Luminal kidney and intestine SLC6 amino acid transporters of B⁰AT-cluster and their tissue distribution in Mus musculus

Abstract

The B 0 transport system mediates the Na ϩ -driven uptake of a broad range of neutral amino acids into epithelial cells of small intestine and kidney proximal tubule. A corresponding transporter was identified in 2004 (A. Broer, K. Klingel, S. Kowalczuk, J. E. Rasko, J. Cavanaugh, and S. Broer. J Biol Chem 279: [24467][24468][24469][24470][24471][24472][24473][24474][24475][24476] 2004) within the SLC6 family and named B 0 AT1 (SLC6A19). A phylogenetically related transporter known as XT3 in human (SLC6A20) and XT3s1 in mouse was shown to function as an imino acid transporter, to localize also to kidney and small intestine and renamed SIT1 or Imino B . Besides these two transporters with known functions, there are two other gene products belonging to the same phylogenetic B 0 AT-cluster, XT2 (SLC6A18) and rodent XT3 that are still "orphans." Quantitative real-time RT-PCR showed that the mRNAs of the four B 0 AT-cluster members are abundant in kidney, whereas only those of B 0 AT1 and XT3s1/SIT1 are elevated in small intestine. In brain, the XT3s1/SIT1 mRNA is more abundant than the other B 0 AT-cluster mRNAs. We show here by immunofluorescence that all four mouse B 0 AT-cluster transporters localize, with differential axial gradients, to the brushborder membrane of proximal kidney tubule and, with the possible exception of XT3, also of intestine. Deglycosylation and Western blotting of brush-border proteins demonstrated the glycosylation and confirmed the luminal localization of B 0 AT1, XT2, and XT3. In summary, this study shows the luminal brush-border localization of the Na ϩ -dependent amino and imino acid transporters B 0 AT1 and XT3s1/SIT1 in kidney and intestine. It also shows that the structurally highly similar orphan transporters XT2 and XT3 have the same luminal but a slightly differing axial localization along the kidney proximal tubule. B 0 AT1; XT2; SIT1; epithelial transporters; small intestine INGESTED DIETARY PROTEINS are cleaved into small oligopeptides and single amino acids that are then absorbed across small intestine enterocytes. Similarly, small oligopeptides and single amino acids are reabsorbed across kidney proximal tubule epithelial cells to prevent their loss in the urine. The first step of this transcellular transport is the influx across the luminal brush-border membrane that is mediated by symporters (cotransporters) and antiporters (exchangers; see Refs. 36). The basolateral efflux of amino acids from the epithelial cells into the extracellular space is mediated by facilitated diffusion pathway(s) and exchangers (36).

Impaired Nutrient Signaling and Body Weight Control in a Na+ Neutral Amino Acid Cotransporter (Slc6a19)-deficient Mouse

“…2) as we and others have reported recently (10,19,20). In wild type kidney, expression of B 0 AT1 was co-localized with the proximal tubule marker Lotus tetragonolobus agglutinin on the apical membranes of proximal tubular epithelial cells (Fig.…”

Orphan Transporter SLC6A18 Is Renal Neutral Amino Acid Transporter B0AT3

“…RNA and Real Time Quantitative Reverse Transcription-PCR-RNA was extracted from frozen kidneys, and real time quantitative reverse transcription-PCR was performed as previously described (3). Three samples per mouse and genotype were run, and the abundance of the target mRNAs was calculated relative to hypoxanthine guanine phosphoribosyltransferase or glyceraldehyde 3-phosphate dehydrogenase as a reference.…”

“…Brush border membrane vesicles were prepared from whole mouse kidney using the Mg 2ϩ precipitation technique, as described elsewhere (15). Immunoblotting of the equivalent of one oocyte or 20 g of brush border membrane vesicles was performed as described elsewhere (3). Primary antibodies were diluted 1:2000 for rabbit affinity-purified (AP) 4 anti-B 0 AT1 ( tems, Minneapolis, MI), and 1:10,000 for mouse anti-␤-actin (Sigma).…”

“…Immunofluorescence-Immunofluorescence was performed as previously described (3). Primary antibodies were diluted 1:200 for rabbit AP anti-mXT2 (3), 1:100 for AP goat antimACE2 (R&D Systems), and 1:1000 for rabbit serum antimCollectrin (antigen peptide: NH 2 -CDPLDMKGGHINDG-FLT-CONH2 as in Ref.…”

“…A cloning study identified, besides the A12 isoform that resembles most the other Slc6 family members, five shorter transcripts, the physiological significance of which is not established (1). In a localization study performed using mouse tissues, we have shown that XT2 is mainly expressed in the kidney, where it localizes to the brush border membrane of the late proximal tubule (S2, S3) in a complementary fashion to B 0 AT1 that localizes to the early proximal tubule (S1) (3).…”