May 2004
Volume 45, Issue 5
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Physiology and Pharmacology  |   May 2004
Retinal Colocalization and In Vitro Interaction of the Glutamate Receptor EAAT3 and the Serum- and Glucocorticoid-Inducible Kinase SGK1
Author Affiliations
  • Roman Schniepp
    From the Department of Physiology I, University of Tübingen, Germany; the
  • Konrad Kohler
    Department of Experimental Ophthalmology, University Eye Hospital, Tübingen, Germany; the
  • Thomas Ladewig
    Department of Experimental Ophthalmology, University Eye Hospital, Tübingen, Germany; the
  • Elke Guenther
    Department of Experimental Ophthalmology, University Eye Hospital, Tübingen, Germany; the
  • Guido Henke
    From the Department of Physiology I, University of Tübingen, Germany; the
  • Monica Palmada
    From the Department of Physiology I, University of Tübingen, Germany; the
  • Christoph Boehmer
    From the Department of Physiology I, University of Tübingen, Germany; the
  • Jeffrey D. Rothstein
    Department of Neurology, John Hopkins University School of Medicine, Baltimore, Maryland; and the
  • Stefan Bröer
    School of Biochemistry and Molecular Biology, Australian National University, Canberra, Australia.
  • Florian Lang
    From the Department of Physiology I, University of Tübingen, Germany; the
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1442-1449. doi:10.1167/iovs.03-0062
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      Roman Schniepp, Konrad Kohler, Thomas Ladewig, Elke Guenther, Guido Henke, Monica Palmada, Christoph Boehmer, Jeffrey D. Rothstein, Stefan Bröer, Florian Lang; Retinal Colocalization and In Vitro Interaction of the Glutamate Receptor EAAT3 and the Serum- and Glucocorticoid-Inducible Kinase SGK1. Invest. Ophthalmol. Vis. Sci. 2004;45(5):1442-1449. doi: 10.1167/iovs.03-0062.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The serum- and glucocorticoid-inducible kinase SGK1 regulates several epithelial channels and transporters, the related protein kinase B (PKB) regulates glucose transport. SGK1 is expressed in the brain and could thus regulate glial and/or neuronal transport processes. The present study explores whether SGK1 is expressed in the retina and whether it regulates EAAT3, a Na+-coupled glutamate transporter. EAAT3 is expressed in retinal ganglion cells and accomplishes the clearance of glutamate from synaptic clefts.

methods. Immunohistochemistry was performed to test for retinal SGK1 expression. For functional analysis, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without additional injection of wild-type SGK1, constitutively active S422DSGK1, inactive K127NSGK1, and/or constitutively active T308D,S473DPKB. Glutamate induced current (IGLU) was taken as a measure for transport.

results. SGK1 is indeed expressed in several retinal cells including retinal ganglion cells where it is colocalized with EAAT3. In EAAT3-expressing Xenopus oocytes, glutamate-induced current was stimulated by coexpression of wild-type SGK1, constitutively active S422DSGK1, and constitutively active T308D,S473DPKB, but not by inactive K127NSGK1.

conclusions. SGK1 and EAAT3 are coexpressed in retinal neurons, and SGK1 serves to stimulate EAAT3. This function is shared by protein kinase B (PKB). The experiments reveal a novel mechanism regulating EAAT3, which may be essential for the function of the retinal ganglion cells.

The serum and glucocorticoid-inducible kinase SGK1 was originally cloned as a glucocorticoid-sensitive gene. 1 2 3 The human orthologue was cloned as a cell-volume–sensitive gene upregulated by osmotic and isotonic cell shrinkage. 4 More recent experiments disclosed the involvement of SGK1 in the regulation of a variety of channels and transporters, 5 such as the renal epithelial Na+ channel ENaC 6 7 8 9 10 11 ; the voltage-gated Na+ channel SCN5A 6 ; the K+ channels ROMK1, 12 KCNE1/KCNQ1, 13 and Kv1.3 14 15 16 ; the Na+/H+ exchanger NHE3 17 ; and Na+/K+-ATPase. 18 19 As shown for the regulation of ENaC, SGK1 is at least partially effective through inhibition of neuronally expressed developmentally downregulated gene Nedd4-2, 20 21 22 a ubiquitin ligase expressed in a wide variety of tissues including the brain. 23 SGK1 is related to PKB, which has been shown to be expressed in retinal tissue 24 and is known to regulate the glucose transporter GLUT4. 25 26 27 28 29 30 Activation of SGK1 requires phosphorylation at Ser422, activation of PKB requires phosphorylation at Thr308 and Ser473. Replacement of those amino acids by aspartate leads to the respective constitutively active kinases S422DSGK1 31 and T308D,S473DPKB. 32 Destruction of the catalytic subunit by replacement of the lysine at position 127 with asparagine leads to the inactive mutant K127NSGK1. 31 In vivo, SGK1 and PKB are activated through a signaling cascade involving phosphatidyl-inositol 3′ kinase (PI3-K) and phosphoinositide-dependent kinase PDK1. 33 SGK1 4 16 and PKB 32 34 35 36 37 are expressed in all human tissues studied thus far, including the brain. Among the stimulators of those kinases is presumably brain-derived neurotrophic factor (BDNF) which has been shown to activate PI3 kinase and PKB in retinal ganglion cells. 38 The present study was performed to explore whether SGK1 is expressed in the retina. We showed strong expression of SGK1 in several neuronal cells, including the retinal ganglion cells. 
