Neutral amino acid transport mediated by ortholog of imino acid transporter SIT1/SLC6A20 in opossum kidney cells

2006 ◽  
Vol 290 (4) ◽  
pp. F880-F887 ◽  
Author(s):  
Zorica Ristic ◽  
Simone M. R. Camargo ◽  
Elisa Romeo ◽  
Susana Bodoy ◽  
Joan Bertran ◽  
...  
Keyword(s):  
Amino Acids ◽  
Small Intestine ◽  
Amino Acid ◽  
Proximal Tubule ◽  
Amino Acid Transport ◽  
Acid Transport ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Imino Acid ◽  
Acid Transporter

Most neutral l-amino acid acids are transported actively across the luminal brush-border membrane of small intestine and kidney proximal tubule epithelial cells by a Na+ cotransport system named B0 that has been recently molecularly identified (B0AT1, SLC6A19). We show here that the opossum kidney-derived cell line OK also displays a Na+-dependent B0-type neutral l-amino acid transport, although with a slightly differing substrate selectivity. We tested the hypothesis that one of the two B0AT1-related transporters, SLC6A18 (ortholog of orphan transporter XT2) or SLC6A20 (ortholog of the recently identified mammalian imino acid transporter SIT1), mediates this transport. Anti-sense RNA to OK SIT1 ( oSIT1) but not to OK XT2 ( oXT2) inhibited Na+-dependent neutral amino acid transport induced by OK mRNA injected in Xenopus laevis oocytes. Furthermore, inhibition of oSIT1 gene expression in OK cells by transfection of siRNA and expression of shRNA selectively reduced the Na+-dependent uptake of neutral l-amino acids. Finally, expression of OK cell oSIT1 cRNA in X. laevis oocytes induced besides the transport of the l-imino acid l-Pro also that of neutral l-amino acids. Taken together, the data indicate that in OK cells SIT1 (SLC6A20) is not only an apical imino acid transporter but also plays a major role as Na+-dependent neutral l-amino acid transporter. A similar double role could be envisaged for SIT1 in mammalian kidney proximal tubule and small intestine.

Expression of mRNA (D2) encoding a protein involved in amino acid transport in S3 proximal tubule

1992 ◽  
Vol 263 (6) ◽  
pp. F1087-F1092 ◽  
Author(s):  
Y. Kanai ◽  
M. G. Stelzner ◽  
W. S. Lee ◽  
R. G. Wells ◽  
D. Brown ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Proximal Tubule ◽  
Amino Acid Transport ◽  
Glucose Transporter ◽  
Rat Kidney ◽  
Expression Cloning ◽  
Acid Transport ◽  
Specific Expression ◽  
High Affinity

A rat kidney- and intestine-specific cDNA (D2) that induces high-affinity, Na(+)-independent uptake of cystine and dibasic and neutral amino acids into cRNA-injected Xenopus oocytes was recently isolated by expression cloning in our laboratory (R. G. Wells and M. A. Hediger. Proc. Natl. Acad. Sci. USA 89: 5596-5600, 1992). At present it is not known whether the D2-encoded protein functions as a transporter or as a transporter activator. To gain more insight into the role of D2 in renal amino acid transport, we studied the site of its expression in the kidney. This was determined by Northern blot analysis and by using a combination of in situ hybridization and immunocytochemistry with antibodies that recognize specific proximal tubule segments. D2 antisense RNA hybridized to the same tubular segments that were strongly positive for anti-ecto-adenosinetriphosphatase but negative for carbonic anhydrase type IV and the facilitated glucose transporter GLUT2. We conclude that D2 mRNA is strongly expressed in the rat kidney proximal tubule S3 segment, although there is weak hybridization to the S1 and S2 segments. The signal is absent in all other parts of the kidney. The S3 specific expression of D2 mRNA coincides with the site of high-affinity transport of cystine and other amino acids, consistent with the proposed involvement of D2 in these processes.

scholarly journals Amino acid sensing by enteroendocrine STC-1 cells: role of the Na+-coupled neutral amino acid transporter 2

