Syntaxin 4 expression affects glucose transporter 8 translocation and embryo survival

Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.     

Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst

We report that a decrease in facilitative glucose transporter (GLUT1) expression and reduced glucose transport trigger apoptosis in the murine blastocyst. Inhibition of GLUT1 expression either by high glucose conditions or with antisense oligodeoxynucleotides significantly lowers protein expression and function of GLUT1 and as a result induces a high rate of apoptosis at the blastocyst stage. Similar to wild-type mice, embryos from streptozotocin-induced diabetic Bax -/- mice experienced a significant decrease in glucose transport compared with embryos from non-diabetic Bax -/- mice. However, despite this decrease, these blastocysts demonstrate significantly fewer apoptotic nuclei as compared with blastocysts from hyperglycemic wild-type mice. This decrease in preimplantation apoptosis correlates with a decrease in resorptions and malformations among the infants of the hyperglycemic Bax -/- mice versus the Bax +/+ and +/- mice. These findings suggest that hyperglycemia by decreasing glucose transport acts as a cell death signal to trigger a BAX-dependent apoptotic cascade in the murine blastocyst. This work also supports the hypothesis that increased apoptosis at a blastocyst stage because of maternal hyperglycemia may result in loss of key progenitor cells and manifest as a resorption or malformation, two adverse pregnancy outcomes more common in diabetic women.     

Gamma delta-T cells are critical for survival and early proinflammatory cytokine gene expression during murine Klebsiella pneumonia

Although cells of the innate inflammatory response, such as macrophages and neutrophils, have been extensively studied in the arena of Gram-negative bacterial pneumonia, a role for T cells remains unknown. To study the role of specific T cell populations in bacterial pneumonia, mice deleted of their TCR beta- and/or delta-chain were intratracheally inoculated with Klebsiella pneumoniae. Gamma delta T cell knockout mice displayed increased mortality at both early and late time points. In contrast, mice specifically lacking only alpha beta-T cells were no more susceptible than wild-type mice. Pulmonary bacterial clearance in gamma delta-T cell knockout mice was unimpaired. Interestingly, these mice displayed increased peripheral blood dissemination. Rapid up-regulation of IFN-gamma and TNF-alpha gene expression, critical during bacterial infections, was markedly impaired in lung and liver tissue from gamma delta-T cell-deficient mice 24 h postinfection. The increased peripheral blood bacterial dissemination correlated with impaired hepatic bacterial clearance following pulmonary infection and increased hepatic injury as measured by plasma aspartate aminotransferase activity. Combined, these data suggest that mice lacking gamma delta-T cells have an impaired ability to resolve disseminated bacterial infections subsequent to the initial pulmonary infection. These data indicate that gamma delta-T cells comprise a critical component of the acute inflammatory response toward extracellular Gram-negative bacterial infections and are vital for the early production of the proinflammatory cytokines IFN-gamma and TNF-alpha.     

