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Characterization of Amino Acid Transporters in the Brain: Molecular and Functional Studies of Members within the Solute Carrier Families SLC38 and SLC6
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology. (Robert Fredriksson)ORCID iD: 0000-0002-6839-3651
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Solute carriers (SLCs) comprise the largest group of transporters in humans and there are currently 52 SLC families. They are embedded in cellular membranes and transport numerous molecules; defects in many of the genes encoding SLCs have been connected to pathological conditions, and several SLCs are potential drug targets.

The SLC38 family has in total eleven members in humans and they encode transporters called SNATs. In paper I and paper II, we reported molecular and functional characterization of Slc38a7 and Slc38a8, two of the previous orphan members in the family which we suggested to be named SNAT7 and SNAT8, respectively. Using in situ hybridization and immunohistochemistry, these transporters showed similar expression pattern and localized to neurons in the brain For functional characterization proteins were overexpressed in X. laevis oocytes and an Uptake Assay and electrophysiological recordings showed preferred transport of L-glutamine, L-histidine, L-alanine, L-asparagine, L-aspartate and L-arginine for SNAT7. A similar pattern was seen for SNAT8 in a slightly different order of affinities. We classified SNAT7 as a system N transporter and SNAT8 as belonging to system A, and suggests that SNAT7 and SNAT8 could play a role in the glutamine/glutamate(GABA) cycle (GGC) in the brain.

Furthermore, we studied the vesicular B0AT3 (Slc6a17) transporter in paper III, and the sodium-coupled amino acid transporter B0AT2 (Slc6a15) in paper IV. Tissue expression studies showed similar localization of Slc6a17 and Slc6a15 mRNA using in situ hybridization and real-time PCR. In paper III, vesicular localization of B0AT2 was shown in both excitatory and inhibitory neurons. When challenging the monoaminergic system with drugs both Slc6a17 and Slc6a15 were upregulated. Suggested roles for the transporters are thereby in synaptic remodeling by regulating the availability of free amino acids used as precursors needed in neurotransmitter synthesis. Moreover, in paper IV, immunohistochemistry showed B0AT3 localization to neurons, astrocytes and epithelial cells of the choroid plexus. Leucine injections caused a smaller reduction of food intake as well as higher neuronal activation in the paraventricular hypothalamic nucleus in Slc6a15 KO mice, compared with wild type mice. This suggests B0AT2 involvement in the anorexigenic effects of leucine.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 960
Keyword [en]
Amino acid transporter, Solute Carrier, Glutamine, Leucine, SNAT, B0AT
National Category
Neurosciences
Research subject
Neuroscience
Identifiers
URN: urn:nbn:se:uu:diva-212610ISBN: 978-91-554-8832-1 (print)OAI: oai:DiVA.org:uu-212610DiVA: diva2:679434
Public defence
2014-02-14, B/B22, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-01-23 Created: 2013-12-12 Last updated: 2014-02-10
List of papers
1. Identification of SLC38A7 (SNAT7) Protein as a Glutamine Transporter Expressed in Neurons
Open this publication in new window or tab >>Identification of SLC38A7 (SNAT7) Protein as a Glutamine Transporter Expressed in Neurons
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 23, 20500-20511 p.Article in journal (Refereed) Published
Abstract [en]

