Publications

• Molecular cloning of the mouse IMINO system: an Na(+)- and Cl(-)-dependent proline transporter.

  Kowalczuk S., Broeer A., Munzinger M., Tietze N., Klingel K., Broeer S.

  Neurotransmitter transporters of the SLC6 family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. Here we demonstrate that the mouse homologue of slc6a20 has all properties of the long-sought IMINO system. The mouse has two ... >> More

  Neurotransmitter transporters of the SLC6 family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. Here we demonstrate that the mouse homologue of slc6a20 has all properties of the long-sought IMINO system. The mouse has two homologues corresponding to the single human SLC6A20 gene: these have been named XT3 and XT3s1. Expression of mouse XT3s1, but not XT3, in Xenopus laevis oocytes induced an electrogenic Na+-and-Cl--dependent transporter for proline, hydroxyproline, betaine, N-methylaminoisobutyric acid and pipecolic acid. Expression of XT3s1 was found in brain, kidney, small intestine, thymus, spleen and lung, whereas XT3 prevailed in kidney and lung. Accordingly we suggest that the two homologues be termed 'XT3s1 IMINO(B)' and 'XT3 IMINO(K)' to indicate the tissue expression of the two genes. << Less

  Biochem. J. 386:417-422(2005) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Human brain-specific L-proline transporter: molecular cloning, functional expression, and chromosomal localization of the gene in human and mouse genomes.

  Shafqat S., Velaz-Faircloth M., Henzi V.A., Whitney K.D., Yang-Feng T.L., Seldin M.F., Fremeau R.T. Jr.

  L-Proline fulfills several of the classic criteria used to identify amino acid neurotransmitters, including the presence of a high affinity, Na(+)-(and Cl-)-dependent synaptosomal transport process and the Ca(2+)-dependent release of exogenously loaded radiolabeled L-proline from brain slices and ... >> More

  L-Proline fulfills several of the classic criteria used to identify amino acid neurotransmitters, including the presence of a high affinity, Na(+)-(and Cl-)-dependent synaptosomal transport process and the Ca(2+)-dependent release of exogenously loaded radiolabeled L-proline from brain slices and synaptosomes after K(+)-induced depolarization. However, studies to define the role of L-proline in discrete pathways in the mammalian brain have been precluded by the inability to block its biosynthesis or high affinity transport in nervous tissue. We report the molecular cloning, functional expression, and chromosomal localization of a human brain-specific high affinity L-proline transporter (hPROT). The pharmacological specificity, kinetic properties, and ionic requirements of hPROT clearly distinguish this carrier from the other Na(+)-dependent plasma membrane carriers that transport L-proline. Multiple tissue Northern blot analysis revealed a prominent approximately 4-kb mRNA transcript in human brain tissue, whereas no specific hybridizing species were detected in peripheral tissue. An antipeptide antiserum directed against the carboxy-terminus of the predicted hPROT protein identified a single, broad immunoreactive protein of 68 kDa on immunoblots of synaptosomal membranes from various human brain regions. In contrast, no specific labeling was detected on immunoblots of membranes from human liver, kidney, or heart. A differential distribution of hPROT mRNA and protein was observed in the human corpus striatum, consistent with the hypothesis that the hPROT protein is synthesized in neuronal cell bodies in an extrastriatal location and axonally transported to the corpus striatum. These findings warrant the consideration of a synaptic regulatory role for this transporter and its presumed natural substrate, L-proline, in the mammalian central nervous system. << Less

  Mol. Pharmacol. 48:219-229(1995) [PubMed] [EuropePMC]

• Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20).

  Broer A., Balkrishna S., Kottra G., Davis S., Oakley A., Broer S.

  The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na(+) and Cl(-)-dependent, but ... >> More

  The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na(+) and Cl(-)-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na(+)-binding sites and a putative Cl(-)-binding site. Mutation of all residues in the two proposed Na(+)-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na(+)-binding site 1) and mutants S20A, S20G, S20G/G405S (Na(+)-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na(+)-binding site 1 caused a decrease of the Na(+)-K(0.5), whereas mutations of site 2 increased the K(0.5). Mutation of Na(+)-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates (36)Cl(-) demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na(+)/1Cl(-)-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria. << Less

  Mol Membr Biol 26:333-346(2009) [PubMed] [EuropePMC]

• Identification of mammalian proline transporter SIT1 (SLC6A20) with characteristics of classical system imino.

