: Jesus Gomeza, Swen Hülsmann, Volker Eulenburg, Wencke Armsen, Koji Ohno, Diethelm W Richter, Bodo Laube, Heinrich BetzAbstract:The Glycine Transporter subtype 2 (GlyT2) is localized in the axon terminals of Glycinergic neurons. Mice deficient in GlyT2 are normal at birth but during the second postnatal week develop a lethal neuromotor deficiency that resembles severe forms of human hyperekplexia (hereditary startle disease) and is characterized by spasticity, tremor, and an inability to right. Histological and immunological analyses failed to reveal anatomical or biochemical abnormalities, but the amplitudes of Glycinergic miniature inhibitory currents (mIPSCs) were strikingly reduced in hypoglossal motoneurons and dissociated spinal neurons from GlyT2-deficient mice. Thus, postnatal GlyT2 function is crucial for efficient transmitter loading of synaptic vesicles in Glycinergic nerve terminals, and the GlyT2 gene constitutes a candidate disease gene in human hyperekplexia patients.
Volker Eulenburg - One of the best experts on this subject based on the ideXlab platform.
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Genetic ablation of VIAAT in Glycinergic neurons causes a severe respiratory phenotype and perinatal death
Brain structure & function, 2014Co-Authors: Jamilur Rahman, Christian Schnell, Johannes Hirrlinger, Volker Eulenburg, Stefanie Besser, Sonja M. Wojcik, Swen HülsmannAbstract:Both Glycinergic and GABAergic neurons require the vesicular inhibitory amino acid Transporter (VIAAT) for synaptic vesicle filling. Presynaptic GABA concentrations are determined by the GABA-synthesizing enzymes glutamate decarboxylase (GAD)65 and GAD67, whereas the presynaptic Glycine content depends on the plasma membrane Glycine Transporter 2 (GlyT2). Although severely impaired, Glycinergic transmission is not completely absent in GlyT2-knockout mice, suggesting that other routes of Glycine uptake or de novo synthesis of Glycine exist in presynaptic terminals. To investigate the consequences of a complete loss of Glycinergic transmission, we generated a mouse line with a conditional ablation of VIAAT in Glycinergic neurons by crossing mice with loxP-flanked VIAAT alleles with a GlyT2-Cre transgenic mouse line. Interestingly, conditional VIAAT knockout (VIAAT cKO) mice were not viable at birth. In addition to the dominant respiratory failure, VIAAT cKO showed an umbilical hernia and a cleft palate. Immunohistochemistry revealed an almost complete depletion of VIAAT in the brainstem. Electrophysiology revealed the absence of both spontaneous Glycinergic and GABAergic inhibitory postsynaptic currents from hypoglossal motoneurons. Our results demonstrate that the deletion of VIAAT in GlyT2-Cre expressing neurons also strongly affects GABAergic transmission and suggest a large overlap of the Glycinergic and the GABAergic neuron population during early development in the caudal parts of the brain.
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development of synaptic inhibition in Glycine Transporter 2 deficient mice
Molecular and Cellular Neuroscience, 2010Co-Authors: Tobias A Latal, Volker Eulenburg, Thomas Kremer, Jesus Gomeza, Swen HülsmannAbstract:Abstract Mice deficient for the neuronal Glycine Transporter subtype 2 (GlyT2) die during the second postnatal week after developing neuromotor deficiencies, which resembles severe forms of human hyperekplexia. This phenotype has been attributed to a dramatic reduction in Glycinergic neurotransmission. In the present study we analyzed the development of GABAergic and Glycinergic synaptic transmission in GlyT2-knockout mice during early postnatal life. Anti-Glycine immunohistochemistry in spinal cord and brainstem slices and whole-cell voltage-clamp recordings of Glycinergic inhibitory postsynaptic currents (IPSCs) from hypoglossal motoneurons revealed strikingly reduced levels of synaptic Glycine already at birth. Since GABA and Glycine use the same vesicular inhibitory amino acid Transporter (VIAAT or VGAT) we also analysed GABAergic neurotransmission. No increase of GABA immunoreactivity was observed in the spinal cord and brainstem of GlyT2−/− mice at any stage of postnatal development. Correspondingly no up-regulation of GABAergic IPSCs was detected in GlyT2−/− hypoglossal motoneurons. These data suggest that in the first postnatal week, loss of the Glycine Transporter 2 is neither compensated by Glycine de-novo synthesis nor by up-regulation of the GABAergic transmission in GlyT2−/− mice.
