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Jonathan Stephan - One of the best experts on this subject based on the ideXlab platform.
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Expression of functional inhibitory Neurotransmitter Transporters GlyT1, GAT-1, and GAT-3 by astrocytes of inferior colliculus and hippocampus
Molecular Brain, 2018Co-Authors: Elsa Ghirardini, Sl Wadle, Vanessa Augustin, Jasmin Becker, Sina Brill, Julia Hammerich, Gerald Seifert, Jonathan StephanAbstract:Neuronal inhibition is mediated by glycine and/or GABA. Inferior colliculus (IC) neurons receive glycinergic and GABAergic inputs, whereas inhibition in hippocampus (HC) predominantly relies on GABA. Astrocytes heterogeneously express Neurotransmitter Transporters and are expected to adapt to the local requirements regarding Neurotransmitter homeostasis. Here we analyzed the expression of inhibitory Neurotransmitter Transporters in IC and HC astrocytes using whole-cell patch-clamp and single-cell reverse transcription-PCR. We show that most astrocytes in both regions expressed functional glycine Transporters (GlyTs). Activation of these Transporters resulted in an inward current ( I _Gly) that was sensitive to the competitive GlyT1 agonist sarcosine. Astrocytes exhibited transcripts for GlyT1 but not for GlyT2. Glycine did not alter the membrane resistance ( R _M) arguing for the absence of functional glycine receptors (GlyRs). Thus, I _Gly was mainly mediated by GlyT1. Similarly, we found expression of functional GABA Transporters (GATs) in all IC astrocytes and about half of the HC astrocytes. These Transporters mediated an inward current ( I _GABA) that was sensitive to the competitive GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively. Accordingly, transcripts for GAT-1 and GAT-3 were found but not for GAT-2 and BGT-1. Only in hippocampal astrocytes, GABA transiently reduced R _M demonstrating the presence of GABA_A receptors (GABA_ARs). However, I _GABA was mainly not contaminated by GABA_AR-mediated currents as R _M changes vanished shortly after GABA application. In both regions, I _GABA was stronger than I _Gly. Furthermore, in HC the I _GABA/ I _Gly ratio was larger compared to IC. Taken together, our results demonstrate that astrocytes are heterogeneous across and within distinct brain areas. Furthermore, we could show that the capacity for glycine and GABA uptake varies between both brain regions.
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functional analysis of the inhibitory Neurotransmitter Transporters glyt1 gat 1 and gat 3 in astrocytes of the lateral superior olive
Glia, 2014Co-Authors: Jonathan Stephan, Eckhard FriaufAbstract:Neurotransmitter clearance from the synaptic cleft is a major function of astrocytes and requires Neurotransmitter Transporters. In the rodent lateral superior olive (LSO), a conspicuous auditory brainstem center, both glycine and GABA mediate synaptic inhibition. However, the main inhibitory input from the medial nucleus of the trapezoid body (MNTB) appears to be glycinergic by postnatal day (P) 14, when circuit maturation is almost accomplished. Using whole-cell patch-clamp recordings at P3-20, we analyzed glycine Transporters (GlyT1) and GABA Transporters (GAT-1, GAT-3) in mouse LSO astrocytes, emphasizing on their developmental regulation. Application of glycine or GABA induced a dose- and age-dependent inward current and a respective depolarization. The GlyT1-specific inhibitor sarcosine reduced the maximal glycine-induced current (IGly (max) ) by about 60%. The GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively, reduced the maximal GABA-induced current (IGABA (max) ) by about 35%. Furthermore, [Cl(-) ]o reduction decreased IGly (max) and IGABA (max) by about 85 to 95%, showing the Cl(-) dependence of GlyT and GAT. IGABA (max) was stronger than IGly (max) , and the ratio increased developmentally from 1.6-fold to 3.7-fold. Together, our results demonstrate the functional presence of the three inhibitory Neurotransmitter Transporters GlyT1, GAT-1, and GAT-3 in LSO astrocytes. Furthermore, the uptake capability for GABA was higher than for glycine, pointing toward eminent GABAergic signaling in the LSO. GABA may originate from another source than the MNTB-LSO synapses, namely from another projection or from reversal of astrocytic GATs. Thus, neuronal signaling in the LSO appears to be more versatile than previously thought. GLIA 2014;62:1992-2003.