2010 ◽  
Vol 298 (6) ◽  
pp. C1401-C1413 ◽  
Author(s):  
Steven H. Young ◽  
Osvaldo Rey ◽  
Catia Sternini ◽  
Enrique Rozengurt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Isobutyric Acid ◽  
Amino Acid Transporter ◽  
Membrane Depolarization ◽  
Neutral Amino Acid ◽  
Acid Transport ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and α-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i). We sought to identify the mechanism by which amino acids induce Ca2+ signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na+-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca2+ signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca2+ signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca2+ signaling in STC-1 cells was suppressed by removing Na+ from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca2+]i; 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca2+ signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca2+]i was associated with membrane depolarization and mediated by Ca2+ influx, since it depended on extracellular Ca2+; 6) the increase in [Ca2+]i in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1–10 μM) or nitrendipine (1 μM), which block L-type voltage-sensitive Ca2+ channels. We hypothesize that the inward current of Na+ associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca2+ channels that mediate Ca2+ influx, thereby leading to an increase in the [Ca2+]i in enteroendocrine STC-1 cells.

Specificity of amino acid transport in renal papilla: microinfusion of Henle's loops and vasa recta

1991 ◽  
Vol 261 (3) ◽  
pp. F495-F504 ◽  
Author(s):  
W. H. Dantzler ◽  
S. Silbernagl
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Proximal Tubule ◽  
Amino Acid Transport ◽  
Renal Papilla ◽  
Acid Transport ◽  
Contralateral Kidney ◽  
Acidic Amino Acids ◽  
Vasa Recta ◽  
Competitive Inhibitors

Amino acids can be reabsorbed distal to tips of Henle's loops and may be recycled between loops and vasa recta in rat papilla. Transport specificity was examined during continuous microinfusions of ascending Henle's loops and vasa recta with radiolabeled amino acids. Percent of recovered radiolabel as intact amino acid was also determined. Previous data indicated that, relative to simultaneously microinfused inulin, 30-40% of radiolabeled L- and D-Ala, L-Glu, L-Glu(NH2), and Gly, but no taurine (Tau) or mannitol, microinfused into Henle's loops was reabsorbed. In the present study, reabsorption was shown to involve intact L- and D-Ala, D-Glu, and L-Ser. L-Phe (50 mM) in infusate had no effect on reabsorption of L-Ala (2.5 mM) or L-Glu(NH2) (42.6 microM), and D-Asp (50 mM) had no effect on reabsorption of L-Glu (1.5 mM). Thus reabsorption from Henle's loops is not stereospecific, not different for neutral and acidic amino acids, and not inhibited by competitive inhibitors of proximal tubule amino acid transport, but it was not completely nonspecific and not a simple leak. Previous vasa recta microinfusions suggested that Ala could move directly from vasa recta to tubules. These studies were extended with simultaneous collections from ipsilateral and contralateral kidneys. Relative to simultaneously microinfused inulin, 40–50% of radiolabeled L- and D-Ala, L-Glu, and L-Glu(NH2) and 30% of L-Ser microinfused into ascending vasa recta appeared intact in urine from ipsilateral kidney, whereas only 1–3% appeared in urine from contralateral kidney. Fifty percent of infused D-Glu was excreted intact by each kidney; 70% of infused Tau was excreted intact by ipsilateral kidney, and 22% was excreted by contralateral kidney. L-Phe (50 mM) in infusate inhibited appearance of L-Ala (2.5 mM) and D-Ala (10 mM) but not L-Glu(NH2) (42.6 microM) in ipsilateral urine. D-Asp (50 mM) inhibited appearance of L-Glu (1.5 mM), and beta-Ala (50 mM) inhibited appearance of Tau (78 microM) in ipsilateral urine. Thus some amino acids can move directly from vasa recta into tubules (probably descending thin limbs of Henle's loops) by a process showing significant specificity.