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generation of a mouse line expressing cre recombinase in Glycinergic interneurons
Genesis, 2010Co-Authors: Naoko Ishihara, Volker Eulenburg, Wencke Armsen, Theofilos Papadopoulos, Heinrich BetzAbstract:In caudal regions of the CNS, Glycine constitutes the major inhibitory neurotransmitter. Here, we describe a mouse line that expresses Cre recombinase under the control of a BAC transgenic Glycine Transporter 2 (GlyT2) promoter fragment. Mating of GlyT2-Cre mice with the Cre reporter mouse lines Rosa26/LacZ and Rosa26/YFP and analysis of double transgenic offsprings revealed strong transgene activity in caudal regions of the central nervous system, i.e., brain stem and spinal cord. Some additional Cre expression was observed in cortical and cerebellar regions. In brain stem and spinal cord, Cre expressing cells were identified as Glycinergic interneurons by staining with GlyT2- and Glycine-immunoreactive antibodies; here, >80% of the Glycine-immunoreactive cells expressed the Cre reporter protein. These data indicate that GlyT2-Cre mice are a useful tool for the genetic manipulation of Glycinergic interneurons.
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deletion of the mouse Glycine Transporter 2 results in a hyperekplexia phenotype and postnatal lethality
Neuron, 2003Co-Authors: Jesus Gomeza, Swen Hülsmann, Volker Eulenburg, Wencke Armsen, Koji Ohno, Diethelm W Richter, Bodo Laube, Heinrich BetzAbstract:The Glycine Transporter subtype 2 (GlyT2) is localized in the axon terminals of Glycinergic neurons. Mice deficient in GlyT2 are normal at birth but during the second postnatal week develop a lethal neuromotor deficiency that resembles severe forms of human hyperekplexia (hereditary startle disease) and is characterized by spasticity, tremor, and an inability to right. Histological and immunological analyses failed to reveal anatomical or biochemical abnormalities, but the amplitudes of Glycinergic miniature inhibitory currents (mIPSCs) were strikingly reduced in hypoglossal motoneurons and dissociated spinal neurons from GlyT2-deficient mice. Thus, postnatal GlyT2 function is crucial for efficient transmitter loading of synaptic vesicles in Glycinergic nerve terminals, and the GlyT2 gene constitutes a candidate disease gene in human hyperekplexia patients.
Beatriz Lopezcorcuera - One of the best experts on this subject based on the ideXlab platform.
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molecular basis of the dominant negative effect of a Glycine Transporter 2 mutation associated with hyperekplexia
Journal of Biological Chemistry, 2015Co-Authors: Carmen Aragon, Beatriz Lopezcorcuera, Jaime De Juansanz, Esther ArribasgonzalezAbstract:Abstract Hyperekplexia or startle disease is a rare clinical syndrome characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. This neurological disorder can have serious consequences in neonates, provoking brain damage and/or sudden death due to apnea episodes and cardiorespiratory failure. Hyperekplexia is caused by defective inhibitory Glycinergic neurotransmission. Mutations in the human SLC6A5 gene encoding the neuronal GlyT2 Glycine Transporter are responsible for the presynaptic form of the disease. GlyT2 mediates synaptic Glycine recycling, which constitutes the main source of releasable transmitter at Glycinergic synapses. Although the majority of GlyT2 mutations detected so far are recessive, a dominant-negative mutant that affects GlyT2 trafficking does exist. In this study, we explore the properties and structural alterations of the S512R mutation in GlyT2. We analyzed its dominant-negative effect that retains wild-type GlyT2 in the endoplasmic reticulum (ER), preventing surface expression. We show that the presence of an arginine rather than serine 512 provoked Transporter misfolding, enhanced association to the ER-chaperone calnexin, altered association with the coat-protein complex II component Sec24D, and thereby impeded ER exit. The S512R mutant formed oligomers with wild-type GlyT2 causing its retention in the ER. Overexpression of calnexin rescued wild-type GlyT2 from the dominant-negative effect of the mutant, increasing the amount of Transporter that reached the plasma membrane and dampening the interaction between the wild-type and mutant GlyT2. The ability of chemical chaperones to overcome the dominant-negative effect of the disease mutation on the wild-type Transporter was demonstrated in heterologous cells and primary neurons.