David E. Krantz - One of the best experts on this subject based on the ideXlab platform.
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Vesicular Neurotransmitter Transporters in Drosophila melanogaster.
Biochimica et biophysica acta. Biomembranes, 2020Co-Authors: Sonali A. Deshpande, Zachary Freyberg, Hakeem O. Lawal, David E. KrantzAbstract:Abstract Drosophila melanogaster express vesicular Transporters for the storage of Neurotransmitters acetylcholine, biogenic amines, GABA, and glutamate. The large array of powerful molecular-genetic tools available in Drosophila enhances the use of this model organism for studying transporter function and regulation.
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Drosophila melanogaster as a genetic model system to study Neurotransmitter Transporters
Neurochemistry international, 2014Co-Authors: Ciara A. Martin, David E. KrantzAbstract:The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of the same Neurotransmitters as mammals and very similar mechanisms of Neurotransmitter storage, release and recycling. This system offers a variety of powerful molecular-genetic methods for the study of Transporters, many of which would be difficult in mammalian models. We review here progress made using Drosophila to understand the function and regulation of Neurotransmitter Transporters and discuss future directions for its use.
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slc18 vesicular Neurotransmitter Transporters for monoamines and acetylcholine
Molecular Aspects of Medicine, 2013Co-Authors: Hakeem O. Lawal, David E. KrantzAbstract:Abstract The exocytotic release of Neurotransmitters requires active transport into synaptic vesicles and other types of secretory vesicles. Members of the SLC18 family perform this function for acetylcholine (SLC18A3, the vesicular acetylcholine transporter or VAChT) and monoamines such as dopamine and serotonin (SLC18A1 and 2, the vesicular monoamine Transporters VMAT1 and 2, respectively). To date, no specific diseases have been attributed to a mutation in an SLC18 family member; however, polymorphisms in SLC18A1 and SLC18A2 may confer risk for some neuropsychiatric disorders. Additional members of this family include SLC18A4, expressed in insects, and SLC18B1, the function of which is not known. SLC18 is part of the Drug:H + Antiporter-1 Family (DHA1, TCID 2.A.1.2) within the Major Facilitator Superfamily (MFS, TCID 2.A.1).
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Trafficking of vesicular Neurotransmitter Transporters.
Traffic (Copenhagen Denmark), 2008Co-Authors: Hao Fei, Anna Grygoruk, Elizabeth S. Brooks, Audrey Chen, David E. KrantzAbstract:Vesicular Neurotransmitter Transporters are required for the storage of all classical and amino acid Neurotransmitters in secretory vesicles. Transporter expression can influence Neurotransmitter storage and release, and trafficking targets the Transporters to different types of secretory vesicles. Vesicular Transporters traffic to synaptic vesicles (SVs) as well as large dense core vesicles and are recycled to SVs at the nerve terminal. Some of the intrinsic signals for these trafficking events have been defined and include a dileucine motif present in multiple transporter subtypes, an acidic cluster in the neural isoform of the vesicular monoamine transporter (VMAT) 2 and a polyproline motif in the vesicular glutamate transporter (VGLUT) 1. The sorting of VMAT2 and the vesicular acetylcholine transporter to secretory vesicles is regulated by phosphorylation. In addition, VGLUT1 uses alternative endocytic pathways for recycling back to SVs following exocytosis. Regulation of these sorting events has the potential to influence synaptic transmission and behavior.
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Membrane trafficking of Neurotransmitter Transporters in the regulation of synaptic transmission
Trends in cell biology, 1999Co-Authors: Yongjian Liu, David E. Krantz, Clarissa Waites, Robert H. EdwardsAbstract:Many psychoactive drugs influence the transport of Neurotransmitters across biological membranes, suggesting that the physiological regulation of Neurotransmitter transport might contribute to normal and perhaps abnormal behaviour. Over the past few years, molecular characterization of the Neurotransmitter Transporters has enabled investigation of their subcellular location and regulation. The analysis of location suggests that membrane trafficking has an important role in the normal function of these proteins. One of the major regulatory mechanisms also involves changes in localization that might contribute to synaptic plasticity. This article discusses recent work on the membrane trafficking of Neurotransmitter Transporters and its role in regulating their activity.
