Both reentrant loops of the sodium-coupled glutamate transporters contain molecular determinants of cation selectivity.

In the brain, glutamate transporters terminate excitatory neurotransmission by removing this neurotransmitter from the synapse via cotransport with three sodium ions into the surrounding cells. Structural studies have identified the binding sites of the three sodium ions in glutamate transporters. The residue side-chains directly interact with the sodium ions at the Na1 and Na3 sites and are fully conserved from archaeal to eukaryotic glutamate transporters. The Na2 site is formed by three main-chain oxygens on the extracellular reentrant hairpin loop HP2 and one on transmembrane helix 7. A glycine residue on HP2 is located closely to the three main-chain oxygens in all glutamate transporters, except for the astroglial transporter GLT-1, which has a serine residue at that position. Unlike for WT GLT-1, substitution of the serine residue to glycine enables sustained glutamate transport also when sodium is replaced by lithium. Here, using functional and simulation studies, we studied the role of this serine/glycine switch on cation selectivity of substrate transport. Our results indicate that the side-chain oxygen of the serine residues can form a hydrogen bond with a main-chain oxygen on transmembrane helix 7. This leads to an expansion of the Na2 site such that water can participate in sodium coordination at Na2. Furthermore, we found other molecular determinants of cation selectivity on the nearby HP1 loop. We conclude that subtle changes in the composition of the two reentrant hairpin loops determine the cation specificity of acidic amino acid transport by glutamate transporters.

Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1.

Glutamate transporters actively take up glutamate into the cell, driven by the co-transport of sodium ions down their transmembrane concentration gradient. It was proposed that glutamate binds to its binding site and is subsequently transported across the membrane in the negatively charged form. With the glutamate binding site being located partially within the membrane domain, the possibility has to be considered that glutamate binding is dependent on the transmembrane potential and, thus, is electrogenic. Experiments presented in this report test this possibility. Rapid application of glutamate to the wild-type glutamate transporter subtype EAAC1 (excitatory amino acid carrier 1) through photo-release from caged glutamate generated a transient inward current, as expected for the electrogenic inward movement of co-transported Na(+) In contrast, glutamate application to a transporter with the mutation A334E induced transient outward current, consistent with movement of negatively charged glutamate into its binding site within the dielectric of the membrane. These results are in agreement with electrostatic calculations, predicting a valence for glutamate binding of -0.27. Control experiments further validate and rule out other possible explanations for the transient outward current. Electrogenic glutamate binding can be isolated in the mutant glutamate transporter because reactions, such as glutamate translocation and/or Na(+) binding to the glutamate-bound state, are inhibited by the A334E substitution. Electrogenic glutamate binding has to be considered together with other voltage-dependent partial reactions to cooperatively determine the voltage dependence of steady-state glutamate uptake and glutamate buffering at the synapse.

Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters.

Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.

Cysteine scanning mutagenesis of transmembrane helix 3 of a brain glutamate transporter reveals two conformationally sensitive positions.

Glutamate transporters in the brain remove the neurotransmitter from the synapse by cotransport with three sodium ions into the surrounding cells. Recent structural work on an archaeal homolog suggests that, during substrate translocation, the transport domain, including the peripheral transmembrane helix 3 (TM3), moves relative to the trimerization domain in an elevator-like process. Moreover, two TM3 residues have been proposed to form part of a transient Na3' site, and another, Tyr-124, appears close to both Na3' and Na1. To obtain independent evidence for the role of TM3 in glutamate transport, each of its 31 amino acid residues from the glial GLT-1 transporter was individually mutated to cysteine. Except for six mutants, substantial transport activity was detected. Aqueous accessibility of the introduced cysteines was probed with membrane-permeant and membrane-impermeant sulfhydryl reagents under a variety of conditions. Transport of six single cysteine mutants, all located on the intracellular side of TM3, was affected by membrane-permeant sulfhydryl reagents. However, only at two positions could ligands modulate the reactivity. A120C reactivity was diminished under conditions expected to favor the outward-facing conformation of the transporter. Sulfhydryl modification of Y124C by 2-aminoethyl methanethiosulfonate, but not by N-ethylmaleimide, was fully protected in the presence of sodium. Our data are consistent with the idea that TM3 moves during transport. Moreover, computational modeling indicated that electrostatic repulsion between the positive charge introduced at position 124 and the sodium ions bound at Na3' and Na1 underlies the protection by sodium.

The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation.

The tuftelin protein isoforms undergo post-translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down-regulation of HIF1α mRNA blocked hypoxia-induced HIF1α expression, and reduced by 89% hypoxia-induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF-mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA-signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the "hypoxic genome," and NGF-induced neurotrophic and angiogenic effects.

Biphasic influence of hypoxia on tuftelin expression in mouse mesenchymal C3H10T1/2 stem cells.

Tuftelin, an acidic protein, thought to play a role in the initial stages of ectodermal enamel mineralization, has since been detected in mesenchymal-derived tissues. During bone/cartilage development and regeneration, mesenchymal stem cells (MSCs) undergo an avascular period in a hypoxic environment, involving induction of hypoxia-inducible factor 1-alpha (HIF-1-alpha), a key component in this process. In the present study we investigated, in a mouse mesenchymal C3H10T1/2 stem cell model, the hypothesis that oxygen stress modulates tuftelin 1 expression in relation to HIF-1-alpha (Hif1a), in a mouse mesenchymal C3H10T1/2 stem cell model. The results of the present study showed a biphasic induction of tuftelin, similar to the pattern of HIF-1-alpha expression, in MSCs subjected to a hypoxic insult of 1% O(2) through a period of 2-24 h. Immunocytochemistry analysis of the cells exposed to hypoxic insult for 2-24 h revealed the same biphasic pattern of tuftelin protein expression. Tuftelin localization appears to be mainly in the cytoplasm, and concentrated at the perinuclear region of the cells by 24 h of hypoxic insult. Based on our previous studies using the neuronal PC12 cell model, in which tuftelin induction was mediated by Hif1a, we propose that tuftelin is a member of oxygen-sensitive genes and implicated in the adaptive mechanisms regulating MSC function.