Biochemical and Biophysical Research Communications
Characterization of a branched-chain amino-acid transporter SBAT1 (SLC6A15) that is expressed in human brain☆
Section snippets
Methods
Isolation of SBAT1 cDNA. To clone human SBAT1, we added 100 ng of random hexamers and 1 μl dNTPs (10 mM) to 0.5 μg of human brain poly(A)+ RNA (Invitrogen) in a total volume of 10 μl, and incubated the mixture for 10 min at 65 °C before it was chilled on ice. We then added 2 μl dithiothreitol (0.1 M), 2 μl of 10× reverse transcriptase buffer, 4 μl MgCl2 (25 mM), and 40 U of RNaseOUT (Invitrogen) before incubating the mixture at 25 °C for 2 min. To initiate cDNA synthesis, we added 200 U of Superscript II-RT
Human SBAT1 is a member of the SLC6 transporter family
Human and rat SBAT1 were previously known as V7-3, NTT73, or v-7-3-2 [10], [17], [18], [19]. One of these reports [17] described the specific CNS localization of human SBAT1, but the present study is the first to report the functional properties of SBAT1. The SLC6 gene family includes the Na+/Cl−-dependent transporters (e.g., GAT1-3, GLYT1-2, and SERT). SBAT1 (SLC6A15) belongs to a new branch of the SLC6 family (Fig. 1), along with SLC6A16 (NTT5), SLC6A17 (Ntt4/Rxt1), SLC6A18 (Xtrp2/ROSIT),
Discussion
We show here that the product of the SLC6A15 gene is a Na+-coupled amino acid transporter, which we named SBAT1. SBAT1 exhibits a preference for the branched-amino acids (BCAA) plus methionine, but is reactive also with each of the hydrophobic, zwitterionic α-amino acids and the imino acid, proline. SBAT1 excludes the aromatic amino acids, charged amino acids, β-amino acids, glycine, and GABA. We also isolated a rat SBAT1 (slc6a15) clone (NM_172321) with 83% amino acid identity to human SBAT1.
Note added in proof
A study of the phylogeny and evolution of the SLC6 gene family [36] appeared after the submission of our manuscript.
Acknowledgments
This research was supported by US Army Grant DAMD17-02-1-0016 to M.A.H. and by a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science to H.T.
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2021, Comparative Biochemistry and Physiology -Part A : Molecular and Integrative PhysiologyCitation Excerpt :B0AT2, also known as SBAT1 (SLC6A15), is a neuron-specific neutral AA transporter. B0AT2 transports several large zwitterionic AAs, but with a preference for the branched-chain AAs, methionine and proline (Km ~ 40–200 μM) (Bröer et al., 2006; Takanaga et al., 2005a). It also accepts glutamine, phenylalanine and alanine with lower affinities (Km ~ 0.6–5.3 mM) (Bröer et al., 2006).
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Abbreviations: BCAA, branched-chain amino acid(s); BCH, 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic-acid; 8-Br-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; ChoCl, choline chloride; DOG, 1,2-dioctanoyl-sn-glycerol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO-Pro, hydroxy-proline; MeAIB, 2-methylaminoisobutyrate; NMDGCl, N-methyl-d-glucamine chloride; NaGlc, sodium gluconate.