Ulrik Gether - One of the best experts on this subject based on the ideXlab platform.
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Elucidating the Mechanism Behind Sodium-Coupled Neurotransmitter Transporters by Reconstitution
Neurochemical Research, 2021Co-Authors: Solveig G. Schmidt, Ulrik Gether, Claus J LolandAbstract:Sodium-coupled Neurotransmitter Transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active Transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today’s single-molecule studies, insights from live functioning Transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled Neurotransmitter Transporters, the Neurotransmitter: sodium symporters and the excitatory amino acid Transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of Neurotransmitter Transporters has been instrumental.
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slc6 Neurotransmitter Transporters structure function and regulation
Pharmacological Reviews, 2011Co-Authors: Anders S Kristensen, Lena Sorensen, Jacob Eriksen, Claus J Loland, Trine N. Jørgensen, Jacob Andersen, Kristian Strømgaard, Ulrik GetherAbstract:The Neurotransmitter Transporters (NTTs) belonging to the solute carrier 6 ( SLC6 ) gene family (also referred to as the Neurotransmitter-sodium-symporter family or Na+/Cl−-dependent Transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane Transporters for the monoamine Neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid Neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating Neurotransmitter signaling and homeostasis by mediating uptake of released Neurotransmitters from the extracellular space into neurons and glial cells. The Transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 Transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of Transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.
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Neurotransmitter Transporters molecular function of important drug targets
Trends in Pharmacological Sciences, 2006Co-Authors: Ulrik Gether, Peter Hongaard Andersen, Orla M Larsson, Arne SchousboeAbstract:The concentration of Neurotransmitters in the extracellular space is tightly controlled by distinct classes of membrane transport proteins. This review focuses on the molecular function of two major classes of Neurotransmitter transporter that are present in the cell membrane of neurons and/or glial cells: the solute carrier (SLC)1 transporter family, which includes the Transporters that mediate the Na + -dependent uptake of glutamate, and the SLC6 transporter family, which includes the Transporters that mediate the Na + -dependent uptake of dopamine, 5-HT, norepinephrine, glycine and GABA. Recent research has provided substantial insight into the structure and function of these Transporters. In particular, the recent crystallizations of bacterial homologs are of the utmost importance, enabling the first reliable structural models of the mammalian Neurotransmitter Transporters to be generated. These models should be an important tool for developing specific drugs that, through selective interaction with Transporters, could improve the treatment of serious neurological and psychiatric disorders.
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Zn2+ modulation of Neurotransmitter Transporters.
Handbook of experimental pharmacology, 2006Co-Authors: Kristine Norgaard-nielsen, Ulrik GetherAbstract:Neurotransmitter Transporters located at the presynaptic or glial cell membrane are responsible for the stringent and rapid clearance of the transmitter from the synapse, and hence they terminate signaling and control the duration of synaptic inputs in the brain. Two distinct families of Neurotransmitter Transporters have been identified based on sequence homology: (1) the Neurotransmitter sodium symporter family (NSS), which includes the Na+/Cl--dependent Transporters for dopamine, norepinephrine, and serotonin; and (2) the dicarboxylate/amino acid cation symporter family (DAACS), which includes the Na+-dependent glutamate Transporters (excitatory amino acid Transporters; EAAT). In this chapter, we describe how the identification of endogenous Zn2+-binding sites, as well as engineering of artificial Zn2+-binding sites both in the Na+/Cl--dependent Transporters and in the EAATs, have proved to be an important tool for studying the molecular function of these proteins. We also interpret the current available data on Zn2+-binding sites in the context of the recently published crystal structures. Moreover, we review how the identification of endogenous Zn2+-binding sites has indirectly suggested the possibility that several of the Transporters are modulated by Zn2+ in vivo, and thus that Zn2+ can play a role as a neuromodulator by affecting the function of Neurotransmitter Transporters.