Additional studies were performed to gain some insight into the functional significance of SGK1. To this end, experiments were performed elucidating the influence of SGK1 on the glutamate transporter EAAT3 (EAAC1), a member of the Na+-coupled glutamate transporter family. 39 40 41 42 43 44 45 46 47 The carrier participates in the clearance of glutamate from the synaptic cleft. 48 49 50 51 52 It is expressed in bipolar, amacrine, and retinal ganglion cells. 53 To test for a role of SGK1 and/or PKB in the regulation of EAAT3, cRNA encoding EAAT3 was injected, with or without cRNA encoding Nedd4-2 and/or constitutively active S422DSGK1, inactive K127NSGK1, or constitutively active T308D,S473DPKB, into Xenopus oocytes. The glutamate-induced currents were taken as a measure of glutamate transport. 
To explore in vivo regulation of glutamate transport in retinal ganglion cells, glutamate induced currents in the presence of glutamate receptor blockers were determined by patch clamp in retinal ganglion cells from BDNF-deficient (BDNF −/−) and in wild-type (BDNF +/+) mice. 
Materials and Methods
Animal Experimentation
All experiments performed in this study were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the guidelines of the ethics committees of the University of Tübingen. 
For immunohistochemistry, retinas from adult (postnatal day [P]92) Brown Norway (BN) rats reared in our animal facilities were used in the study. The animals were kept under a 12-hour light-dark cycle, with an illumination of 10 lux. Tissue samples were obtained between 9 AM and 5 PM, during the light phase. 
Glutamate transporter currents in retinal ganglion cells were studied in homozygous BDNF-deficient (BDNF −/−) and wild-type (BDNF +/+) mice 54 at P3 and P4. The exact genotype of each tested mouse was determined by Southern blot analysis. 
Immunohistochemistry
The animals were killed with CO2 and decapitated. Their eyes were enucleated and the anterior poles removed. The remaining posterior eyecups were fixed in 4% paraformaldehyde (PFA) in phosphate buffer (PB; 0.2 M NaH2PO4/Na2HPO4; pH 7.4) for 30 minutes at 4°C. After being washed in PB, the eyecups were cryoprotected by immersion in 30% sucrose in PB overnight at 4°C and embedded (Tissue Tek; Leica, Nussloch, Germany). Vertical cryosections of 10 μm thickness were collected on gelatin-coated glass slides, dried for 3 hours and stored at −80°C until use. 
To reduce background staining, sections were incubated for 30 minutes with 20% normal goat serum (NGS; Sigma-Aldrich, Taufkirchen, Germany) in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 (PBST). For immunohistochemical detection of SGK1, a specific polyclonal antiserum raised in rabbit was used. 16 A monoclonal mouse anti-EAAT3 antibody was purchased from Chemicon (Temecula, CA). The antibodies were diluted in PBST (1:250 and 1:100, respectively) and used to incubate sections overnight at 4°C. After they were washed with PBS, the sections were incubated for 1 hour with the appropriate secondary antibodies (goat anti-rabbit or goat anti-mouse; dilution 1:200; Sigma-Aldrich) conjugated to Alexa Fluor 488 or conjugated to Cy3 (Molecular Probes, Eugene, OR). Double-labeling studies were performed by simultaneously applying the primary antibodies followed by a mixture of secondary antibodies. Labeled sections were washed in PBS and coverslipped with glycerol. For determination of the specificity of the antibody-reaction, negative control were performed by omitting the primary antibody. 
All histologic examinations and photodocumentation were performed by microscope (model AX70; Olympus, Tokyo, Japan). Photographs were digitized and adjusted for brightness and contrast (Photoshop; Mountain View, CA). 
Construction of Xenopus laevis Expression Vectors
cRNA encoding human EAAT3, 39 40 human Nedd4-2, 20 wild-type SGK1, 4 constitutively active human S422DSGK1, 31 inactive K127NSGK1, 31 and constitutively active human T308D,S473DPKB 32 were synthesized as described. 55  
Two-Electrode Voltage Clamp
Dissection of Xenopus laevis ovaries and collection and handling of the oocytes have been described in detail elsewhere. 55 Oocytes were injected with 7.5 ng S422DSGK1, K127NSGK1, T308D,S473DPKB cRNA, and/or 5 ng Nedd4-2 cRNA on the first day after preparation of the oocytes and with 10 ng EAAT3 cRNA on the second day after preparation. Inactive K127NSGK1 served as the control for the active kinases. When no cRNA was injected, water was injected as a control to avoid any bias by the injection itself. All experiments were performed at room temperature 4 days after the second injection. Two-electrode voltage-clamp recordings were performed at a holding potential of −60 mV. The data were filtered at 10 Hz, and recorded with an A/D-D/A converter (MacLab; ADInstruments, Castle Hill, Australia) and software for data acquisition and analysis (ADInstruments). The control solution (superfusate/ND96) contained (in mM) 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 5 HEPES [pH 7.4]. The final solutions were titrated to the pH indicated using HCl or NaOH. The flow rate of the superfusion was 20 mL/min, and a complete exchange of the bath solution was reached within approximately 10 seconds. 