  Takanaga H., Mackenzie B., Suzuki Y., Hediger M.A.

  Amino acid homeostasis depends on specific amino acid transport systems, many of which have been characterized at the molecular level. However, the classical System IMINO, defined as the Na+-dependent proline transport activity that escapes inhibition by alanine, had not been identified at the mol ... >> More

  Amino acid homeostasis depends on specific amino acid transport systems, many of which have been characterized at the molecular level. However, the classical System IMINO, defined as the Na+-dependent proline transport activity that escapes inhibition by alanine, had not been identified at the molecular level. We report here the functional characteristics and tissue distribution of Sodium/Imino-acid Transporter 1 (SIT1), which exhibits the properties of classical System IMINO. SIT1, the product of the slc6a20 gene, is a member of the SLC6 Na+- and Cl--dependent neurotransmitter transporter family whose function has remained unknown. When expressed in Xenopus oocytes, rat SIT1 mediated the uptake of imino acids such as proline (K0.5 approximately 0.2 mM) and pipecolate, as well as N-methylated amino acids (e.g. MeAIB, sarcosine). SIT1-mediated proline transport was pH-independent and insensitive to inhibition by alanine or lysine. Proline transport was Na+-dependent, Cl--stimulated, and voltage-dependent. Li+, but not H+, could substitute for Na+. Human SIT1 also functioned as a Na+-dependent proline transporter. Rat SIT1 mRNA was expressed in epithelial cells of duodenum, jejunum, ileum, stomach, cecum, colon, and kidney proximal tubule S 3 segments. SIT1 mRNA was also expressed in the choroid plexus, microglia, and meninges of the brain and in the ovary. Previous reports have documented the marked urinary hyperexcretion of proline in newborn rodents and man. We found that SIT1 was dramatically up-regulated in the kidneys of 3-day-old mice, accounting for the maturation of proline reabsorption in the mouse. The human slc6a20 gene coding SIT1 is an appropriate target for investigation of hereditary forms of iminoaciduria in man. << Less

  J. Biol. Chem. 280:8974-8984(2005) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• L-proline and L-pipecolate induce enkephalin-sensitive currents in human embryonic kidney 293 cells transfected with the high-affinity mammalian brain L-proline transporter.

  Galli A., Jayanthi L.D., Ramsey I.S., Miller J.W., Fremeau R.T. Jr., DeFelice L.J.

  The high-affinity mammalian brain L-proline transporter (PROT) belongs to the GAT1 gene family, which includes Na- and Cl-dependent plasma membrane carriers for neurotransmitters, osmolites, and metabolites. These transporters couple substrate flux to transmembrane electrochemical gradients, parti ... >> More

  The high-affinity mammalian brain L-proline transporter (PROT) belongs to the GAT1 gene family, which includes Na- and Cl-dependent plasma membrane carriers for neurotransmitters, osmolites, and metabolites. These transporters couple substrate flux to transmembrane electrochemical gradients, particularly the Na gradient. In the nervous system, transporters clear synapses and help to replenish transmitters in nerve terminals. The localization of PROT to specific excitatory terminals in rat forebrain suggests a role for this carrier in excitatory transmission (). We investigated the voltage regulation and electrogenicity of this novel transporter, using human embryonic kidney (HEK) 293 cells stably transfected with rat PROT cDNA. In physiological solutions between -140 and -40 mV, L-proline (PRO) and its six-member ring congener L-pipecolate (PIP) induced inward current. The current-voltage relationship and the variance of current fluctuations were similar for PRO- and PIP-induced current, and the ratio of induced variance to the mean current ranged from 20 to 60 fA. Des-Tyr-Leu-enkephalin (GGFL), a competitive peptide inhibitor of PROT, reduced the rat PROT-associated current to control levels. GGFL alone did not elicit currents, and the GGFL-sensitive substrate-induced current was absent in nontransfected cells. Finally, GGFL inhibited PROT-mediated transport only when applied to the extracellular face of PROT. These data suggest that (1) PROT uptake is electrogenic, (2) individual transporter currents are voltage-independent, and (3) GGFL is a nonsubstrate inhibitor that interacts either with an extracellular domain of PROT or in an externally accessible pore. << Less

  J. Neurosci. 19:6290-6297(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.