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The monoamine Neurotransmitter Transporters: structure, conformational changes and molecular gating.
Current opinion in drug discovery & development, 2001Co-Authors: Lene Norregaard, Ulrik GetherAbstract:Monoamine Transporters are primary targets for the action of many psychoactive compounds including the most commonly used antidepressants and widely abused drugs, such as cocaine and amphetamine. Consequently, these Transporters are the focus of continuous intensive research. Over the last couple of years, these efforts have resulted in significant progress both in our understanding of their role in drug abuse mechanisms and of the structural basis that underlies their capability as Transporters to translocate their substrate across the plasma membrane. The aim of this review is to describe both the current awareness regarding the structural organization of the monoamine Neurotransmitter Transporters as well as the molecular mechanisms responsible for function, with specific emphasis on conformational changes and putative gating mechanisms.
Randy D. Blakely - One of the best experts on this subject based on the ideXlab platform.
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Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters
Pharmacological reviews, 2016Co-Authors: Daniel P. Bermingham, Randy D. BlakelyAbstract:Modulation of neurotransmission by the monoamines dopamine (DA), norepinephrine (NE), and serotonin (5-HT) is critical for normal nervous system function. Precise temporal and spatial control of this signaling in mediated in large part by the actions of monoamine Transporters (DAT, NET, and SERT, respectively). These Transporters act to recapture their respective Neurotransmitters after release, and disruption of clearance and reuptake has significant effects on physiology and behavior and has been linked to a number of neuropsychiatric disorders. To ensure adequate and dynamic control of these Transporters, multiple modes of control have evolved to regulate their activity and trafficking. Central to many of these modes of control are the actions of protein kinases, whose actions can be direct or indirectly mediated by kinase-modulated protein interactions. Here, we summarize the current state of our understanding of how protein kinases regulate monoamine Transporters through changes in activity, trafficking, phosphorylation state, and interacting partners. We highlight genetic, biochemical, and pharmacological evidence for kinase-linked control of DAT, NET, and SERT and, where applicable, provide evidence for endogenous activators of these pathways. We hope our discussion can lead to a more nuanced and integrated understanding of how Neurotransmitter Transporters are controlled and may contribute to disorders that feature perturbed monoamine signaling, with an ultimate goal of developing better therapeutic strategies.
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Bound to be different: Neurotransmitter Transporters meet their bacterial cousins.
Molecular interventions, 2007Co-Authors: L. K. Henry, Jens Meiler, Randy D. BlakelyAbstract:The Neurotransmitter Transporters belonging to the solute carrier 6 (SLC6) family, including the gamma-aminobutyric acid (GAT), norepinephrine (NET), serotonin (SERT) and dopamine (DAT) Transporters are extremely important drug targets of great clinical relevance. These Na+, Cl(-)-dependent Transporters primarily function following neurotransmission to reset neuronal signaling by transporting Neurotransmitter out of the synapse and back into the pre-synaptic neuron. Recent studies have tracked down an elusive binding site for Cl(-) that facilitates Neurotransmitter transport using structural differences evident with bacterial family members (e.g., the Aquifex aeolicus leucine transporter LeuT Aa) that lack Cl(-) dependence. Additionally, the crystal structures of antidepressant-bound LeuT Aa reveals a surprising mode of drug interaction that may have relevance for medication development. The study of sequence and structural divergence between LeuT Aa and human SLC6 family Transporters can thus inform us as to how and why Neurotransmitter Transporters evolved a reliance on extracellular Cl(-) to propel the transport cycle; what residue changes and helical rearrangements give rise to recognition of different substrates; and how drugs such as antidepressants, cocaine, and amphetamines halt (or reverse) the transport process.
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All aglow about presynaptic receptor regulation of Neurotransmitter Transporters.