Glucose deprivation and hexose transporter polypeptides of murine fibroblasts

The effect of Glc deprivation (starvation) on hexose transporter (GT) polypeptide(s) (pp) was studied in 3T3-C2 murine fibroblasts. Cells deprived of Glc exhibit 5-fold increases in hexose transport and Glc-displaceable cytochalasin B binding. Immunoblots of membranes reveal a Mr 55,000 GT pp in fed (4 g of Glc/liter) cells and Mr 55,000 and Mr 42,000 GT pp in starved cells. A 10-40-fold increase in total GT pp occurs upon Glc deprivation; part of this accumulation (2-5-fold) is in the Mr 55,000 GT pp, and the remaining increase is in the Mr 42,000 GT pp. During the first 12 h of Glc deprivation only the Mr 55,000 GT pp accumulates. At later times (24-72 h) the Mr 42,000 GT pp appears and constitutes a larger fraction of the total accumulation. Similarly, the Glc concentration dependence of these phenomena reveals that the Mr 55,000 GT pp accumulates at higher concentrations of Glc (less than or equal to g/liter) than the Mr 42,000 GT pp (less than or equal to 0.5 g/liter). Using alternative nutrients, sugar analogs, and inhibitors we observed that the accumulation of total GT pp is dependent upon both hexose phosphate metabolism and the interaction of substrate with the GT. The role(s) of oligosaccharide biosynthesis, protein synthesis, and the transport process itself in the Glc deprivation-induced accumulation of GT pp were examined. The appearance of the Mr 42,000 GT pp but not the Mr 55,000 GT pp was dependent upon protein synthesis. The Glc deprivation-induced accumulation of GT pp is reversible upon refeeding with Glc (4 g/liter, 12 h). This reversal was dependent upon protein synthesis. The electrophoretic mobility of the Mr 42,000 GT pp is similar to the GT pp observed after tunicamycin treatment. The Mr 55,000 but not the Mr 42,000 GT pp binds specifically to agarose-bound wheat germ agglutinin and is sensitive to endoglycosidase F digestion. Oligosaccharide-stripped GT pp and the Mr 42,000 GT pp have the same Mr. The results suggest that the accumulation of total GT pp induced by Glc deprivation is partially independent of the effect of Glc deprivation on glycoprotein biogenesis. The appearance of the Mr 42,000 GT pp with aglyco characteristics is the result of the latter. The accumulation of total GT pp, however, is the result of a specialized and sensitive adaptation of the cell to Glc deprivation. The GT pp synthesized during chronic Glc deprivation has an Mr of 42,000; fed cells synthesize the Mr 55,000 GT pp. Neither the level of in vitro translatable GT mRNA nor the rate of GT pp synthesis are increased by Glc deprivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glucose transporter expression in human skeletal muscle fibers

The present study was initiated to investigate GLUT-1 through -5 expression in developing and mature human skeletal muscle. To bypass the problems inherent in techniques using tissue homogenates, we applied an immunocytochemical approach, employing the sensitive enhanced tyramide signal amplification (TSA) technique to detect the localization of glucose transporter expression in human skeletal muscle. We found expression of GLUT-1, GLUT-3, and GLUT-4 in developing human muscle fibers showing a distinct expression pattern. 1) GLUT-1 is expressed in human skeletal muscle cells during gestation, but its expression is markedly reduced around birth and is further reduced to undetectable levels within the first year of life; 2) GLUT-3 protein expression appears at 18 wk of gestation and disappears after birth; and 3) GLUT-4 protein is diffusely expressed in muscle cells throughout gestation, whereas after birth, the characteristic subcellular localization is as seen in adult muscle fibers. Our results show that GLUT-1, GLUT-3, and GLUT-4 seem to be of importance during muscle fiber growth and development. GLUT-5 protein was undetectable in fetal and adult skeletal muscle fibers. In adult muscle fibers, only GLUT-4 was expressed at significant levels. GLUT-1 immunoreactivity was below the detection limit in muscle fibers, indicating that this glucose transporter is of minor importance for muscle glucose supply. Thus we hypothesize that GLUT-4 also mediates basal glucose transport in muscle fibers, possibly through constant exposure to tonal contraction and basal insulin levels.     

Glucose transporter expression in rat mammary gland.

The expression of different glucose transporter isoforms was measured during the development and differentiation of the rat mammary gland. Before conception, when the mammary gland is mainly composed of adipocytes, Glut 4 and Glut 1 mRNAs and proteins were present. During pregnancy, the expression of Glut 4 decreased progressively, whereas that of Glut 1 increased. In the lactating mammary gland only Glut 1 was present, and was expressed at a high level. The absence of Glut 4 suggests that glucose transport is not regulated by insulin in the lactating rat mammary gland.     

F-spondin plays a critical role in murine neuroblastoma survival by maintaining IL-6 expression

F-spondin is associated with the regulation of axonal growth and the development of the nervous system. Its mechanism of action, however, is not clearly understood. In this study, we found that murine neuroblastoma Neuro-2a cells expressed a significant level of IL-6, but only trace amounts of IL-12, tumor necrosis factor α and nitric oxide. Knock-down of F-spondin mRNA in murine neuroblastoma NB41A3 and Neuro-2a cells using small interfering RNAs led to decreased IL-6 levels along with lower resistance to serum starvation and cytotoxic amyloid β_1–42 (Aβ_1–42) peptide. Restoring decline of F-spondin or IL-6 induced by F-spondin knock-down through adding exogenous F-spondin, IL-6 or over-expressing F-spondin reversed the cell death induced by Aβ_1–42 peptide or serum starvation. The decrease of IL-6 level was positively correlated with decrease of NF-κB and inhibition of p38 mitogen-activated protein kinase (MAPK). Over-expressing MEKK, a kinase activator of the p38 MAPK pathway, increased IL-6 production, restored the decrease of p38 induced by F-spondin knock-down, and rescued the cells from death caused by Aβ_1–42 peptide. Taken together, these results suggest that F-spondin may play a critical role in murine neuroblastoma survival under adverse conditions by maintaining IL-6 level via a MEKK/p38 MAPK/NF-κB-dependent pathway.     

