Neuronal and glial glycine transporters have different stoichiometries.
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A neurotransmitter transporter can potentially mediate uptake or release of substrate, and its stoichiometry is a key factor that controls the driving force and thus the neurotransmitter flux direction. We have used a combination of electrophysiology and radio-tracing techniques to evaluate the stoichiometries of two glycine transporters involved in glycinergic or glutamatergic transmission. We show that GlyT2a, a transporter present in glycinergic boutons, has a stoichiometry of 3 Na+/Cl-/glycine, which predicts effective glycine accumulation in all physiological conditions. GlyT1b, a glial transporter, has a stoichiometry of 2 Na+/Cl-/ glycine, which predicts that glycine can be exported or imported, depending on physiological conditions. GlyT1b may thus modulate glutamatergic synapses by increasing or decreasing the glycine concentration around N-methyl-D-aspartate receptors (NMDARs). (+info)
Characterization of the interactions between the glycine transporters GLYT1 and GLYT2 and the SNARE protein syntaxin 1A.
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In this study we have examined the effect of the SNARE protein syntaxin 1A on the glycine transporters GLYT1 and GLYT2. Our results demonstrate a functional and physical interaction between both glycine transporters and syntaxin 1A. Co-transfection of syntaxin 1A with GLYT1 or GLYT2 in COS cells resulted in approximately 40% inhibition in glycine transport. This inhibition was reversed by the syntaxin 1A-binding protein, Munc18. Furthermore, immunoprecipitation studies showed a physical interaction between syntaxin 1A and both transporters in COS cells and in rat brain tissue. Finally, we conclude that this physical interaction resulted in a partial removal of the glycine transporters from the plasma membrane as demonstrated by biotinylation studies. (+info)
A study of AroP-PheP chimeric proteins and identification of a residue involved in tryptophan transport.
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In vivo recombination has been used to make a series of AroP-PheP chimeric proteins. Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan. Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP. Phenylalanine is the residue at the corresponding position in PheP. The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane. Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P. These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110. Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins. (+info)
Transport properties of a system y+L neutral and basic amino acid transporter. Insights into the mechanisms of substrate recognition.
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The properties of system y(+)L-mediated transport were investigated on rat system y(+)L transporter, ry(+)LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopus oocytes. ry(+)LAT1-mediated transport of basic amino acids was Na(+)-independent, whereas that of neutral amino acids, although not completely, was dependent on Na(+), as is typical of system y(+)L-mediated transport. In the absence of Na(+), lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H(+), besides Na(+) and Li(+), is capable of supporting neutral amino acid transport. Na(+) and H(+) augmented leucine transport by decreasing the apparent K(m) values, without affecting the V(max) values. We demonstrate that although ry(+)LAT1-mediated transport of [(14)C]l-leucine was accompanied by the cotransport of (22)Na(+), that of [(14)C]l-lysine was not. The Na(+) to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na(+). ry(+)LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry(+)LAT1 and 4F2 heavy chain in the absence of Na(+), but not in the presence of physiological concentrations of Na(+), indicating that cotransport of H(+) with leucine occurred in the absence of Na(+). Therefore, for the substrate recognition by ry(+)LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na(+) and H(+), which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry(+)LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids. (+info)
Transmembrane domain III plays an important role in ion binding and permeation in the glycine transporter GLYT2.
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The neuronal glycine transporter GLYT2 takes up glycine from the extracellular space by an electrogenic process where this neurotransmitter is co-transported with sodium and chloride ions. We report in this paper that tyrosine at position 289 of GLYT2a is crucial for ion coupling, glycine affinity and sodium selectivity, stressing the essential role played by this residue of transmembrane domain III in the mechanism of transport. Substitution to tryptophan (Y289W), phenylalanine (Y289F), or serine (Y289S), renders transporters unable to catalyze glycine uptake. Measurements of glycine evoked steady-state currents in transfected HEK-293 cells reveal EC(50) values for glycine 17-fold (Y289F) and 45-fold (Y289S) higher than that of the wild type transporter. Sodium dependence is severely altered in tyrosine 289 mutants, both at the level of apparent affinity and cooperativity, with the more dramatic change corresponding to the less conservative substitution (Y289S). Accordingly, sodium selectivity is gradually lost in Y289F and Y289S mutants, and chloride dependence of glycine evoked currents is markedly decreased in Y289F and Y289S mutants. In the absence of three-dimensional information from these transporters, these results provide experimental evidence supporting the hypothesis of transmembrane domain III being part of a common permeation pathway for substrate and co-transported ions. (+info)
Effects of pH on the uncoupled, coupled and pre-steady-state currents at the amino acid transporter KAAT1 expressed in Xenopus oocytes.
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The effects of pH on the different kinds of currents occurring at the lepidopteran amino acid cotransporter KAAT1 have been examined using heterologous expression in Xenopus oocytes and voltage clamp. Acidic pH (5.5-4.5) caused a slight depression of the uncoupled current and a complete inhibition of the coupled and transient currents, in the presence of either Na+ or K+, the two ions physiologically relevant to the transport process. Conversely, at alkaline pH (9) no statistically significant effects could be observed on uncoupled, coupled and transient currents compared to the effects at pH 7.6. These effects of pH indicate that operation of the transporter is maximal in the physiologically alkaline native environment. The dose-response curves for the inhibition of coupled and transient currents were similar, with respective pKa values of 6. 29 +/- 0.05 and 6.40 +/- 0.03 and respective Hill coefficient values (nH) of 0.93 +/- 0.07 and 1.08 +/- 0.08, suggesting that the two effects can be explained by a single proton binding to the same site in the transporter. (+info)
Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter.
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We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic. (+info)
Amino acid transport system A resembles system N in sequence but differs in mechanism.
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Classical amino acid transport System A accounts for most of the Na(+)-dependent neutral amino acid uptake by mammalian cells. System A has also provided a paradigm for short- and long-term regulation by physiological stimuli. We now report the isolation of a cDNA encoding System A that shows close similarity to the recently identified System N transporter (SN1). The System A transporter (SA1) and SN1 share many functional characteristics, including a marked sensitivity to low pH, but, unlike SN1, SA1 does not mediate proton exchange. Transport mediated by SA1 is also electrogenic. Amino acid transport Systems A and N thus appear closely related in function as well as structure, but exhibit important differences in ionic coupling. (+info)