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presynaptic control of Glycine Transporter 2 glyt2 by physical and functional association with plasma membrane ca2 atpase pmca and na ca2 exchanger ncx
Journal of Biological Chemistry, 2014Co-Authors: Jaime De Juansanz, Enrique Nunez, Beatriz Lopezcorcuera, Francisco Zafra, Maria Berrocal, Isaac Corbacho, Ignacio Ibanez, Esther Arribasgonzalez, Daniel Marcos, Ana M MataAbstract:Fast inhibitory Glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, Glycine is recovered back to the presynaptic terminal by the neuronal Glycine Transporter 2 (GlyT2) to maintain quantal Glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of Glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca2+-ATPase (PMCA) isoforms 2 and 3, and Na+/Ca2+-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2–3·NCX complex would help Na+/K+-ATPase in controlling local Na+ increases derived from GlyT2 activity after neurotransmitter release.
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calnexin assisted biogenesis of the neuronal Glycine Transporter 2 glyt2
PLOS ONE, 2013Co-Authors: Carmen Aragon, Beatriz Lopezcorcuera, Esther Arribasgonzalez, Pablo AlonsotorresAbstract:The neuronal Transporter GlyT2 is a polytopic, 12-transmembrane domain, plasma membrane glycoprotein involved in the removal and recycling of synaptic Glycine from inhibitory synapses. Mutations in the human GlyT2 gene (SLC6A5) that cause deficient Glycine transport or defective GlyT2 trafficking are the second most common cause of hyperekplexia or startle disease. In this study we examined several aspects of GlyT2 biogenesis that involve the endoplasmic reticulum chaperone calnexin (CNX). CNX binds transiently to an intermediate under-glycosylated Transporter precursor and facilitates GlyT2 processing. In cells expressing GlyT2, Transporter accumulation and transport activity were attenuated by siRNA-mediated CNX knockdown and enhanced by CNX overexpression. GlyT2 binding to CNX was mediated by glycan and polypeptide-based interactions as revealed by pharmacological approaches and the behavior of GlyT2 N-glycan-deficient mutants. Moreover, Transporter folding appeared to be stabilized by N-glycans. Co-expression of CNX and a fully non-glycosylated mutant rescues Glycine transport but not mutant surface expression. Hence, CNX discriminates between different conformational states of GlyT2 displaying a lectin-independent chaperone activity. GlyT2 wild-type and mutant Transporters were finally degraded in the lysosome. Our findings provide further insight into GlyT2 biogenesis, and a useful framework for the study of newly synthesized GlyT2 Transporters bearing hyperekplexia mutations.