Activins are critical modulators of growth and survival

Activins betaA and betaB (encoded by Inhba and Inhbb genes, respectively) are related members of the TGF-beta superfamily. Previously, we generated mice with an Inhba knock-in allele (InhbaBK) that directs the expression of activin betaB protein in the spatiotemporal pattern of activin betaA. These mice were small and had shortened life spans, both influenced by the dose of the hypomorphic InhbaBK allele. To understand the mechanism(s) underlying these abnormalities, we now examine growth plates, liver, and kidney and analyze IGF-I, GH, and major urinary proteins. Our studies show that activins modulate the biological effects of IGF-I without substantial effects on GH, and that activin signaling deficiency also has modest effects on hepatic and renal function. To assess the relative influences of activin betaA and activin betaB, we produced mice that express activin betaB from the InhbaBK allele, and not from its endogenous Inhbb locus. InhbaBK/BK, Inhbb-/- mice have failure of eyelid fusion at birth and demonstrate more severe effects on somatic growth and survival than either of the corresponding single homozygous mutants, showing that somatic growth and life span are supported by both activins betaA and betaB, although activin betaA plays a more substantial role.     

Glucose transporter gene expression in early mouse embryos.

The glucose transporter (GLUT) isoforms responsible for glucose uptake in early mouse embryos have been identified. GLUT 1, the isoform present in nearly every tissue examined including adult brain and erythrocytes, is expressed throughout preimplantation development. GLUT 2, which is normally present in adult liver, kidney, intestine and pancreatic beta cells is expressed from the 8-cell stage onward. GLUT 4, an insulin-recruitable isoform, which is expressed in adult fat and muscle, is not expressed at any stage of preimplantation development or in early postimplantation stage embryos. Genetic mapping studies of glucose transporters in the mouse show that Glut-1 is located on chromosome 4, Glut-2 on chromosome 3, Glut-3 on chromosome 6, and Glut-4 on chromosome 11.     

Glucose transporter expression in developing fetal lungs and lung neoplasms.

Glucose uptake and metabolism are essential for proliferation and survival of cells, and are supposed to be enhanced in actively proliferating cell systems such as embryonic and cancer tissues. Glucose uptake is usually carried out through glucose transporters. In the developing fetal lung, metabolism of glucose is thought to be an important process in cell proliferation, differentiation and maturation. Active glucose uptake could result in accumulation of glycogen in epithelial cells, and utilization of glycogen could be a critical phenomenon for lung epithelial development. In hamsters, although facilitative glucose transporter isoform 1 (GLUT1) and isoform 4 (GLUT4) are not detected in adult lungs, expression of them is detected with immunohistochemical and Western blot analyses in the developing fetal lungs. In human lung carcinomas, GLUT1 expression is seen in most cases of lung carcinoma, and is seen especially frequently in squamous cell carcinoma. GLUT1 expression in adenocarcinoma of the lung is correlated with reduced cell differentiation, larger tumor size and positive lymph node metastasis. A few cases of lung carcinoma show positive staining for GLUT3 and GLUT4. Thus, expression of some facilitative glucose transporter isoforms is detected in developing fetal epithelium and in lung carcinomas. Overexpression of them could enhance uptake of glucose into these cells, and the increased influx of glucose could be involved in active cell proliferation, which is a common character of the developing lung epithelium and carcinoma.     