Patch Clamp
To determine glutamate transporter currents in retinal ganglion cells from homozygous BDNF-deficient (BDNF −/−) and wild-type mice (BDNF +/+) at P3 and P4, postnatal C57BL6BDNF mice pups were killed by decapitation. After removal of the eyeballs, retinas were dissected, cut into strips, and kept at room temperature in continuously bubbled (95% O2, 5% CO2) bicarbonate-buffered ACSF, containing (in mM) 125 NaCl, 3.5 KCl, 1 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, and 25 glucose, at pH 7.4, adjusted with 1 M NaOH. Recordings were made between 2 and 5 hours after retinal dissection. All measurements were performed at a temperature of 23°C to 25°C. Cells were chosen for recording according to position in the ganglion cell layer and soma size. Only large-diameter ganglion cells were selected, because type I retinal ganglion cells have been shown to have much larger cell somata than displaced amacrine cells and other types of retinal ganglion cells. Therefore, we assume that we mainly recorded from this ganglion cell type. 
For the isolation of glutamate transporter currents, ACSF was replaced with a solution with lowered calcium, containing (in mM): 125 NaCl, 3.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 0.2 CaCl2, 1 MgCl2, and 25 glucose [pH 7.4, adjusted with 1 M NaOH]. Both solutions were held at room temperature and continuously bubbled with carbogen during the whole experiment. The pipette solution (ic) contained (in mM): 140 KCl2, 2 MgCl2, 10 EGTA, 4 Na2-ATP, 0.44 Na-GTP, and 10 HEPES [pH 7.2, adjusted with 1 M KOH]. The inhibition of different glutamate current types in rat retinal ganglion cells was based on pharmacological properties. Drugs were applied to the bath in the following concentrations CNQX (25 μM), DAP-5 (10 μM), MCPG (500 μM). Remaining spontaneous activity was blocked by the addition of strychnine (200 μM) and picrotoxin (200 μM). Glutamate (1 mM) was applied by a six-barrel superfusion pipette. All drugs were purchased from Sigma-Aldrich (Deisenhofen, Germany) and dissolved in bath solution just before use. Drug solutions were bubbled with 95% O2-5% CO2 at room temperature immediately before application to retinal slices. 
For electrophysiological recordings, wholemount strips were transferred into the recording chamber on a microscope stage (Axioskop; Carl Zeiss Meditec, Oberkochen, Germany) and fixed with a small grid made of fine nylon strings tightened between a U-shaped platinum wire. The recording chamber was superfused with 1.5- to 2-mL/min ACSF. Patch pipettes were pulled out of borosilicate capillaries. Pipettes were filled with intracellular solution (ic). When filled, they displayed resistances of 2.0 to 3.5 MΩ. Recordings were performed with an EPC-7 amplifier (Heka Elektronik, Lambrecht, Germany), using optimal series resistance compensation. Series resistance compensation was usually 60% to 80%. Cells were selected for study if they had series resistances less than 15 MΩ. Whole-cell currents were recorded with sampling frequencies of 100 and 5 kHz. Data were not corrected for the liquid junction. Cells were normally kept at a holding potential of −60 mV. 
A commercial software program (Clampex, ver. 8.1.0; Axon Instruments, Union City, CA) was used for data acquisition and analysis, and another (Sigmaplot; SPSS Inc., Chicago, IL) was used for curve fitting and plotting. Time constants of decay (τ) were determined by fitting the single exponential function A n · exp(tK)/ n + C to the data, where A is the amplitude relative to offset evaluated at the start of the fit region (n), C is the steady state asymptote, and (tK) is the time, set to zero at the beginning (K) of the fit region. Fitting procedures were performed on computer (Clampfit; Axon Instruments) and IGOR (Wavemetrics, Lake Oswego, OR) software routines. The amplitude was determined by measuring the time from the beginning of the current until the peak current was reached. 
Statistical Analysis
Data are provided as arithmetic means ± SEM, and n represents the number of oocytes investigated. All experiments were repeated in at least three batches of oocytes. In all repetitions, qualitatively similar data were obtained. All data were tested for significance by ANOVA, and only results with P < 0.05 were considered statistically significant. 
Results
SGK1 Expression and Colocalization with EAAT3 in Rat Retina
To examine cell-specific protein localization, we performed immunohistochemical stainings in adult rat retina sections using our polyclonal anti-SGK1 antiserum. 16 By using this antiserum, we observed a strong staining of most neurons in the ganglion cell layer (GCL; 1 , arrows). Individual cell bodies of different sizes were labeled with a particular strong immunoreaction in cells with a diameter larger than 12 μm. The fiber layer built by the ganglion cell axons did not show any SGK1 immunoreactivity. As approximately half of the cells in the GCL of the rodent retina are displaced amacrine cells, both ganglion cells and displaced amacrine cells showed a positive SGK1 immunoreactivity. 
In addition to the cells in the ganglion cell layer, staining was found in the cell bodies in the inner nuclear layer (INL). A first subset of cells in the innermost row of the INL along the border to the inner plexiform layer (IPL) was immunoreactive (1 , triangle and double arrows). Because of their position, the cells can be addressed as amacrine cells. An immunoreaction was also visible in three narrow, stratified bands in the IPL 1 . Band one was running in the outermost IPL along the border to the INL and band two at 25% and band three at 50% depth between the INL and GCL. However, we do not yet know which of the cell types from the GCL and the INL are connected to which of the three different sublayers in the IPL. No staining was present in the outer plexiform layer (OPL). No staining was visible when the SGK1 antibody was omitted (data not shown). 
