Glutamate transport by the excitatory amino acid carrier EAAC1 is known to be reversible. Thus, glutamate can either be taken up into cells, or it can be released from cells through reverse transport, depending on the electrochemical gradient of the co-and countertransported ions. However, it is unknown how fast and by which reverse transport mechanism glutamate can be released from cells. Here, we determined the steady-and pre-steady-state kinetics of reverse glutamate transport with submillisecond time resolution. First, our results suggest that glutamate and Na ؉ dissociate from their cytoplasmic binding sites sequentially, with glutamate dissociating first, followed by the three cotransported Na ؉ ions. Second, the kinetics of glutamate transport depend strongly on transport direction, with reverse transport being faster but less voltage-dependent than forward transport. Third, electrogenicity is distributed over several reverse transport steps, including intracellular Na ؉ binding, reverse translocation, and reverse relocation of the K ؉ -bound EAAC1. We propose a kinetic model, which is based on a ''first-in-first-out'' mechanism, suggesting that glutamate association, with its extracellular binding site as well as dissociation from its intracellular binding site, precedes association and dissociation of at least one Na ؉ ion. Our model can be used to predict rates of glutamate release from neurons under physiological and pathophysiological conditions. excitatory amino acid transporter ͉ electrophysiology ͉ reverse transport ͉ patch-clamp ͉ caged compounds G lutamate transporters belong to the class of Na ϩ -driven secondary-active transporters. They couple the uphill uptake of glutamate into cells to the movement of three Na ϩ ions down their ion concentration gradient (1). Neurons, like many other cells, express glutamate transporters, allowing them to keep a 10 6 -fold glutamate concentration gradient across their cell membranes (2). This steep concentration gradient is essential for neuronal signaling, because it ensures submicromolar resting concentrations of extracellular glutamate.Glutamate transporters are not strictly unidirectional and are able to change the direction of glutamate transport (3). Under physiological conditions, forward transport from the extracellular side to the cytosol is favored. However, if the driving force for glutamate uptake decreases, glutamate can be released from cells through reverse glutamate transport (3, 4). This situation may arise in oxygen-deprived cells when the Na ϩ concentration gradient across the membrane runs down, and/or when cells depolarize. In ischemic neurons, the majority of glutamate release upon oxygen/glucose deprivation was shown to be caused by reverse glutamate transport and not by vesicular release (5, 6). Considering the severe neurotoxic effects of elevated extracellular glutamate concentrations, it is of major importance to understand the mechanism of how glutamate is released through reverse transport.Here, we investigated with high time re...

Sodium-glucose cotransporters (SGLTs) are secondary active transporters belonging to the SLC5 gene family. SGLT1, a well-characterized member of this family, electrogenically transports glucose and galactose. Human SGLT3 (hSGLT3), despite sharing a high amino acid identity with human SGLT1 (hSGLT1), does not transport sugar, although functions as a sugar sensor. In contrast to humans, two different genes in mice and rats code for two different SGLT3 proteins, SGLT3a and SGLT3b. We previously cloned and characterized mouse SGLT3b (mSGLT3b) and showed that, while it does transport sugar like SGLT1, it likely functions as a physiological sugar sensor like hSGLT3. In this study, we cloned mouse SGLT3a (mSGLT3a) and characterized it by expressing it in Xenopus laevis oocytes and performing electrophysiology and sugar transport assays. mSGLT3a did not transport sugar, and sugars did not induce currents at pH 7.4, though acidic pH induced inward currents that increased in the presence of sugar. Moreover, mutation of residue 457 from glutamate to glutamine resulted in a Na(+)-dependent transport of sugar that was inhibited by phlorizin. To corroborate our results in oocytes, we expressed and characterized mSGLT3a in mammalian cells and confirmed our findings. In addition, we cloned, expressed, and characterized rat SGLT3a in oocytes and found characteristics similar to mSGLT3a. In summary, acidic pH induces currents in mSGLT3a, and sugar-induced currents are increased at acidic pH, but wild-type SGLT3a does not transport sugar.