The SLC38 family of transporters has in total 11 members in humans and they encode amino acid transporters called sodium-coupled amino acid transporters (SNAT). To date, five SNATs have been characterized and functionally subdivided into systems A (SLC38A1, SLC38A2, and SLC38A4) and N (SLC38A3 and SLC38A5) showing the highest transport for glutamine and alanine. Here we present identification of a novel glutamine transporter encoded by the Slc38a7 gene, which we propose should be named SNAT7. This transporter has L-glutamine as the preferred substrate but also transports other amino acids with polar side chains, as well as L-histidine and L-alanine. The expression pattern and substrate profile for SLC38A7 shows highest similarity to the known system N transporters. Therefore, we propose that SLC38A7 is a novel member of this system. We used in situ hybridization and immunohistochemistry with a custom-made antibody to show that SLC38A7 is expressed in all neurons, but not in astrocytes, in the mouse brain. SLC38A7 is unique in being the first system N transporter expressed in GABAergic and also other neurons. The preferred substrate and axonal localization of SLC38A7 close to the synaptic cleft indicates that SLC38A7 could have an important function for the reuptake and recycling of glutamate.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-155238 (URN)10.1074/jbc.M110.162404 (DOI)000291267600037 ()
Available from: 2011-06-21 Created: 2011-06-20 Last updated: 2017-12-11Bibliographically approved
2. Transport of L-glutamine, L-alanine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS.: SNAT8 is a neuronal glutamine transporter.
Open this publication in new window or tab >>Transport of L-glutamine, L-alanine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS.: SNAT8 is a neuronal glutamine transporter.
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(English)Article in journal (Refereed) Submitted
Keyword
Glutamine/glutamate(GABA) cycle, Glutamine transporter, SLC38A8, SNAT8, Xenopus laevis oocytes
National Category
Cell and Molecular Biology
Research subject
Neuroscience
Identifiers
urn:nbn:se:uu:diva-212592 (URN)
Funder
Swedish Society for Medical Research (SSMF)Swedish Research Council
Available from: 2013-12-12 Created: 2013-12-12 Last updated: 2014-02-10Bibliographically approved
3. Characterization of the transporterB0AT3 (Slc6a17) in the rodent central nervous system
Open this publication in new window or tab >>Characterization of the transporterB0AT3 (Slc6a17) in the rodent central nervous system
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2013 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 14, 54- p.Article in journal (Refereed) Published
Abstract [en]

Background: The vesicular B(0)AT3 transporter (SLC6A17), one of the members of the SLC6 family, is a transporter for neutral amino acids and is exclusively expressed in brain. Here we provide a comprehensive expression profile of B(0)AT3 in mouse brain using in situ hybridization and immunohistochemistry. Results: We confirmed previous expression data from rat brain and used a novel custom made antibody to obtain detailed co-labelling with several cell type specific markers. B(0)AT3 was highly expressed in both inhibitory and excitatory neurons. The B(0)AT3 expression was highly overlapping with those of vesicular glutamate transporter 2 (VGLUT2) and vesicular glutamate transporter 1 (VGLUT1). We also show here that Slc6a17mRNA is up-regulated in animals subjected to short term food deprivation as well as animals treated with the serotonin reuptake inhibitor fluoxetine and the dopamine/noradrenaline reuptake inhibitor bupropion. Conclusions: This suggests that the B(0)AT3 transporter have a role in regulation of monoaminergic as well as glutamatergic synapses.

National Category
Neurosciences Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-205338 (URN)10.1186/1471-2202-14-54 (DOI)000320714200001 ()
Available from: 2013-08-16 Created: 2013-08-16 Last updated: 2017-12-06Bibliographically approved
4. B(0)AT2 (SLC6A15) is localized to neurons and astrocytes, and is involved in mediating the effect of leucine in the brain
Open this publication in new window or tab >>B(0)AT2 (SLC6A15) is localized to neurons and astrocytes, and is involved in mediating the effect of leucine in the brain
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 3, e58651- p.Article in journal (Refereed) Published
Abstract [en]

The B(0)AT2 protein is a product of the SLC6A15 gene belonging to the SLC6 subfamily and has been shown to be a transporter of essential branched-chain amino acids. We aimed to further characterize the B(0)AT2 transporter in CNS, and to use Slc6a15 knock out (KO) mice to investigate whether B(0)AT2 is important for mediating the anorexigenic effect of leucine. We used the Slc6a15 KO mice to investigate the role of B(0)AT2 in brain in response to leucine and in particular the effect on food intake. Slc6a15 KO mice show lower reduction of food intake as well as lower neuronal activation in the ventromedial hypothalamic nucleus (VMH) in response to leucine injections compared to wild type mice. We also used RT-PCR on rat tissues, in situ hybridization and immunohistochemistry on mouse CNS tissues to document in detail the distribution of SLC6A15 on gene and protein levels. We showed that B(0)AT2 immunoreactivity is mainly neuronal, including localization in many GABAergic neurons and spinal cord motor neurons. B(0)AT2 immunoreactivity was also found in astrocytes close to ventricles, and co-localized with cytokeratin and diazepam binding inhibitor (DBI) in epithelial cells of the choroid plexus. The data suggest that B(0)AT2 play a role in leucine homeostasis in the brain.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-199769 (URN)10.1371/journal.pone.0058651 (DOI)000318334500110 ()23505546 (PubMedID)
Available from: 2013-05-13 Created: 2013-05-13 Last updated: 2017-12-06Bibliographically approved

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