Staining with an EAAT3-specific antibody revealed expression of the transporter in apparently all cells of the GCL 1 . Arrows denote prominent immunoreactivity of the GCL composed of ganglion and displaced amacrine cells. EAAT3 expression was also found in the cell bodies in the inner nuclear layer. The INL shows partial immunoreactivity with most intensive staining of a subset of cells in close proximity to the inner plexiform layer (1 , triangle and double arrows). These cells can be assigned as amacrine cells. Antibody staining occurred also in the OPL 1 and in the IPL. EAAT3 immunoreactivity was more uniformly distributed within the IPL, with an alleviated reaction in the inner part of the IPL toward the GCL 1 . Control experiments omitting the first antibody resulted in a complete absence of immunoreactivity over the entire retinal tissue (data not shown). 
Double labeling with both the SGK1 and the EAAT3 antibody 1 , demonstrated an overlap of SGK1 and EAAT3 expression in amacrine cells in the inner row of the INL (1 , triangle) and in cells in the GCL (1 , arrows). Besides colocalization, cells without a clear sign of an overlap were present in the INL and in the GCL (1 , double arrows). Whereas the OPL did not show any coexpression, a colocalization within the INL is very likely, even though a distinct layering for the EAAT3 antibody corresponding to that of SGK1 was missing. However, both antibodies did not stain (or had at least a markedly reduced immunoreactivity in) the inner part of the IPL. Moreover, the border of the internal IPL staining for EAAT3 corresponded with the one of the innermost SGK1 band 1
Regulation of EAAT3 by SGK1
In Xenopus oocytes expressing the glutamate transporter EAAT3, but not in water injected oocytes, the addition of 1 mM glutamate to the bath induced an inward current (IGLU) approaching −21.6 ± 1.4 nA (n = 30) at a holding potential of −60 mV. As shown in 2 , coexpression of wild-type SGK1 significantly increased the EAAT3 mediated current by 55.1% ± 6.2% (n = 26). 
The effect of wild-type SGK1 was mimicked by the constitutively active S422DSGK1 3 , which increased the EAAT3 mediated current by 55.0% ± 8.2% (n = 13). In contrast, the inactive K127NSGK1 did not significantly modify IGLU (+4.8% ± 7.9%, n = 13). Similar to SGK1 and S422DSGK1, the constitutively active T308D,S473DPKB stimulated IGLU in EAAT3-expressing oocytes 3 . Coexpression of T308D,S473DPKB increased IGLU by 75.83% ± 5.8% (n = 26). 
IGLU was not significantly altered (−5.2% ± 3.1%, n = 11) after coexpression of Nedd4-2 3 . Moreover, the effects of SGK1, S422DSGK1, and T308D,S473DPKB were not significantly modified by coexpression of Nedd4-2 3 . In Xenopus oocytes coexpressing EAAT3 together with SGK1, S422DSGK1, or T308D,S473DPKB and Nedd4-2, IGLU was not significantly different from IGLU in oocytes expressing EAAT3 with the respective kinases alone but significantly larger than IGLU in Xenopus oocytes expressing EAAT3 alone. 
As illustrated in 4 addition of glutamate to mouse retinal ganglion cells in the presence of the glutamate receptor blockers CNQX (25 μM), DAP-5 (10 μM), and MCPG (500 μM), as well as strychnine (200 μM) and picrotoxin (200 μM), induced a current that was most likely generated by electrogenic glutamate uptake. The current was significantly smaller in retinal ganglion cells from BDNF-deficient mice (BDNF −/−) compared with the current in retinal ganglion cells from wild-type mice (BDNF +/+). 
Discussion
EAAT3 encodes for a carrier protein mediating the electrogenic, Na+-coupled transport of glutamate. 39 40 As a transport of negatively charged glutamate is coupled to the translocation of 3 Na+ ions, transport of glutamate through EAAT3 is coupled to the translocation of charge. 39 40 56 57 The current generated by addition of substrate is a measure of glutamate transport. Xenopus oocytes do not express endogenous electrogenic glutamate transport—that is, the current is solely generated by the heterologously expressed transport protein. 
The present observations reveal a novel mechanism for regulation of EAAT3—that is, the stimulation by SGK1 and the PKB. The kinases stimulate EAAT3 in the absence of heterologously expressed Nedd4-2, and their effect is not modified by the additional expression of Nedd4-2. Moreover, Nedd4-2 does not significantly modify EAAT3 activity. Thus, in contrast to what has previously been shown for the regulation by SGK1 of the voltage gated cardiac Na+ channel 58 and the epithelial Na+ channel ENaC, 20 21 the kinases do not regulate EAAT3 through inhibition of Nedd4-2 in this system. Other signaling molecules involved in the regulation of ENaC include the small G protein K-Ras 2A and the channel activating protein (CAP)-1. 59 60 The regulation of renal outer medullary K+ channel ROMK1 12 and of the Na+/H+ exchanger NHE3 by SGK1 involves the Na+/H+ exchanger regulating factor NHERF2. Moreover, regulation of ROMK1 involves direct phosphorylation of the channel protein. 61 Thus, several candidate mechanisms may mediate the effect of SGK1 on EAAT3. 