Glucose transporter expression in English sparrows (Passer domesticus)

Patterns of glucose transporter expression have been well-characterized in mammals. However, data for birds is currently restricted to isolated cells, domestic chickens and chicks, and ducklings. Therefore, in the present study, protein and gene expression of various glucose transporters (GLUTs) in English sparrow extensor digitorum communis, gastrocnemius and pectoralis muscles as well as heart, kidney, and brain tissues were examined. The hypothesis is that the expression pattern of avian GLUTs differs from mammals to maintain the high plasma glucose levels of birds and insulin insensitivity. Our studies failed to identify a GLUT4-like insulin responsive transporter in sparrows. GLUT1 gene expression was identified in all tissues examined and shares 88% homology with chicken and 84% homology with human GLUT1. Compared to the rat control, GLUT1 immunostaining of sparrow extensor digitorum communis muscle was weak and appeared to be localized to blood vessels whereas immunostaining of gastrocnemius muscles was comparable to rat muscle controls. Gene expression of GLUT3 was identified in all tissues examined and shares 90% gene sequence homology with chicken embryonic fibroblast and 75% homology with human GLUT3. Protein expression of GLUT3 was not determined as an avian antibody is not available. Moreover, the C-terminus of the mammalian GLUT3 transporter, against which antibodies are typically designed, differs significantly among species. The predominant difference of chicken and sparrow GLUT expression patterns from that of mammals is the lack of an avian GLUT4. The absence of this insulin responsive GLUT in birds may be a contributing factor to the observed high blood glucose levels and insulin insensitivity.     

Aspartate 345 of the dopamine transporter is critical for conformational changes in substrate translocation and cocaine binding

The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na+/Cl(-)-dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low Vmax and Km values for DA uptake, converted the inhibitory effect of Zn2+ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn2+ but not by Na+. These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K+ substitution for Na+. Thus, cocaine binding to D345N likely induces a different and Na(+)-dependent conformational change, which may contribute to its Na(+)-dependent uptake inhibitory activity.     

Determination of residues in the norepinephrine transporter that are critical for tricyclic antidepressant affinity

The norepinephrine (NET) and dopamine (DAT) transporters are highly homologous proteins, displaying many pharmacological similarities. Both transport dopamine with higher affinity than norepinephrine and are targets for the psychostimulants cocaine and amphetamine. However, they strikingly contrast in their affinities for tricyclic antidepressants (TCA). Previous studies, based on chimeric proteins between DAT and NET suggest that domains ranging from putative transmembrane domain (TMD) 5 to 8 are involved in the high affinity binding of TCA to NET. We substituted 24 amino acids within this region in the human NET with their counterparts in the human DAT, resulting in 22 different mutants. Mutations of residues located in extra- or intracytoplasmic loops have no effect on binding affinity of neither TCA nor cocaine. Three point mutations in TMD6 (F316C), -7 (V356S), and -8 (G400L) induced a loss of TCA binding affinity of 8-, 5-, and 4-fold, respectively, without affecting the affinity of cocaine. The triple mutation F316C/V356S/G400L produced a 40-fold shift in desipramine affinity. These three residues are strongly conserved in all TCA-sensitive transporters cloned in mammalian and nonmammalian species. A strong shift in TCA affinity (IC(50)) was also observed for double mutants F316C/D336T (35-fold) and S399P/G400L (80-fold for nortriptyline and 1000-fold for desipramine). Reverse mutations P401S/L402G in hDAT did not elicit any gain in TCA affinities, whereas C318F and S358V resulted in a 3- and 10-fold increase in affinity, respectively. Our results clearly indicate that two residues located in TMD6 and -7 of hNET may play an important role in TCA interaction and that a critical region in TMD8 is likely to be involved in the tertiary structure allowing the high affinity binding of TCA.     

Phosphatidylinositol 3-kinase activity is critical for glucose metabolism and embryo survival in murine blastocysts

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is a well known mediator of cell growth, proliferation, and survival signals. Whereas the expression and function of this pathway has been documented during mammalian development, evidence demonstrating the physiologic importance of this pathway in murine preimplantation embryos is beginning to emerge. This study demonstrates that inhibition of the PI3K pathway leads to the induction of apoptosis in both murine blastocysts and trophoblast stem cells. The apoptosis induced in both model systems correlates with a decrease in the expression of the glucose transporter GLUT1 at the plasma membrane. In addition, blastocysts cultured in the presence of the PI3K inhibitor LY-294002 display a decrease in both 2-deoxyglucose uptake and hexokinase activity as compared with control blastocysts. To determine the impact of PI3K inhibition on pregnancy outcome, embryo transfer experiments were performed. Blastocysts cultured in the presence of LY-294002 demonstrate a dramatic increase in fetal resorptions as compared with control embryos. Finally, we demonstrate that impairment of glucose metabolism via iodoacetate, a glyceraldehyde-3-phosphate dehydrogenase inhibitor, is sufficient to induce apoptosis in both blastocysts and trophoblast stem cells. Moreover, blastocysts treated with iodoacetate result in poor pregnancy outcome as determined by embryo transfer experiments. Taken together these data demonstrate the critical importance of the PI3K pathway in preimplantation embryo survival and pregnancy outcome and further emphasize the importance of glucose utilization and metabolism in cell survival pathways.     

