The polyamine transporter Slc18b1(VPAT) is important for both short and long time memory and for regulation of polyamine content in the brain.

SLC18B1 is a sister gene to the vesicular monoamine and acetylcholine transporters, and the only known polyamine transporter, with unknown physiological role. We reveal that Slc18b1 knock out mice has significantly reduced polyamine content in the brain providing the first evidence that Slc18b1 is functionally required for regulating polyamine levels. We found that this mouse has impaired short and long term memory in novel object recognition, radial arm maze and self-administration paradigms. We also show that Slc18b1 KO mice have altered expression of genes involved in Long Term Potentiation, plasticity, calcium signalling and synaptic functions and that expression of components of GABA and glutamate signalling are changed. We further observe a partial resistance to diazepam, manifested as significantly lowered reduction in locomotion after diazepam treatment. We suggest that removal of Slc18b1 leads to reduction of polyamine contents in neurons, resulting in reduced GABA signalling due to long-term reduction in glutamatergic signalling.

The neuronal and astrocytic protein SLC38A10 transports glutamine, glutamate, and aspartate, suggesting a role in neurotransmission.

In brain cells, glutamine transporters are vital to monitor and control the levels of glutamate and GABA. There are 11 members of the SLC38 family of amino acid transporters of which eight have been functionally characterized. Here, we report the first histological and functional characterization of the previously orphan member, SLC38A10. We used pairwise global sequence alignments to determine the sequence identity between the SLC38 family members. SLC38A10 was found to share 20-25% transmembrane sequence identity with several family members, and was predicted to have 11 transmembrane helices. SLC38A10 immunostaining was abundant in mouse brain using a custom-made anti-SLC38A10 antibody and colocalization of SLC38A10 immunoreactivity with markers for neurons and astrocytes was detected. Using Xenopus laevis oocytes overexpressing SLC38A10, we show that SLC38A10 mediates bidirectional transport of l-glutamine, l-alanine, l-glutamate, and d-aspartate, and efflux of l-serine. This profile mostly resembles system A members of the SLC38 family. In conclusion, the bidirectional transport of glutamine, glutamate, and aspartate by SLC38A10, and the immunostaining detected in neurons and astrocytes, suggest that SLC38A10 plays a role in pathways involved in neurotransmission.

Transport of L-glutamine, L-alanine, L-arginine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS.

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.

The G protein-coupled receptor GPR162 is widely distributed in the CNS and highly expressed in the hypothalamus and in hedonic feeding areas.

The Rhodopsin family is a class of integral membrane proteins belonging to G protein-coupled receptors (GPCRs). To date, several orphan GPCRs are still uncharacterized and in this study we present an anatomical characterization of the GPR162 protein and an attempt to describe its functional role. Our results show that GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in areas related to energy homeostasis and hedonic feeding such as hypothalamus, amygdala and ventral tegmental area, regions known to be involved in the regulation of palatable food consumption.

Obesity-linked homologues TfAP-2 and Twz establish meal frequency in Drosophila melanogaster.

In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2ß and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2ß and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2ß and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2ß may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior.

Characterization of the transporterB0AT3 (Slc6a17) in the rodent central nervous system.

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.

B(0)AT2 (SLC6A15) is localized to neurons and astrocytes, and is involved in mediating the effect of leucine in the brain.

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.

Evolutionary origin of amino acid transporter families SLC32, SLC36 and SLC38 and physiological, pathological and therapeutic aspects.

About 25% of all solute carriers (SLCs) are likely to transport amino acids as their primary substrate. One of the major phylogenetic clusters of amino acid transporters from the SLC family is the ß-family, which is part of the PFAM APC clan. The ß-family includes three SLC families, SLC32, SLC36 and SLC38 with one, four and eleven members in humans, respectively. The most well characterized genes within these families are the vesicular inhibitory amino acid transporter (VIAAT, SLC32A1), PAT1 (SLC36A1), PAT2 (SLC36A2), PAT4 (SLC36A4), SNAT1 (SLC38A1), SNAT2 (SLC38A2), SNAT3 (SLC38A3), and SNAT4 (SLC38A4). Here we review the structural characteristics and functional role of these transporters. We also mined the complete protein sequence datasets for nine different genomes to clarify the evolutionary history of the ß-family of transporters. We show that all three main branches of the this family are found as far back as green algae suggesting that genes from these families existed in the early eukaryote before the split of animals and plants and that they are present in most animal species. We also address the potential of further drug development within this field highlighting the important role of these transporters in neurotransmission and transport of amino acids as nutrients.

Identification of SLC38A7 (SNAT7) protein as a glutamine transporter expressed in neurons.

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.

The solute carrier (SLC) complement of the human genome: phylogenetic classification reveals four major families.

Solute carriers (SLCs) is the largest group of transporters, embracing transporters for inorganic ions, amino acids, neurotransmitters, sugars, purines and fatty acids among other substrates. We mined the finished assembly of the human genome using Hidden Markov Models (HMMs) obtaining a total of 384 unique SLC sequences. Detailed clustering and phylogenetic analysis of the entire SLC family showed that 15 of the families place into four large phylogenetic clusters with the largest containing eight SLC families, suggesting that many of the distinct families of SLCs have a common evolutionary origin. This study represents the first overall genomic roadmap of the SLCs providing large sequence sets and clarifies the phylogenetic relationships among the families of the second largest group of membrane proteins.