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the second intracellular loop of the Glycine Transporter 2 contains crucial residues for Glycine transport and phorbol ester induced regulation
Journal of Biological Chemistry, 2004Co-Authors: Amparo Fornes, Enrique Nunez, Carmen Aragon, Beatriz LopezcorcueraAbstract:Na+ and Cl(-)-coupled Glycine Transporters control the availability of Glycine neurotransmitter in the synaptic cleft of inhibitory Glycinergic pathways. In this report, we have investigated the involvement of the second intracellular loop of the neuronal Glycine Transporter 2 (GLYT2) on the protein conformational equilibrium and the regulation by 4alpha-phorbol 12 myristate 13-acetate (PMA). By substituting several charged (Lys-415, Lys-418, and Lys-422) and polar (Thr-419 and Ser-420) residues for different amino acids and monitoring plasma membrane expression and kinetic behavior, we found that residue Lys-422 is crucial for Glycine transport. The introduction of a negative charge in 422, and to a lower extent in neighboring N-terminal residues, dramatically increases Transporter voltage dependence as assessed by response to high potassium depolarizing conditions. In addition, [2-(trimethylammonium)ethyl] methanethiosulfonate accessibility revealed a conformational connection between Lys-422 and the Glycine binding/permeation site. Finally, we show that the mutation of positions Thr-419, Ser-420, and mainly Lys-422 to acidic residues abolishes the PMA-induced inhibition of transport activity and the plasma membrane Transporter internalization. Our results establish a new structural basis for the action of PMA on GLYT2 and suggest a complex nature of the PMA action on this Glycine Transporter.
Carmen Aragon - One of the best experts on this subject based on the ideXlab platform.
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structural determinants of the neuronal Glycine Transporter 2 for the selective inhibitors alx1393 and org25543
ACS Chemical Neuroscience, 2021Co-Authors: Enrique Nunez, Cristina Benitomunoz, Almudena Perona, Raquel Felipe, Gonzalo Perezsiles, Carmen AragonAbstract:The neuronal Glycine Transporter GlyT2 modulates inhibitory Glycinergic neurotransmission by controlling the extracellular concentration of synaptic Glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord Glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing Glycine extracellular levels, so it has analgesic action. O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the Transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine Transporter from Drosophila melanogaster. Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the Transporter in outward-facing conformation and hence inhibit Glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.
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molecular basis of the dominant negative effect of a Glycine Transporter 2 mutation associated with hyperekplexia
Journal of Biological Chemistry, 2015Co-Authors: Carmen Aragon, Beatriz Lopezcorcuera, Jaime De Juansanz, Esther ArribasgonzalezAbstract:Abstract Hyperekplexia or startle disease is a rare clinical syndrome characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. This neurological disorder can have serious consequences in neonates, provoking brain damage and/or sudden death due to apnea episodes and cardiorespiratory failure. Hyperekplexia is caused by defective inhibitory Glycinergic neurotransmission. Mutations in the human SLC6A5 gene encoding the neuronal GlyT2 Glycine Transporter are responsible for the presynaptic form of the disease. GlyT2 mediates synaptic Glycine recycling, which constitutes the main source of releasable transmitter at Glycinergic synapses. Although the majority of GlyT2 mutations detected so far are recessive, a dominant-negative mutant that affects GlyT2 trafficking does exist. In this study, we explore the properties and structural alterations of the S512R mutation in GlyT2. We analyzed its dominant-negative effect that retains wild-type GlyT2 in the endoplasmic reticulum (ER), preventing surface expression. We show that the presence of an arginine rather than serine 512 provoked Transporter misfolding, enhanced association to the ER-chaperone calnexin, altered association with the coat-protein complex II component Sec24D, and thereby impeded ER exit. The S512R mutant formed oligomers with wild-type GlyT2 causing its retention in the ER. Overexpression of calnexin rescued wild-type GlyT2 from the dominant-negative effect of the mutant, increasing the amount of Transporter that reached the plasma membrane and dampening the interaction between the wild-type and mutant GlyT2. The ability of chemical chaperones to overcome the dominant-negative effect of the disease mutation on the wild-type Transporter was demonstrated in heterologous cells and primary neurons.