Transactivating DNA-binding protein-43 (TDP-43) inclusions and the accumulation of phosphorylated and ubiquitinated tau proteins (p-tau) have been identified in postmortem brain specimens from patients with chronic traumatic encephalopathy (CTE). To examine whether these proteins contribute to the development of CTE, we utilized an in vitro trauma system known to reproduce many of the findings observed in humans and experimental animals with traumatic brain injury. Accordingly, we examined the role of TDP-43 and Tau in an in vitro model of trauma, and determined whether these proteins contribute to the defective neuronal integrity associated with CNS trauma. Single or multiple episodes of trauma to cultured neurons resulted in a time-dependent increase in cytosolic levels of phosphorylated TDP-43 (p-TDP-43). Trauma to cultured neurons also caused an increase in levels of casein kinase 1 epsilon (CK1ε), and ubiquitinated p-TDP-43, along with a decrease in importin-β (all factors known to mediate the "TDP-43 proteinopathy"). Defective neuronal integrity, as evidenced by a reduction in levels of the NR1 subunit of the NMDA receptor, and in PSD95, along with increased levels of phosphorylated tau were also observed. Additionally, increased levels of intra- and extracellular thrombospondin-1 (TSP-1) (a factor known to regulate neuronal integrity) were observed in cultured astrocytes at early stages of trauma, while at later stages decreased levels were identified. The addition of recombinant TSP-1, conditioned media from cultured astrocytes at early stages of trauma, or the CK1ε inhibitor PF4800567 hydrochloride to traumatized cultured neurons reduced levels of p-TDP-43, and reversed the trauma-induced decline in NR1 subunit of the NMDA receptor and PSD95 levels. These findings suggest that a trauma-induced increase in TDP-43 phosphorylation contributes to defective neuronal integrity, and that increasing TSP-1 levels may represent a useful therapeutic approach for the prevention of the neuronal TDP-43 proteinopathy associated with CTE. Read the Editorial Highlight for this article on page 531.

SGLT1 is a sodium/glucose cotransporter that moves two Na ؉ ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. Members of the SLC5 cotransporter family present highly diverse functions. They are capable of cotransporting Na ϩ with glucose (SGLT1 and SGLT2), with myoinositol (SMIT), with iodide (NIS), or with choline (CHT), among other substrates (reviewed in Ref. 1). In fact, there is one family member, human SGLT3, that is not a transporter but is a glucose sensor (2, 3).The crystal structures of Na ϩ symporters from different families reveal that they share a core of 10 transmembrane segments composed of an inverted repeat of five segments (4). The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) 2 clearly resolved the residues of the sugar binding site (5). One residue that directly interacts with the sugar is glutamine 428, which is equivalent to amino acid 457 in mammalian SGLT proteins. Notably, the identity of the amino acid at position 457 in human SGLT1 (hSGLT1) has proven to have a dramatic effect on the function of the protein. Structure-function studies on hSGLT1 suggest that residue 457 interacts with sugar (6), and mutations of this residue cause glucose-galactose malabsorption (7,8).The crystal structure of vSGLT did not reveal the position of the single Na ϩ -binding site. SGLT1 and other transporters like LeuT, and presumably SGLT3, have two Na ϩ -binding sites. Based on structural homology to LeuT, whose two Na ϩ -binding sites were identified, Na1 and Na2 (9), a Na ϩ -binding site in vSGLT ϳ 10 Å away from the sugar binding site was proposed and corresponds to Na2 in LeuT (5). The location of the other Na ϩ binding site in SGLT has not been identified, but it may correspond to Na1 in LeuT. These Na ϩ -binding sites are likely to be conserved in SGLT1 and SGLT3 proteins.Despite ϳ70% amino acid identity between hSGLT3 and hSGLT1, there are significant differences in their function, besides the inability of hSGLT3 to transport sugar. hSGLT3 also has a weaker apparent glucose affinity, no visible presteady-state currents and a different tissue distribution than SGLT1 (2, 10). In terms of amino acid sequence, all SGLT1 and SGLT2 proteins have a glutamine at residue 457, whereas most SGLT3 proteins have a glutamate. There are two exceptions, glycine in mouse SGLT3b and serine in rat SGLT3b (Fig. 1).Because of the strict conservation of amino acid 457 among the SGLT1 and SGLT2 glucose transporters and the difference with the SGLT3 glucose sensors, we explored whether the identity of this residue plays a major role in the differences in function between the different protein...