ABSTRACT
BACKGROUND: The vesicular B0AT3 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 B0AT3 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. B0AT3 was highly expressed in both inhibitory and excitatory neurons. The B0AT3 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 B0AT3 transporter have a role in regulation of monoaminergic as well as glutamatergic synapses.
Subject(s)
Central Nervous System/physiology , Gene Expression Regulation/physiology , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Animals , Antidepressive Agents/pharmacology , Cells, Cultured , Central Nervous System/cytology , Central Nervous System/drug effects , Embryo, Mammalian , Female , Food Deprivation , Gene Expression Regulation/drug effects , Male , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Neurons/drug effects , Neurons/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Pregnancy , Protein Transport/physiology , Rats , Rats, Sprague-Dawley , Rats, Wistar , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/metabolismABSTRACT
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.
Subject(s)
Axons/metabolism , Brain/metabolism , Carrier Proteins/metabolism , Glutamine/metabolism , Nerve Tissue Proteins/metabolism , Organic Cation Transport Proteins/metabolism , Animals , Carrier Proteins/genetics , Gene Expression Regulation/physiology , Glutamine/genetics , Ion Transport/physiology , Mice , Nerve Tissue Proteins/genetics , Organic Cation Transport Proteins/geneticsABSTRACT
Glutamine transporters are important for regulating levels of glutamate and GABA in the brain. To date, six members of the SLC38 family (SNATs) have been characterized and functionally subdivided them into System A (SNAT1, SNAT2 and SNAT4) and System N (SNAT3, SNAT5 and SNAT7). Here we present the first functional characterization of SLC38A8, one of the previous orphan transporters from the family, and we suggest that the encoded protein should be named SNAT8 to adhere with the SNAT nomenclature. We show that SLC38A8 has preference for transporting L-glutamine, L-alanine, L-arginine, L-histidine and L-aspartate using a Na+-dependent transport mechanism and that the functional characteristics of SNAT8 have highest similarity to the known System A transporters. We also provide a comprehensive central nervous system expression profile in mouse brain for the Slc38a8 gene and the SNAT8 protein. We show that Slc38a8 (SNAT8) is expressed in all neurons, both excitatory and inhibitory, in mouse brain using in situ hybridization and immunohistochemistry. Furthermore, proximity ligation assay shows highly similar subcellular expression of SNAT7 and SNAT8. In conclusion, the neuronal SLC38A8 has a broad amino acid transport profile and is the first identified neuronal System A transporter. This suggests a key role of SNAT8 in the glutamine/glutamate (GABA) cycle in the brain.
Subject(s)
Alanine/metabolism , Amino Acid Transport Systems, Neutral/metabolism , Arginine/metabolism , Brain/metabolism , Glutamine/metabolism , Histidine/metabolism , Neurons/metabolism , Amino Acid Transport Systems, Neutral/genetics , Animals , Blotting, Western , Brain/cytology , Cells, Cultured , Electrophysiology , Female , Fluorescent Antibody Technique , Immunoenzyme Techniques , In Situ Hybridization , Ion Transport , Male , Mice , Mice, Inbred C57BL , Neurons/cytology , Oocytes/cytology , Oocytes/metabolism , Phylogeny , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sodium/metabolism , Xenopus laevisABSTRACT
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.