The effects of SGK1 and PKB on the glutamate transporter EAAT3 presumably participate in the clearance of glutamate from the extracellular space and thus in the termination of the excitatory signal. As shown in the present study, SGK1 is expressed in several retinal cells, including retinal ganglion cells, and PKB has been shown to be expressed in retinal tissue. 24 The decreased glutamate induced currents in retinal ganglion cells from BDNF-deficient mice points to a role of BDNF in the regulation of glutamate transport. Because BDNF signals through PI3 kinase and PKB in retinal ganglion cells, 38 it is expected similarly to activate SGK1. The two kinases could thus contribute to the effects of BDNF in those cells. 
In theory, stimulation of glutamate transport could provide some protection against excessive activation by glutamate. Experimental evidence points to a role for glutamate in apoptosis after nerve crush 62 and in mutant quails, an animal model for glaucoma, glutamate concentrations increase immediately before cell death. 38 External application of glutamate is known to trigger retinal ganglion cell death, 63 64 65 66 67 at least partially, by increase of cytosolic Ca2+ activity, 68 activation of caspases, 69 70 and subsequent apoptosis. 71 Conversely, PKB activity has been shown to correlate with retinal cell survival. 24 62 Accordingly, dephosphorylation and thus deactivation of PKB by inhibition of PI3 kinase triggers apoptosis of retinal cells. 24 Notably, this maneuver not only disrupts PKB activity but similarly downregulates SGK1 activity. The effect is not reversed by activation of PKA and subsequent phosphorylation of Bad. Thus, the authors concluded that the antiapoptotic effect of PKB was not due to phosphorylation of Bad. 24 In theory, the mechanism could involve regulation of glutamate uptake by PKB and SGK1. Nothing is known, however, about the specific role of EAAT3 in the survival of retinal ganglion cells. Antisense knockdown of EAAT3 in mice did not lead to severe neurodegeneration but, besides dicarboxylic amino aciduria, caused some subtle behavioral abnormalities and seizures. 72 Dicarboxylic amino aciduria in humans may be similarly paralleled by neurologic abnormalities. 73 The lack of severe neurodegeneration could be due to glutamate uptake through other glutamate transporters which may be similarly sensitive to SGK1 and PKB. 58 Taken together, presently available evidence does not allow the speculation that SGK1- or PKB-dependent regulation of EAAT3 participates in the regulation of retinal cell survival. 
In conclusion, the present observations demonstrated that SGK1 is expressed in retinal neurons and disclosed a novel function of this kinase—that is, the regulation of the glutamate transporter EAAT3. The effect of SGK1 is shared by PKB. The mechanism is likely to participate in the regulation of glutamate clearance from the extracellular space. 
Figure 1.
 
SGK1 and EAAT3 protein expression in neurons of the rat retina: radial sections of adult rat retina simultaneously labeled with SGK1 (A) and EAAT3 (B) antibodies and their colocalization (C). (A) Cellular localization of SGK1 protein: cell bodies are marked in the GCL (arrows) and in the inner row of the INL (triangle; small double arrows). A positive immunoreaction is present in three distinct bands in the IPL. (B) Cellular localization of EAAT3 protein. Similar to SGK1 immunostaining, cell bodies in the GCL (arrows) and in the inner row of the INL are marked (triangle, small double arrows in the INL). Both plexiform layers, OPL and IPL, showed an EAAT3-positive reaction. In contrast to the ribbon-like SGK1 distribution (A) EAAT3 was more uniformly distributed over the IPL. (C) Merged images of (A) and (B). Colocalization of SGK1 and EAAT3 is indicated by a yellow stain and was found in amacrine cells (triangle) and in most of the cells in the GCL (arrows). Cells without a clear sign for a colocalization of both proteins were present in the INL and GCL (A, B, C: small double arrows). Whereas the OPL did not show any coexpression, colocalization in the IPL is very likely, even though a distinct layering for the EAAT3 antibody corresponding to that of SGK1 is missing. Scale bar, 25 μm.
Figure 1.
 
SGK1 and EAAT3 protein expression in neurons of the rat retina: radial sections of adult rat retina simultaneously labeled with SGK1 (A) and EAAT3 (B) antibodies and their colocalization (C). (A) Cellular localization of SGK1 protein: cell bodies are marked in the GCL (arrows) and in the inner row of the INL (triangle; small double arrows). A positive immunoreaction is present in three distinct bands in the IPL. (B) Cellular localization of EAAT3 protein. Similar to SGK1 immunostaining, cell bodies in the GCL (arrows) and in the inner row of the INL are marked (triangle, small double arrows in the INL). Both plexiform layers, OPL and IPL, showed an EAAT3-positive reaction. In contrast to the ribbon-like SGK1 distribution (A) EAAT3 was more uniformly distributed over the IPL. (C) Merged images of (A) and (B). Colocalization of SGK1 and EAAT3 is indicated by a yellow stain and was found in amacrine cells (triangle) and in most of the cells in the GCL (arrows). Cells without a clear sign for a colocalization of both proteins were present in the INL and GCL (A, B, C: small double arrows). Whereas the OPL did not show any coexpression, colocalization in the IPL is very likely, even though a distinct layering for the EAAT3 antibody corresponding to that of SGK1 is missing. Scale bar, 25 μm.
Figure 2.
 
EAAT3 transport activity was upregulated by SGK1. (A) Original tracings. Solid rectangles: administration of 1 mM glutamate. (B) Arithmetic means ± SEM. *Statistically significant compared with currents in Xenopus oocytes expressing EAAT3+H2O.
Figure 2.
