Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-μs time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 μM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na+-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at −90 mV). Applying step changes to the transmembrane potential, V m, of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a τ of ∼15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<−40 mV) and activation at positive V m (>0 mV). A similar inhibitory effect at V m < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V m curve. Jumping the glutamate concentration to 100 μM generated biphasic, saturable transient transport and anion currents (K m ∼ 5 μM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1–3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.

Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. Here, we have tested the effect of multimerization on transporter function. To do so, we coexpressed EAAC1 WT with the mutant transporter EAAC1 R446Q , which transports glutamine, but not glutamate. Application of 50 μM glutamate or 50 μM glutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 pA and 130 pA, respectively. Application of both substrates at the same time generated an anion current of 297 pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purely additive. This result is unexpected for anion permeation through a central pore, but could be explained by anion permeation through independently-functioning subunits. To further test the subunit independence, we coexpressed EAAC1 WT and EAAC1 H295K , a transporter with a 90-fold reduced glutamate affinity as compared to EAAC1 WT , and determined the glutamate concentration dependence of currents of the mixed transporter population. The data were consistent with two independent populations of transporters with apparent glutamate affinities similar to those of EAAC1 H295K and EAAC1 WT , respectively. Finally, we coexpressed EAAC1 WT with the pH-independent mutant transporter EAAC1 E373Q , showing two independent populations of transporters, one being pH dependent, the other being pH-independent. In conclusion, we propose that EAAC1 assembles as trimers of identical subunits, but that the individual subunits in the trimer function independently of each other.Plasma membrane glutamate transporters actively remove glutamate from the synaptic cleft after excitatory neurotransmission is complete. Uptake into the cells surrounding the synapse against a glutamate concentration gradient is achieved by these transporters by coupling transmembrane glutamate movement to the cotransport of three sodium ions and one proton, and the countertransport of one potassium ion (1,2). In addition to the movement of ions across the membrane being directly coupled to glutamate transport, glutamate transporters also catalyze uncoupled transmembrane flux of anions (3). This anion conductance is thought to be an integral property of the transporters and is not mediated by indirect coupling of transport to a secondary anion channel (3-5).Address correspondence to: Christof Grewer, PhD, Department of Physiology and Biophysics, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136; Phone: (305) 243-1021; Fax: (305) The mammalian glutamate transporters belong to a large family of membrane transport proteins that comprise also neutral amino acid transporters, such as the alanine serine cysteine transporters (ASCTs (6,7)), and dicarboxylate transporters (8,9). A large number of biochemical data from both mammalian (10,11) and bacterial glutamate transporters (12,13), as well as recent crystallographic evidence f...

Transcriptional targeting using a tissue-specific cellular promoter is proving to be a powerful means for restricting transgene expression in targeted tissues. In the context of cancer suicide gene therapy, this approach may lead to cytotoxic effects in both cancer and nontarget normal cells. Considering microRNA (miRNA) function in post-transcriptional regulation of gene expression, we have developed a viral vector platform combining cellular promoter-based transcriptional targeting with miRNA regulation for a glioma suicide gene therapy in the mouse brain. The therapy employed, in a single baculoviral vector, a glial fibrillary acidic protein (GFAP) gene promoter and the repeated target sequences of three miRNAs that are enriched in astrocytes but downregulated in glioblastoma cells to control the expression of the herpes simplex virus thymidine kinase (HSVtk) gene. This resulted in significantly improved in vivo selectivity over the use of a control vector without miRNA regulation, enabling effective elimination of human glioma xenografts while producing negligible toxic effects on normal astrocytes. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of gene delivery vectors with high targeting specificity for cancer therapy.

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Achievement of specific tumor cell targeting remains a challenge for glioma gene therapy. We observed that the human high mobility group box2 (HMGB2) gene had a low level of expression in normal human brain tissues, but was significantly upregulated in glioblastoma tissues. With progressive truncation of a 5'-upstream sequence of the HMGB2 gene, we identified a 0.5-kb fragment displaying a high transcriptional activity in glioblastoma cells, but a low activity in normal brain cells. To test the feasibility of using the HMGB2 promoter sequence in targeted cancer therapy, we constructed a baculoviral vector expressing the herpes simplex virus thymidine kinase (HSVtk) gene driven by the HMGB2 promoter. Transduction with the viral vector induced cell death in glioblastoma cell lines in the presence of ganciclovir (GCV), but did not affect the survival of human astrocytes and neurons. In a mouse xenograft model, intratumor injection of the baculoviral vector suppressed the growth of human glioblastoma cells and prolonged the survival of tumor-bearing mice. Our results suggest that the novel 5' sequence of HMGB2 gene has a potential to be used as an efficient, tumor-selective promoter in targeted vectors for glioblastoma gene therapy.

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Treatment of aortic smooth muscle cells with PDGF induces the upregulation of protein tyrosine phosphatase 1B (PTP1B). PTP1B, in turn, decreases the function of several growth factor receptors, thus completing a negative feedback loop. Studies have reported that PDGF induces the downregulation of PKG as part of a repertoire of dedifferentiation of vascular smooth muscle cells. Other studies have reported that chronic nitric oxide (NO) treatment also induces the downregulation of PKG. In the present study, we tested the hypothesis that the downregulation of PKG by PDGF or NO in differentiated rat aortic smooth muscle cells can be attributed to the upregulation of PTP1B. We found that treatment with PDGF or NO induced an upregulation of PTP1B levels. Overexpression of PTP1B induced a marked downregulation of PKG mRNA and protein levels, whereas the expression of dominant negative PTP1B or short interfering RNA directed against PTP1B blocked the capacity of PDGF or NO to decrease PKG levels. We conclude that the upregulation of PTP1B by PDGF or NO is both necessary and sufficient to induce the downregulation of PKG via an effect on PKG mRNA levels.

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