Characterization of a mouse colonic system B0+ amino acid transporter related to amino acid absorption in colon

2001 ◽  
Vol 281 (2) ◽  
pp. G365-G370 ◽  
Author(s):  
Shinya Ugawa ◽  
Yoko Sunouchi ◽  
Takashi Ueda ◽  
Eri Takahashi ◽  
Yoshitsugu Saishin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Transport System ◽  
Amino Acid Transport ◽  
Amino Acid Transporter ◽  
Acid Transport ◽  
Amino Acid Transport System ◽  
Acid Transporter

Previous experiments have shown that an amino acid transport system B0+ transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B0+ transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB0+. Northern blot analysis revealed that mCATB0+ was selectively expressed in the large intestine. In situ hybridization showed the mCATB0+ mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB0+ exhibited a Na+-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B0+. In vivo [3H]glycine uptake assay demonstrated that a system B0+-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB0+ may absorb amino acids from the intestinal contents in the colon.

scholarly journals A new family of proteins (rBAT and 4F2hc) involved in cationic and zwitterionic amino acid transport: a tale of two proteins in search of a transport function.

1994 ◽  
Vol 196 (1) ◽  
pp. 123-137 ◽  
Author(s):  
M Palacín
Keyword(s):  
Amino Acids ◽  
Small Intestine ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Transport Systems ◽  
Amino Acid Transporters ◽  
Cdna Clones ◽  
Missense Mutations ◽  
Acid Transport ◽  
High Affinity

The currently identified cDNA clones of mammalian amino acid transporters can be grouped into five different families. One family is composed of the proteins rBAT and the heavy chain (hc) of the cell surface antigen 4F2. RNAs encoding these two proteins induce a system b(o,+)-like (rBAT) and a system y+L-like (4F2hc) activity in Xenopus oocytes. Surprisingly, rBAT and 4F2hc do not seem to be pore-forming proteins. This finding supports the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transport systems. Expression of rBAT in oocytes induces high-affinity transport of cystine, which is shared with transport of cationic and zwitterionic amino acids. The rBAT gene is expressed mainly in kidney and small intestine. The rBAT protein is localized to the microvilli of proximal straight tubules of the kidney and mucosa from the small intestine. This finding is consistent with the involvement of rBAT in a high-affinity resorption system for cystine in the proximal straight tubule of the nephron. All of these characteristics suggest that rBAT is a good candidate for a cystinuria gene. Cystinuria is an inheritable defect in high-affinity transport of cystine, shared with cationic amino acids, through epithelial cells of the renal tubule and intestinal tract. Very recently, point missense mutations have been found in the rBAT gene of cystinuria patients. The most frequent rBAT mutation, M467T (threonine substitution of methionine at residue 467) nearly abolished the amino acid transport activity elicited by rBAT in oocytes. This result offers convincing evidence that rBAT is a cystinuria gene. Biochemical, cytological and genetic approaches are now needed to delineate the mechanism of action of rBAT and 4F2hc in the transport of amino acids.

scholarly journals Identification and Functional Characterization of the Lactococcus lactis CodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

2006 ◽  
Vol 188 (9) ◽  
pp. 3280-3289 ◽  
Author(s):  
Chris D. den Hengst ◽  
Maarten Groeneveld ◽  
Oscar P. Kuipers ◽  
Jan Kok
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
Amino Acid Transport ◽  
Amino Acid Transporter ◽  
Acid Transport ◽  
Amino Acid Permease ◽  
Branched Chain Amino Acid ◽  
Acid Transporter ◽  
Branched Chain

ABSTRACT Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation. CtrA is required for optimal growth in media containing free amino acids as the only amino acid source. Amino acid transport studies showed that ctrA encodes a secondary amino acid transport system that is specific for branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and methionine, which is in disagreement with its previously proposed function (a cationic amino acid transporter), which was assigned based on homology. We propose to rename CtrA BcaP, for branched-chain amino acid permease. BcaP is a member of a group of conserved transport systems, as homologs are widely distributed among gram-positive bacteria. Deletion of bcaP resulted in the loss of most of the BCAA uptake activity of L. lactis, indicating that BcaP is the major BCAA carrier of this organism. Deletion of bcaP together with a second (putative) BCAA permease, encoded by brnQ, further reduced the viability of the strain. DNA microarray analysis showed that deletion of bcaP predominantly affects genes belonging to the regulons of the transcriptional regulator CodY, which is involved in global nitrogen metabolism and needs BCAAs for its activation, and of CmbR, which is involved in sulfur amino acid metabolism.

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