Glucose and insulin are needed for optimal defensin expression in human cell lines

Many infections are associated with diabetes, as the ability of the body to fight pathogens is impaired. Recently, low levels of defensins have been found in diabetic rodents. However, whether hyperglycemia and/or insulin deficiency/insensitivity is the reason for the reduced defensin levels is still unknown. To study the functionality of the innate immune system during hyperglycemia, the expression levels of human beta-defensin-1 (hBD-1) was measured in human embryonic kidney (HEK-293) and colon adenocarcinoma (HCT-116) cells treated with different concentrations of glucose and insulin. Increasing concentrations of glucose enhanced hBD-1 expression and these levels were further elevated after insulin treatment. Insulin treatment also led to the up-regulation of human sodium/glucose transporter 1 (hSGLT1), which further increases intracellular glucose levels. Thus, our findings suggest for the first time that insulin signaling is important for hBD-1 optimal expression by elevating intracellular glucose levels and by mediating gene expression.     

The C-terminus is critical for the functional expression of the human serotonin transporter

The plasma membrane serotonin transporter (SERT) has an important role in terminating serotonergic neurotransmission by re-uptake of 5-HT from the synaptic cleft. The expression of SERT on the cell surface is therefore a critical factor. In this study, we examined the role of the carboxyl terminus of SERT in trafficking to the plasma membrane. 5-HT uptake activity was used to measure the effects of systematic deletions or alanine substitutions in the C-terminus. We found that deletion of 16 amino acids in the distal C-terminus had no effect on uptake activity, whereas further deletion was detrimental for the function of SERT. Cell surface biotinylation was used to determine the role of the C-terminus in localization and trafficking. We showed that the C-terminus is crucial for the delivery of SERT to the plasma membrane and that the deletion of this part of the transporter results in a lack of mature glycosylation and impaired trafficking to the plasma membrane. Furthermore, the C-terminally truncated mutants were shown to have a dominant negative effect on wild-type SERT uptake activity.     

Glucose uptake mediated by glucose transporter 1 is essential for early tooth morphogenesis and size determination of murine molars

Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1-4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination.     

Two conformational changes are associated with glutamate translocation by the glutamate transporter EAAC1

Glutamate is transported across membranes by means of a carrier mechanism that is thought to require conformational changes of the transport protein. In this work, we have determined the thermodynamic parameters of glutamate and the Na+ binding steps to their extracellular binding sites along with the activation parameters of rapid, glutamate-induced processes in the transport cycle by analyzing the temperature dependence of glutamate transport at steady state and pre-steady state. Our results suggest that glutamate binding to the transporter is driven by a negative reaction enthalpy (DeltaH0 = -33 kJ/mol), whereas the tighter binding of the non-transportable inhibitor TBOA is caused by an additional increase in entropy. Processes linked to the binding of glutamate and Na+ to the transporter are associated with low activation barriers, indicative of diffusion-controlled reactions. The activation enthalpies of two processes in the glutamate translocation branch of the transport cycle were DeltaH++ = 95 kJ/mol and DeltaH++ = 120 kJ/mol, respectively. Such large values of DeltaH++ suggest that these processes are rate-limited by conformational changes of the transporter. We also found a large activation barrier for steady-state glutamate transport, which is rate-limited by the K+-dependent relocation of the empty transporter. Together, these results suggest that two conformational changes accompany glutamate translocation and at least one conformational change accompanies the relocation of the empty transporter. We interpret the data with an alternating access model that includes the closing and opening of an extracellular and an intracellular gate, respectively, in analogy to a hypothetical model proposed previously on the basis of the crystal structure of the bacterial glutamate transporter GltPh.