Molecular pharmacology, 2007Co-Authors: Randy D. Blakely, Louis J. DefeliceAbstract:Mounting evidence supports the idea that Neurotransmitter Transporters are subject to many forms of post-translational regulation typically associated with receptors and ion channels, including receptor and kinase-mediated changes in transporter phosphorylation, cell surface trafficking, and/or catalytic activation. Although hints of this regulation can be achieved with traditional radiolabeled substrate flux techniques, higher resolution methods are needed that can localize transporter function in situ as well as permit real-time monitoring of transport function without confounds associated with coincident receptor activation. The elegant study by Bolan et al. (p. 1222) capitalizes on the fluorescent properties of a recently introduced substrate for the dopamine (DA) transporter (DAT), termed 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+), to illuminate a pertussis toxin-sensitive, extracellular signal-regulated kinase (ERK1/2)-dependent pathway by which presynaptic DA D(2) receptors regulate DATs.
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biogenic amine Neurotransmitter Transporters just when you thought you knew them
Physiology, 2005Co-Authors: Randy D. Blakely, Louis J. Defelice, Aurelio GalliAbstract:Plasma membrane Transporters have long been known to support the reuptake of biogenic amine Neurotransmitters following release in the central and peripheral nervous systems. Using high-resolution imaging, patch-clamp and amperometric approaches, as well as molecular manipulations of transporter-regulatory pathways, surprising new details have been uncovered as to how Transporters work and are influenced by signaling pathways and psychostimulants.
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Distinct, developmentally regulated brain mRNAs direct the synthesis of Neurotransmitter Transporters.
Journal of neurochemistry, 1991Co-Authors: Randy D. Blakely, Janet A. Clark, Tadeusz Pacholczyk, Susan G. AmaraAbstract:The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of Neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent Neurotransmitter Transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte Transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine Transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA Transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid Neurotransmitter uptake systems in the rodent CNS.
Nathan Nelson - One of the best experts on this subject based on the ideXlab platform.
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the family of na cl Neurotransmitter Transporters
Journal of Neurochemistry, 2002Co-Authors: Nathan NelsonAbstract:Abstract: The termination of neurotransmission is achieved by rapid uptake of the released Neurotransmitter by specific high-affinity Neurotransmitter Transporters. Most of these Transporters are encoded by a family of genes (Na+/Cl− Transporters) having a similar membrane topography of 12 transmembrane helices. An evolutionary tree revealed five distinct subfamilies: γ-aminobutyric acid Transporters, monoamine Transporters, amino acid Transporters, “orphan” Transporters, and the recently discovered bacterial Transporters. The bacterial Transporters that belong to this family may help to develop heterologous expression systems with the aim of solving the three-dimensional structure of these membrane proteins. Some of the Neurotransmitter Transporters have been implicated as important sites for drug action. Monoamine Transporters, for example, are targeted by major classes of antidepressants, psychostimulants, and antihypertensive drugs. Localization of individual Transporters in specific cells and brain areas is pertinent to understanding their contribution to neurotransmission and their potential as targets for drugs. The most important questions in the field include resolving the mechanism of Neurotransmitter transport, the structure of the Transporters, and the interaction of each transporter in complex neurological activities.
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Porters and Neurotransmitter Transporters.
The Journal of Experimental Biology, 1994Co-Authors: Nathan Nelson, Holger LillAbstract:Uptake of Neurotransmitters involves multiple Transporters acting in different brain locations under different physiological conditions. The vesicular Transporters are driven by a proton-motive force generated by a V-ATPase and their substrates are taken up via proton/substrate exchange. The plasma membrane Transporters are driven by an electrochemical gradient of sodium generated by a Na+/K(+)-ATPase. Two distinct families of Transporters were identified in this group. One cotransports sodium with glutamate and other amino acids and requires additionally an outwardly directed potassium gradient. The second cotransports sodium, chloride and a variety of Neurotransmitters, including gamma-aminobutyric acid (GABA), glycine and monoamines. Genes and cDNA encoding several members of the latter family have been cloned and studied in detail. The structure and function as well as the evolutionary relationships among these Neurotransmitter Transporters are discussed.