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calnexin assisted biogenesis of the neuronal Glycine Transporter 2 glyt2
PLOS ONE, 2013Co-Authors: Carmen Aragon, Beatriz Lopezcorcuera, Esther Arribasgonzalez, Pablo AlonsotorresAbstract:The neuronal Transporter GlyT2 is a polytopic, 12-transmembrane domain, plasma membrane glycoprotein involved in the removal and recycling of synaptic Glycine from inhibitory synapses. Mutations in the human GlyT2 gene (SLC6A5) that cause deficient Glycine transport or defective GlyT2 trafficking are the second most common cause of hyperekplexia or startle disease. In this study we examined several aspects of GlyT2 biogenesis that involve the endoplasmic reticulum chaperone calnexin (CNX). CNX binds transiently to an intermediate under-glycosylated Transporter precursor and facilitates GlyT2 processing. In cells expressing GlyT2, Transporter accumulation and transport activity were attenuated by siRNA-mediated CNX knockdown and enhanced by CNX overexpression. GlyT2 binding to CNX was mediated by glycan and polypeptide-based interactions as revealed by pharmacological approaches and the behavior of GlyT2 N-glycan-deficient mutants. Moreover, Transporter folding appeared to be stabilized by N-glycans. Co-expression of CNX and a fully non-glycosylated mutant rescues Glycine transport but not mutant surface expression. Hence, CNX discriminates between different conformational states of GlyT2 displaying a lectin-independent chaperone activity. GlyT2 wild-type and mutant Transporters were finally degraded in the lysosome. Our findings provide further insight into GlyT2 biogenesis, and a useful framework for the study of newly synthesized GlyT2 Transporters bearing hyperekplexia mutations.
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the second intracellular loop of the Glycine Transporter 2 contains crucial residues for Glycine transport and phorbol ester induced regulation
Journal of Biological Chemistry, 2004Co-Authors: Amparo Fornes, Enrique Nunez, Carmen Aragon, Beatriz LopezcorcueraAbstract:Na+ and Cl(-)-coupled Glycine Transporters control the availability of Glycine neurotransmitter in the synaptic cleft of inhibitory Glycinergic pathways. In this report, we have investigated the involvement of the second intracellular loop of the neuronal Glycine Transporter 2 (GLYT2) on the protein conformational equilibrium and the regulation by 4alpha-phorbol 12 myristate 13-acetate (PMA). By substituting several charged (Lys-415, Lys-418, and Lys-422) and polar (Thr-419 and Ser-420) residues for different amino acids and monitoring plasma membrane expression and kinetic behavior, we found that residue Lys-422 is crucial for Glycine transport. The introduction of a negative charge in 422, and to a lower extent in neighboring N-terminal residues, dramatically increases Transporter voltage dependence as assessed by response to high potassium depolarizing conditions. In addition, [2-(trimethylammonium)ethyl] methanethiosulfonate accessibility revealed a conformational connection between Lys-422 and the Glycine binding/permeation site. Finally, we show that the mutation of positions Thr-419, Ser-420, and mainly Lys-422 to acidic residues abolishes the PMA-induced inhibition of transport activity and the plasma membrane Transporter internalization. Our results establish a new structural basis for the action of PMA on GLYT2 and suggest a complex nature of the PMA action on this Glycine Transporter.
Heinrich Betz - One of the best experts on this subject based on the ideXlab platform.
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generation of a mouse line expressing cre recombinase in Glycinergic interneurons
Genesis, 2010Co-Authors: Naoko Ishihara, Volker Eulenburg, Wencke Armsen, Theofilos Papadopoulos, Heinrich BetzAbstract:In caudal regions of the CNS, Glycine constitutes the major inhibitory neurotransmitter. Here, we describe a mouse line that expresses Cre recombinase under the control of a BAC transgenic Glycine Transporter 2 (GlyT2) promoter fragment. Mating of GlyT2-Cre mice with the Cre reporter mouse lines Rosa26/LacZ and Rosa26/YFP and analysis of double transgenic offsprings revealed strong transgene activity in caudal regions of the central nervous system, i.e., brain stem and spinal cord. Some additional Cre expression was observed in cortical and cerebellar regions. In brain stem and spinal cord, Cre expressing cells were identified as Glycinergic interneurons by staining with GlyT2- and Glycine-immunoreactive antibodies; here, >80% of the Glycine-immunoreactive cells expressed the Cre reporter protein. These data indicate that GlyT2-Cre mice are a useful tool for the genetic manipulation of Glycinergic interneurons.