 
EAAT3 transport activity was upregulated by SGK1. (A) Original tracings. Solid rectangles: administration of 1 mM glutamate. (B) Arithmetic means ± SEM. *Statistically significant compared with currents in Xenopus oocytes expressing EAAT3+H2O.
Figure 3.
 
(A) EAAT3 transport activity was upregulated by the constitutively active S422DSGK but not by the inactive mutant K127NSGK1. Similar to SGK1, the constitutively active T308D,S473DPKB increased EAAT3 currents. (B) The ubiquitin ligase Nedd4-2 did not modify EAAT3 activity. On coexpression of Nedd4-2, glutamate-induced currents remained unchanged compared with the expression of EAAT3 alone. Moreover, the stimulating effect of the protein kinases SGK1, S422DSGK1, and T308D,S473DPKB on EAAT3 were not significantly different in the presence of Nedd4-2 than with expression of the protein kinases alone with EAAT3. Arithmetic means ± SEM. *Statistically significant compared with currents in Xenopus oocytes expressing EAAT3+H2O.
Figure 3.
 
(A) EAAT3 transport activity was upregulated by the constitutively active S422DSGK but not by the inactive mutant K127NSGK1. Similar to SGK1, the constitutively active T308D,S473DPKB increased EAAT3 currents. (B) The ubiquitin ligase Nedd4-2 did not modify EAAT3 activity. On coexpression of Nedd4-2, glutamate-induced currents remained unchanged compared with the expression of EAAT3 alone. Moreover, the stimulating effect of the protein kinases SGK1, S422DSGK1, and T308D,S473DPKB on EAAT3 were not significantly different in the presence of Nedd4-2 than with expression of the protein kinases alone with EAAT3. Arithmetic means ± SEM. *Statistically significant compared with currents in Xenopus oocytes expressing EAAT3+H2O.
Figure 4.
 
Blunted glutamate-induced current in retinal ganglion cells from BDNF-deficient mice (BDNF−/−) and wild-type (BDNF+/+) mice. The glutamate (1 mM)-induced currents were determined in retinal ganglion cells in slices of retinal tissue under blockage of glutamate receptors. (A) Original tracings; (B) arithmetic means ± SEM (n = 4) of the time constants (top) and maximum currents (bottom). *Statistically significant value in (BDNF−/−) compared with wild-type (BDNF+/+) mice.
Figure 4.
 
Blunted glutamate-induced current in retinal ganglion cells from BDNF-deficient mice (BDNF−/−) and wild-type (BDNF+/+) mice. The glutamate (1 mM)-induced currents were determined in retinal ganglion cells in slices of retinal tissue under blockage of glutamate receptors. (A) Original tracings; (B) arithmetic means ± SEM (n = 4) of the time constants (top) and maximum currents (bottom). *Statistically significant value in (BDNF−/−) compared with wild-type (BDNF+/+) mice.
 
The authors thank Sir Philip Cohen for providing the constructs of active S422DSGK1, inactive K127NSGK1, and constitutively active T308D,S473DPKB, and Birgitta Noll, Sylvia Bolz, and Gudrun Härer for expert technical assistance. 
Firestone GL, Giampaolo JR, O’Keeffe BA. Stimulus-dependent regulation of the serum and glucocorticoid inducible protein kinase (Sgk) transcription, subcellular localization and enzymatic activity. Cell Physiol Biochem. 2003;13:1–12.
Webster MK, Goya L, Firestone GL. Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine/threonine protein kinase. J Biol Chem. 1993;268:11482–11485.
Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL. Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol. 1993;13:2031–2040.
Waldegger S, Barth P, Raber G, Lang F. Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA. 1997;94:4440–4445.
Lang F, Henke G, Embark HM, et al. Regulation of channels by the serum and glucocorticoid-inducible kinase: implications for transport, excitability and cell proliferation. Cell Physiol Biochem. 2003;13:41–50.
Böhmer C, Wagner CA, Beck S, et al. The shrinkage-activated Na+ conductance of rat hepatocytes and its possible correlation to rENaC. Cell Physiol Biochem. 2000;10:187–194.
Chen SY, Bhargava A, Mastroberardino L, et al. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA. 1999;96:2514–2519.
Lang F, Klingel K, Wagner CA, et al. Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc Natl Acad Sci USA. 2000;97:8157–8162.
Naray-Fejes-Toth A, Canessa C, Cleaveland ES, Aldrich G, Fejes-Toth G. sgk is an aldosterone-induced kinase in the renal collecting duct: effects on epithelial na+ channels. J Biol Chem. 1999;274:16973–16978.
Pearce D. SGK1 regulation of epithelial sodium transport. Cell Physiol Biochem. 2003;13:13–20.
Shigaev A, Asher C, Latter H, Garty H, Reuveny E. Regulation of sgk by aldosterone and its effects on the epithelial Na+ channel. Am J Physiol. 2000;278:F613–F619.
Yun CC, Palmada M, Embark HM, et al. The serum and glucocorticoid-inducible kinase SGK1 and the Na+/H+ exchange regulating factor NHERF2 synergize to stimulate the renal outer medullary K+ channel ROMK1. J Am Soc Nephrol. 2002;13:2823–2830.
Embark HM, Bohmer C, Vallon V, Luft F, Lang F. Regulation of KCNE1-dependent K+ current by the serum and glucocorticoid-inducible kinase (SGK) isoforms. Pflugers Arch. 2003;445:601–606.