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Structure, function and brain localization of Neurotransmitter Transporters
The Journal of experimental biology, 1994Co-Authors: F. Jursky, Hannah Nelson, Sreekala Mandiyan, Shigehiko Tamura, A. Tamura, Nathan NelsonAbstract:We studied four different cDNAs encoding GABA Transporters and three different cDNAs encoding glycine Transporters in mouse and rat brains. A genomic clone of two of the glycine Transporters (GLYT1a and GLYT1b) revealed that they derive from differential splicing of a single gene. The third glycine transporter (GLYT2) is encoded by a separate gene. Antibodies were raised against seven of these Neurotransmitter Transporters and their cytochemical localization in the mouse brain was studied. In general, we observed a deviation from the classical separation of neuronal and glial Transporters. It seems that each of the Neurotransmitter Transporters is present in specific places in the brain and is expressed in a different way in very specific areas. For example, the GABA transporter GAT4, which also transports beta-alanine, was localized to neurons. However, GAT1, which is specific for GABA, was localized not only to neurons but also to glial cells. The recently discovered glycine transporter GLYT2 was of particular interest because of its deviation from the general structure by a very extended N terminus containing multiple potential phosphorylation sites. Western analysis and immunocytochemistry in frozen sections of mouse brain demonstrated a clear caudal-rostral gradient of GLYT2 distribution, with massive accumulation in the spinal cord and brainstem and less in the cerebellum. Its distribution is typically neuronal and it is present in processes with varicosities. A correlation as observed between the pattern we obtained and that observed previously from strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission at the classical inhibitory system in the hindbrain. The availability of four different GABA Transporters made it possible to look for specific binding sites upon the Neurotransmitter Transporters. An extensive program of site-directed mutagenesis led us to identify a potential Neurotransmitter binding site on the GABA Transporters.
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Cloning and expression of a cDNA encoding the transporter of taurine and beta-alanine in mouse brain.
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Qing-rong Liu, Beatriz López-corcuera, Hannah Nelson, Sreekala Mandiyan, Nathan NelsonAbstract:A taurine/beta-alanine transporter was cloned from a mouse brain cDNA library by screening with a partial cDNA probe of the glycine transporter at low stringency. The deduced amino acid sequence predicts 590 amino acids with typical characteristics of the sodium-dependent Neurotransmitter Transporters such as sequence homology and membrane topography. However, the calculated isoelectric point of the taurine/beta-alanine transporter is more acidic (pI = 5.98) than those (pI > 8.0) of other cloned Neurotransmitter Transporters. Xenopus oocytes injected with cRNA of the cloned transporter expressed uptake activities with Km = 4.5 microM for taurine and Km = 56 microM for beta-alanine. Northern hybridization showed a single transcript of 7.5 kilobases that was highly enriched in kidney and distributed evenly in various parts of the brain. In situ hybridization showed the mRNA of the taurine/beta-alanine transporter to be localized in the corpus callosum, striatum, and anterior commisure. Specific localization of the taurine/beta-alanine transporter in mouse brain suggests a potential function for taurine and beta-alanine as Neurotransmitters.
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A family of genes encoding Neurotransmitter Transporters.
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Qing-rong Liu, Hannah Nelson, Sreekala Mandiyan, Nathan NelsonAbstract:Abstract The genomic and cDNA clones of the mouse gamma-aminobutyric acid transporter were sequenced and analyzed. The genomic clone contains 12 introns including 1 intron prior to the initiator methionine. The second intron comes immediately after the stretch of amino acids that is most conserved among the Neurotransmitter Transporters sequenced so far. By using a probe constructed according to this conserved region, several partial genomic clones were isolated. Sequence analysis of those clones reveals not only homology to the family of Neurotransmitter Transporters within the reading frame but also an identical location of an exon-intron junction after the conserved region. A search of the GenBank data base (April 1991) revealed that two invertebrate genes exhibit homology to the conserved sequence of the above family. One, a Drosophila melanogaster gene, encoded the N-terminal part of a protein homologous to Neurotransmitter Transporters and the second was in Caenorhabditis elegans. The Drosophila gene contains an intron that starts at a position identical to the corresponding positions of all the mammalian genes of the family.