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cellular localization and subcellular distribution of unc 33 like protein 6 a brain specific protein of the collapsin response mediator protein family that interacts with the neuronal Glycine Transporter 2
Journal of Neurochemistry, 2005Co-Authors: Masahisa Horiuchi, Sven Loebrich, Johann Helmut Brandstaetter, Matthias Kneussel, Heinrich BetzAbstract:Unc-33-like protein (Ulip)6, a brain-specific phosphoprotein of the Ulip/collapsin response mediator protein family, was originally identified in our laboratory by yeast two-hybrid screening using the cytoplasmic N-terminal domain of the neuronal Glycine Transporter, Glycine Transporter (GlyT) 2, as a bait. Here, the interaction of Ulip6 with the N-terminal domain of GlyT2 was found to be specific for this member of the Ulip/collapsin response mediator protein family and to involve amino acids 135-184 of GlyT2. In pull-down assays and coimmunoprecipitation experiments with rat spinal cord extract, the presence of phosphatase inhibitors significantly enhanced binding of Ulip6 to GlyT2. Subcellular fractionation of spinal cord and retina homogenates at different developmental stages showed Ulip6 immunoreactivity to be associated with light vesicles that were distinct from GlyT2-containing and synaptic vesicles. Immunocytochemistry revealed punctate Ulip6 immunoreactivity in both somatic regions and processes of cultured spinal neurones; no colocalization with GlyT2 or other synaptic marker proteins was found. In retina, which expresses only GlyT1 but not GlyT2, Ulip6 was detected in the inner plexiform layer and along the somata and processes of selected bipolar, amacrine and ganglion cells. Our data support a model in which Ulip6 transiently interacts with GlyT2 in a phosphorylation-dependent manner.
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the neuronal Glycine Transporter 2 interacts with the pdz domain protein syntenin 1
Molecular and Cellular Neuroscience, 2004Co-Authors: Koji Ohno, Jesus Gomeza, Michael Koroll, Oussama El Far, Petra Scholze, Heinrich BetzAbstract:The Glycine Transporter subtype 2 (GlyT2) is localized at Glycinergic axon terminals where it mediates the re-uptake of Glycine from the extracellular space. In this study, we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic carboxy terminal tail region of GlyT2. Screening of a rat brain cDNA library identified the PDZ domain protein syntenin-1 as an intracellular binding partner of GlyT2. In pull-down experiments, the interaction between GlyT2 and syntenin-1 was found to involve the C-terminal amino acid residues of GlyT2 and the PDZ2 domain of syntenin-1. Syntenin-1 is widely expressed in brain and co-localizes with GlyT2 in brainstem sections. Furthermore, syntenin-1 binds syntaxin 1A, which is known to regulate the plasma membrane insertion of GlyT2. Thus, syntenin-1 may be an in vivo binding partner of GlyT2 that regulates its trafficking and/or presynaptic localization in Glycinergic neurons.
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deletion of the mouse Glycine Transporter 2 results in a hyperekplexia phenotype and postnatal lethality
Neuron, 2003Co-Authors: Jesus Gomeza, Swen Hülsmann, Volker Eulenburg, Wencke Armsen, Koji Ohno, Diethelm W Richter, Bodo Laube, Heinrich BetzAbstract:The Glycine Transporter subtype 2 (GlyT2) is localized in the axon terminals of Glycinergic neurons. Mice deficient in GlyT2 are normal at birth but during the second postnatal week develop a lethal neuromotor deficiency that resembles severe forms of human hyperekplexia (hereditary startle disease) and is characterized by spasticity, tremor, and an inability to right. Histological and immunological analyses failed to reveal anatomical or biochemical abnormalities, but the amplitudes of Glycinergic miniature inhibitory currents (mIPSCs) were strikingly reduced in hypoglossal motoneurons and dissociated spinal neurons from GlyT2-deficient mice. Thus, postnatal GlyT2 function is crucial for efficient transmitter loading of synaptic vesicles in Glycinergic nerve terminals, and the GlyT2 gene constitutes a candidate disease gene in human hyperekplexia patients.