The neuronal and astrocytic proteinSLC38A10 transports glutamine, glutamate, and aspartate, suggesting a role in neurotransmission

Abstract

In brain cells, glutamine transporters are vital to monitor and control the levels of glutamate and GABA . There are 11 members of the SLC 38 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, SLC 38A10. We used pairwise global sequence alignments to determine the sequence identity between the SLC 38 family members. SLC 38A10 was found to share 20–25% transmembrane sequence identity with several family members, and was predicted to have 11 transmembrane helices. SLC 38A10 immunostaining was abundant in mouse brain using a custom‐made anti‐ SLC 38A10 antibody and colocalization of SLC 38A10 immunoreactivity with markers for neurons and astrocytes was detected. Using Xenopus laevis oocytes overexpressing SLC 38A10, we show that SLC 38A10 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 SLC 38 family. In conclusion, the bidirectional transport of glutamine, glutamate, and aspartate by SLC 38A10, and the immunostaining detected in neurons and astrocytes, suggest that SLC 38A10 plays a role in pathways involved in neurotransmission.

Control of amino acid transport into Chinese hamster ovary cells

“…In CHO cells, the predominant uniport–symport loaders are SLC1A3 (anionic amino acids), SLC6A9, SLC38A2, SLC38A7 (neutral amino acids), SLC7A1 (cationic amino acids), SLC36A4 (Trp, Pro), and SLC43A2 (selected essential amino acids). The predominant harmonizing antiporters are SLC1A4 (Ala, Ser, Cys,), SLC1A5 (Gln, Ala, Ser, Cys, and other amino acids), SLC7A5–SLC3A2 (neutral amino acids), SLC7A6–SLC3A2 (Arg, Lys, His [in], Gln, Leu Met [out]), SLC7A11 (Cys [in], Glu [out]) and SLC38A10 (Gln, Ala, Glu, Asp [in–out], Ser [out]; Hellsten et al, ). Therefore, our data suggest that the majority of essential proteinogenic branched chain, aromatic amino acids, which generally exhibit uptake rates that correlate with cell‐specific growth rate (Figure ), are predominantly loaded by SLC43A2 and harmonized by SLC7A5–SLC3A2.…”

“…Exponential phase CHO cell cultures were seeded at 2 × 10 5 cells/ml in 10 ml of CD‐CHO medium in 50‐ml vented TubeSpin ® Bioreactors (TPP, Trasadingen, Switzerland) maintained at 170 rpm, 37°C, and 5% (vol/vol) CO 2 . After 24 hr, cells were recovered by centrifugation at 200 g for 5 min and the medium was replaced with fresh CD‐CHO medium containing well‐characterized inhibitors targeting highly expressed AATs in CHO and cancer cells (Bhutia et al, ; Kyriakopoulos et al, ; Table ), specifically (a) 2‐(methylamino)isobutyric acid (MeAIB; Sigma‐Aldrich, Poole, UK), which inhibits members of the SLC38 family— SLC38A1, SLC38A2, SLC38A4, and SLC38A10 (Hellsten, Hägglund, Eriksson, & Fredriksson, ; Sugawara et al, ; Varoqui, Zhu, Yao, Ming, & Erickson, ; Yao et al, ); (b) l ‐γ‐glutamyl‐p‐nitroanilide (GPNA; Sigma‐Aldrich) which inhibits SLC1A5 (Esslinger, Cybulski, & Rhoderick, ; Nicklin et al, ); (c) 2‐amino‐2‐norbornanecarboxylic acid (BCH; Sigma‐Aldrich), which inhibits SLC7A5, SLC7A8, SLC43A1, and SLC43A2 (Babu et al, ; Bodoy et al, ; Kanai et al, ; Pineda et al, ); (d) sulfasalazine (SAS; Sigma‐Aldrich) which inhibits SLC7A11 (Chung et al, ; Gout, Buckley, Simms, & Bruchovsky, ; Timmerman et al, ); and (e) N ‐[(3 R )‐3‐([1,1′‐Biphenyl]‐4‐yloxy)‐3‐(4‐fluorophenyl)propyl]‐ N ‐methylglycine hydrochloride (ALX‐5407; Tocris, Bristol, UK) which inhibits SLC6A5 and SLC6A9 (Atkinson et al, ). GPNA, SAS, and ALX‐5407 HCl were all solubilized in 0.2% (vol/vol) dimethyl sulfoxide (Sigma‐Aldrich) before addition to CD‐CHO medium at their final concentrations.…”

“…Duplicate RNA-seq analyses are shown at Day 4 and Day 8 sample points for each cell population. General amino acid transport specificity was compiled utilizing Bröer and Bröer (2017), Fotiadis, Kanai, and Palacín (2013), the IUPHAR Guide to Pharmacology transporter database (http://www.guidetopharmacology.org/), and more specifically Hellsten et al (2017) and Krall et al (2016), indicated by *1 and *2, respectively. The specificity of inhibitors of amino acid transport utilized in this study is indicated, specifically ALX-5407, MeAIB, GPNA, BCH, and SAS.…”

“…Our data reveal that whilst CHO cells utilize endogenous feedback controls to regulate the activity of predominant cell growth-associated amino transporters, the activity of other transporters associated with MAb production is not generally subject to cellular feedback control, indicating that a necessary adaptation to MAb production is selection of cell clones with an elevated expression of specific AATs. Transport of different subsets of amino acids was associated with either cell growth or MAb production, where transport of amino acids utilizing (Hellsten, Hägglund, Eriksson, & Fredriksson, 2017;Sugawara et al, 2000;Yao et al, 2000); (b) L-γ-glutamyl-p-nitroanilide (GPNA; Sigma-Aldrich) which inhibits SLC1A5 (Esslinger, Cybulski, & Rhoderick, 2005;Nicklin et al, 2009); (c) 2-amino-2-norbornanecarboxylic acid (BCH; Sigma-Aldrich), which inhibits SLC7A5, SLC7A8, SLC43A1, and SLC43A2 (Babu et al, 2003;Bodoy et al, 2005;Kanai et al, 1998;Pineda et al, 1999) Stationary phase cultures were seeded at 2 × 10 5 cells/ml in 140 ml of CD-CHO medium in 500-ml vented Erlenmeyer flasks maintained at 140 rpm, 37°C, and 5% (vol/vol) CO 2 and grown until stationary phase was reached on Days 5-6. Culture aliquots containing 5 × 10 6 cells were then transferred to 50-ml vented TubeSpin Bioreactors, recovered by centrifugation, and cultured as described above in conditioned (Days 5-6) culture medium containing solubilized inhibitors.…”

Colocalization of MOR1 and GAD67 in Mouse Nucleus Accumbens

“…Specifically, the NAcc efferent projections are believed to be GABAergic to the ventral tegmental area (VTA) and ventral pallidum (VP) 2 and thus we expected a robust immunostaining of the GABA neurons in the NAcc. To identify GABAergic neurons, we used GAD67 as the neuronal marker 16,17 . Figure 1 (A,B,C) shows GAD67 immunostaining in the NAcc neurons using the primary mouse anti-GAD67 antibody visualized with a rhodamine red TM -X-conjugated secondary antibody.…”