Gamper N, Fillon S, Feng Y, et al. K+ channel activation by all three isoforms of serum- and glucocorticoid-dependent protein kinase SGK. Pflugers Arch. 2002;445:60–66.
Gamper N, Fillon S, Huber SM, et al. IGF-1 up-regulates K+ channels via PI3-kinase, PDK1 and SGK1. Pflugers Arch. 2002;443:625–634.
Wärntges S, Friedrich B, Henke G, et al. Cerebral localization and regulation of the cell volume-sensitive serum- and glucocorticoid-dependent kinase SGK1. Pflugers Arch. 2002;443:617–624.
Yun CC, Chen Y, Lang F. Glucocorticoid activation of Na+/H+ exchanger isoform 3 revisited: the roles of SGK1 and NHERF2. J Biol Chem. 2002;277:7676–7683.
Setiawan I, Henke G, Feng Y, et al. Stimulation of Xenopus oocyte Na+,K+ATPase by the serum and glucocorticoid-dependent kinase sgk1. Pflugers Arch. 2002;444:426–431.
Henke G, Setiawan I, Bohmer C, Lang F. Activation of Na+/K+-ATPase by the serum and glucocorticoid-dependent kinase isoforms. Kidney Blood Press Res. 2002;25:370–374.
Debonneville C, Flores SY, Kamynina E, et al. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+ channel cell surface expression. EMBO J. 2001;20:7052–7059.
Snyder PM, Olson DR, Thomas BC. Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel. J Biol Chem. 2002;277:5–8.
Verrey F, Loffing J, Zecevic M, Heitzmann D, Staub O. SGK1: aldosterone-induced relay of Na+ transport regulation in distal kidney nephron cells. Cell Physiol Biochem. 2003;13:21–28.
Anan T, Nagata Y, Koga H, et al. Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes. Genes Cells. 1998;3:751–763.
Campos CB, Bedard PA, Linden R. Selective involvement of the PI3K/PKB/bad pathway in retinal cell death. J Neurobiol. 2003;56:171–177.
Calera MR, Martinez C, Liu H, Jack AK, Birnbaum MJ, Pilch PF. Insulin increases the association of Akt-2 with Glut4-containing vesicles. J Biol Chem. 1998;273:7201–7204.
Foran PG, Fletcher LM, Oatey PB, Mohammed N, Dolly JO, Tavare JM. Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3–L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin. J Biol Chem. 1999;274:28087–28095.
Hajduch E, Alessi DR, Hemmings BA, Hundal HS. Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells. Diabetes. 1998;47:1006–1013.
Kohn AD, Summers SA, Birnbaum MJ, Roth RA. Expression of a constitutively active Akt Ser/Thr kinase in 3T3–L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem. 1996;271:31372–31378.
Tanti JF, Grillo S, Gremeaux T, Coffer PJ, Van Obberghen E, Marchand-Brustel Y. Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes. Endocrinology. 1997;138:2005–2010.
Wang Q, Somwar R, Bilan PJ, et al. Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. Mol Cell Biol. 1999;19:4008–4018.
Kobayashi T, Cohen P. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J. 1999;339:319–328.
Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996;15:6541–6551.
Lang F, Cohen P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Sci STKE. 2001;2001:RE17.
Altomare DA, Lyons GE, Mitsuuchi Y, Cheng JQ, Testa JR. Akt2 mRNA is highly expressed in embryonic brown fat and the AKT2 kinase is activated by insulin. Oncogene. 1998;16:2407–2411.
Kim SO, Hasham MI, Katz S, Pelech SL. Insulin-regulated protein kinases during postnatal development of rat heart. J Cell Biochem. 1998;71:328–339.
Meier R, Alessi DR, Cron P, Andjelkovic M, Hemmings BA. Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bbeta. J Biol Chem. 1997;272:30491–30497.
Zinda MJ, Vlahos CJ, Lai MT. Ceramide induces the dephosphorylation and inhibition of constitutively activated Akt in PTEN negative U87mg cells. Biochem Biophys Res Commun. 2001;280:1107–1115.
Dkhissi O, Chanut E, Wasowicz M, et al. Retinal TUNEL-positive cells and high glutamate levels in vitreous humor of mutant quail with a glaucoma-like disorder. Invest Ophthalmol Vis Sci. 1999;40:990–995.
Dowd LA, Robinson MB. Rapid stimulation of EAAC1-mediated Na+-dependent L-glutamate transport activity in C6 glioma cells by phorbol ester. J Neurochem. 1996;67:508–516.
Dowd LA, Coyle AJ, Rothstein JD, Pritchett DB, Robinson MB. Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6 glioma, and Xenopus oocytes expressing excitatory amino acid carrier 1 (EAAC1). Mol Pharmacol. 1996;49:465–473.
Hediger MA. Glutamate transporters in kidney and brain. Am J Physiol. 1999;277:F487–F492.
Kanai Y, Hediger MA. Primary structure and functional characterization of a high-affinity glutamate transporter. Nature. 1992;360:467–471.
Kanai Y, Smith CP, Hediger MA. The elusive transporters with a high affinity for glutamate. Trends Neurosci. 1993;16:365–370.
Peghini P, Janzen J, Stoffel W. Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration. EMBO J. 1997;16:3822–3832.
Shashidharan P, Huntley GW, Meyer T, Morrison JH, Plaitakis A. Neuron-specific human glutamate transporter: molecular cloning, characterization and expression in human brain. Brain Res. 1994;662:245–250.
