Functional Characterization of Two Novel Mammalian Electrogenic Proton-dependent Amino Acid Cotransporters (original) (raw)
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A cluster of proton/amino acid transporter genes in the human and mouse genomes☆
Genomics, 2003
We recently cloned and functionally characterized two novel proton/amino acid transporters (PAT1 and PAT2) from mouse. Here we report the isolation of the corresponding cDNAs of the human orthologues and one additional mouse and human PAT-like transporter cDNA, designated PAT3. The PAT proteins comprise 470 to 483 amino acids. The mouse PAT3 mRNA is expressed in testis of adult mice. In the human and mouse genomes the genes of the PAT transporters (designated SLC36A1-3 and Slc36a1-3, respectively) are clustered on human chromosome 5q33.1 and in the syntenic region of mouse chromosome 11B1.3. PAT-like transporter genes are present as well in the genomes of other eukaryotic organisms such as Drosophila melanogaster and Caenorhabditis elegans. For the PAT3 subtype transporter, we could not yet identify its function. The human PAT1 and PAT2 transporters when functionally expressed in Xenopus laevis oocytes show characteristics similar to those of their mouse counterparts.
British Journal of Pharmacology, 2009
1 Functional characteristics and substrate specificity of the rat proton-coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrateevoked current using the two-electrode voltage-clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2. 2 Amino (and imino) acid transport via rPAT2 was pH-dependent, Na þ -independent and electrogenic. At extracellular pH 5.5 (in Na þ -free conditions) proline uptake was saturable (Km 172741 mM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter. 3 PAT2 preferred substrates are L-a-amino acids with small aliphatic side chains (e.g. the methyl group in alanine) and 4-or 5-membered heterocyclic amino and imino acids such as 2-azetidinecarboxylate, proline and cycloserine, where both D-and L-enantiomers are transported. 4 The major restrictions on transport are side chain size (the ethyl group of a-aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH 2 groups, as in b-alanine, is enough to reduce transport. Methylation of the amino group is tolerated (e.g. sarcosine) but increasing methylation, as in betaine, decreases transport. A free carboxyl group is preferred as O-methyl esters show either reduced transport (alanine-O-methyl ester) or are excluded. 5 The structural characteristics that determine the substrate specificity of rPAT2 have been identified. This information should prove valuable in the design of selective substrates/inhibitors for PAT1 and PAT2. -Omethyl ester; O-Me-b-Ala, b-alanine-O-methyl ester; O-Me-Gly, glycine-O-methyl ester; O-Me-Pro, proline-Omethyl ester; PAT1, proton-coupled amino acid transporter 1; PAT2, proton-coupled amino acid transporter 2; pH i , intracellular pH; rPAT2, rat proton-coupled amino acid transporter 2; SLC36, solute carrier family 36; Slc36a2, solute carrier family 36 member 2; Tramdorin 1, transmembrane domain-rich protein 1
Adaptation of plasma membrane amino acid transport mechanisms to physiological demands
Pfl�gers Archiv European Journal of Physiology, 2002
The molecular identification of almost all physiologically characterized amino acid transporters in recent years has facilitated the functional analysis of this important class of transport proteins. The picture that emerges from these studies is that antiport is the prevalent mode of amino acid transport rather than a combination of uniporters and cotransporters. Mainly neurotransmitters and osmolytes are transported by complex cotransport mechanisms that allow a high intracellular accumulation. Antiport mechanisms almost invariably include the nonessential amino acids alanine and glutamine, which are used as exchange substrates. The intracellular level of both amino acids is well regulated by Na + /amino acid cotransporters. Transport mechanisms are not conserved within families and may change with mutation of even a single amino acid residue in the transport protein. Thus transport mechanisms are easily adapted to physiological demands during evolution.
Structure, tissue expression pattern, and function of the amino acid transporter rat PAT2
Biochemical and Biophysical Research Communications, 2003
The second member of the PAT (proton-coupled amino acid transporter) family of H þ -coupled, pH-dependent, Na þ -independent amino acid transporters was isolated from a rat lung cDNA library. The cDNA for rat PAT2 is 2396 bp in length, including 70 bp of 5 0 UTR and a poly(A) tail. The transporter gene, consisting of 10 exons, is located on rat chromosome 10q22. The cDNA codes for a protein of 481 amino acids with 72% identity (over 449 amino acids) with rat PAT1. Tissue expression studies demonstrate that mRNA abundance is generally low with highest levels being detected in lung and spleen, with lower levels in the brain, heart, kidney, and skeletal muscle. Functional expression in either mammalian cells or Xenopus laevis oocytes demonstrates that rat PAT2 mediates pH-dependent, Na þ -independent uptake of glycine, proline, and a(methyl)aminoisobutyric acid (MeAIB). In conclusion PAT2 has a limited tissue distribution, higher affinity (Michaelis-Menten constant for glycine uptake between 0.49 and 0.69 mM), and distinct substrate specificity compared to PAT1.
Journal of Biological Chemistry, 1993
A cDNA has been isolated from human hippocampus that appears to encode a novel Na+-dependent, Cl-independent, neutral amino acid transporter. The putative protein, designated SA", is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na+-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATT's apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines. Carrier-mediated amino acid transport has evolved to maintain transmembrane fluxes of amino acids for cellular nutrition and metabolism. In eukaryotic cells, a large number of distinct amino acid transport systems have been distinguished based on differences in substrate specificity, kinetic properties, and ionic dependence (1, 2). Our knowledge of the structural properties of amino acid transporters is limited. However, recent advances in the cloning of amino acid transporters have begun to provide insights into the molecular basis of carrier-mediated amino acid transport. A cDNA clone, SAAT1, has recently been described that exhibits structural and sequence similarity with
Electrogenic amino acid exchange via the rBAT transporter
FEBS Letters, 1994
A cDNA clone was isolated from rabbit renal cortex using DNA-mediated expression cloning, which caused alanine-dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na-independent amino acid transporter previously cloned elsewhere. Exposure of cDNA-injected oocytes to neutral amino acids led to voltage-dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut-open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.
Characterization of mouse amino acid transporter B0AT1 (slc6a19)
Biochemical Journal, 2005
The mechanism of the mouse (m)B0AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB0AT1. Substrates were transported with K0.5 values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na+–amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na+ concentration reduced the K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the t...