Smith CP, Weremowicz S, Kanai Y, Stelzner M, Morton CC, Hediger MA. Assignment of the gene coding for the human high-affinity glutamate transporter EAAC1 to 9p24: potential role in dicarboxylic aminoaciduria and neurodegenerative disorders. Genomics. 1994;20:335–336.
Velaz-Faircloth M, McGraw TS, Alandro MS, Fremeau RT, Jr, Kilberg MS, Anderson KJ. Characterization and distribution of the neuronal glutamate transporter EAAC1 in rat brain. Am J Physiol. 1996;270:C67–C75.
Rothstein JD, Martin L, Levey AI, et al. Localization of neuronal and glial glutamate transporters. Neuron. 1994;13:713–725.
Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, Danbolt NC. Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci. 1995;15:1835–1853.
Lehre KP, Danbolt NC. The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci. 1998;18:8751–8757.
Kugler P, Schmitt A. Glutamate transporter EAAC1 is expressed in neurons and glial cells in the rat nervous system. Glia. 1999;27:129–142.
Dehnes Y, Chaudhry FA, Ullensvang K, Lehre KP, Storm-Mathisen J, Danbolt NC. The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia. J Neurosci. 1998;18:3606–3619.
Schultz K, Stell WK. Immunocytochemical localization of the high-affinity glutamate transporter, EAAC1, in the retina of representative vertebrate species. Neurosci Lett. 1996;211:191–194.
Rothe T, Bahring R, Carroll P, Grantyn R. Repetitive firing deficits and reduced sodium current density in retinal ganglion cells developing in the absence of BDNF. J Neurobiol. 1999;40:407–419.
Wagner CA, Friedrich B, Setiawan I, Lang F, Broer S. The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem. 2000;10:1–12.
Otis TS, Kavanaugh MP. Isolation of current components and partial reaction cycles in the glial glutamate transporter EAAT2. J Neurosci. 2000;20:2749–2757.
Vandenberg RJ, Arriza JL, Amara SG, Kavanaugh MP. Constitutive ion fluxes and substrate binding domains of human glutamate transporters. J Biol Chem. 1995;270:17668–17671.
Boehmer C, Wilhelm V, Palmada M, et al. Serum and glucocorticoid inducible kinases in the regulation of the cardiac sodium channel SCN5A. Cardiovasc Res. 2003;57:1079–1084.
Gormley K, Dong Y, Sagnella GA. Regulation of the epithelial sodium channel by accessory proteins. Biochem J. 2003;371:1–14.
Vuagniaux G, Vallet V, Jaeger NF, Hummler E, Rossier BC. Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus oocytes. J Gen Physiol. 2002;120:191–201.
Yoo D, Kim BY, Campo CK, et al. Cell surface expression of the ROMK (Kir 1.1) channel is regulated by the aldosterone-induced kinase, SGK-1 and PKA. J Biol Chem. 2003;278:23066–23075.
Klocker N, Kermer P, Weishaupt JH, Labes M, Ankerhold R, Bahr M. Brain-derived neurotrophic factor-mediated neuroprotection of adult rat retinal ganglion cells in vivo does not exclusively depend on phosphatidyl-inositol-3′-kinase/protein kinase B signaling. J Neurosci. 2000;20:6962–6967.
Goto W, Ota T, Morikawa N, et al. Protective effects of timolol against the neuronal damage induced by glutamate and ischemia in the rat retina. Brain Res. 2002;958:10–19.
Gross RL, Hensley SH, Gao F, Yang XL, Dai SC, Wu SM. Effects of betaxolol on light responses and membrane conductance in retinal ganglion cells. Invest Ophthalmol Vis Sci. 2000;41:722–728.
Luo X, Heidinger V, Picaud S, et al. Selective excitotoxic degeneration of adult pig retinal ganglion cells in vitro. Invest Ophthalmol Vis Sci. 2001;42:1096–1106.
Romano C, Price MT, Almli T, Olney JW. Excitotoxic neurodegeneration induced by deprivation of oxygen and glucose in isolated retina. Invest Ophthalmol Vis Sci. 1998;39:416–423.
Schori H, Kipnis J, Yoles E, et al. Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: implications for glaucoma. Proc Natl Acad Sci USA. 2001;98:3398–3403.
Otori Y, Kusaka S, Kawasaki A, Morimura H, Miki A, Tano Y. Protective effect of nilvadipine against glutamate neurotoxicity in purified retinal ganglion cells. Brain Res. 2003;961:213–219.
Chen TA, Yang F, Cole GM, Chan SO. Inhibition of caspase-3-like activity reduces glutamate induced cell death in adult rat retina. Brain Res. 2001;904:177–188.
Li Y, Schlamp CL, Poulsen GL, Jackson MW, Griep AE, Nickells RW. p53 regulates apoptotic retinal ganglion cell death induced by N-methyl-D-aspartate. Mol Vis. 2002;8:341–350.
Li Y, Schlamp CL, Nickells RW. Experimental induction of retinal ganglion cell death in adult mice. Invest Ophthalmol Vis Sci. 1999;40:1004–1008.
Rothstein JD, Dykes-Hoberg M, Pardo CA, et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron. 1996;16:675–686.
Melancon SB, Dallaire L, Lemieux B, Robitaille P, Potier M. Dicarboxylic aminoaciduria: an inborn error of amino acid conservation. J Pediatr. 1977;91:422–427.
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