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. 2014 Jun 5;9(6):e98469.
doi: 10.1371/journal.pone.0098469. eCollection 2014.

Role of Na,K-ATPase α1 and α2 isoforms in the support of astrocyte glutamate uptake

Nina B Illarionova  1 , Hjalmar Brismar  2 , Anita Aperia  1 , Eli Gunnarson  1
Affiliations

Role of Na,K-ATPase α1 and α2 isoforms in the support of astrocyte glutamate uptake

Nina B Illarionova et al. PLoS One. .

Erratum in

  • PLoS One. 2014;9(9): doi/10.1371/journal.pone.0110095. Illarionava, Nina B [corrected to Illarionova, Nina B]

Abstract

Glutamate released during neuronal activity is cleared from the synaptic space via the astrocytic glutamate/Na(+) co-transporters. This transport is driven by the transmembrane Na(+) gradient mediated by Na,K-ATPase. Astrocytes express two isoforms of the catalytic Na,K-ATPase α subunits; the ubiquitously expressed α1 subunit and the α2 subunit that has a more specific expression profile. In the brain α2 is predominantly expressed in astrocytes. The isoforms differ with regard to Na+ affinity, which is lower for α2. The relative roles of the α1 and α2 isoforms in astrocytes are not well understood. Here we present evidence that the presence of the α2 isoform may contribute to a more efficient restoration of glutamate triggered increases in intracellular sodium concentration [Na(+)]i. Studies were performed on primary astrocytes derived from E17 rat striatum expressing Na,K-ATPase α1 and α2 and the glutamate/Na(+) co-transporter GLAST. Selective inhibition of α2 resulted in a modest increase of [Na(+)]i accompanied by a disproportionately large decrease in uptake of aspartate, an indicator of glutamate uptake. To compare the capacity of α1 and α2 to handle increases in [Na(+)]i triggered by glutamate, primary astrocytes overexpressing either α1 or α2 were used. Exposure to glutamate 200 µM caused a significantly larger increase in [Na(+)]i in α1 than in α2 overexpressing cells, and as a consequence restoration of [Na(+)]i, after glutamate exposure was discontinued, took longer time in α1 than in α2 overexpressing cells. Both α1 and α2 interacted with astrocyte glutamate/Na(+) co-transporters via the 1st intracellular loop.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Aspartate uptake, effect of inhibition of Na,K-ATPase α2 and of α2+ α1.
A. GFAP immunostaining of primary astrocytes with multiple thin processes. Astrocytes were derived from E17 rat striatum and cultured for 9–12 days in medium enriched with neuronal conditioned medium (NCM) during the last 24 h. B. Immunoblotting of Na,K-ATPase α1 and α2, GLAST and GLT-1 in whole brain lysate and in primary astrocyte culture treated with NCM (Astrocytes +NCM) or without NCM (Astrocytes -NCM). The expression of the glutamate transporter GLAST in primary astrocyte culture increased by 46% after application of NCM for 24 h (One-way ANOVA, N = 3 experiments, P<0.01). The expression of GLT-1, Na,K-ATPase α1 and α2 did not change. C. Increase in astrocyte [Na+]i (Δ[Na+]i) following treatment with ouabain 1 µM or 2000 µM for 15 min (One-way ANOVA, N = 38 cells, P<0.001). D. Decrease in D-Asp uptake following treatment with ouabain 1 µM or ouabain 2000 µM for 15 min (One-way ANOVA, N = 9 experiments, P<0.001).
Figure 2
Figure 2. Astrocytes overexpressing Na,K-ATPase α1 or α2, characterization of a model.
A. Basal [Na+]i in astrocytes overexpressing Na,K-ATPase α1 (white) or Na,K-ATPase α2 (grey). B. Increase in [Na+]i (Δ[Na+]i) in rat primary astrocytes expressing α1 (white) or α2 (grey) in response to treatment with ouabain 1 µM and 2000 µM for 5 min, respectively (Repeated Measures ANOVA, N = 30 cells, **P<0.01 and ***P<0.001). C. Images of immunostaining with GLAST (left image) in primary cultured astrocytes transfected with mCherry IRES Na,K-ATPase α1 or α2 (middle image). Merged image of GLAST and mCherry IRES (right image). Regions of interest were selected in plasma membrane (#1 and #2) and cytosol (#3 and #4) of transfected and non-transfected cells in the same field of view. Ratio of GLAST fluorescence intensities was estimated between region #1/#2 and #3/#4 (for details see Materials and Methods). Values were compared between α1 and α2 transfected cells. Scale bar 10 µm. D. GLAST immunofluorescence intensity ratios (transfected vs. non-transfected cells (WT)) in plasma membrane and cytosol of primary astrocytes expressing Na,K-ATPase α1 (white) or α2 (grey). There were no significant differences in GLAST expression between α1 and α2 expressing cells. All data are presented as mean values (bar) and SEM (whiskers). E. Primary astrocytes expressing Na,K-ATPase α1 or α2 with an extracellular pH sensitive tag - superecliptic pHluorin. The mean fluorescent signal was measured for the whole cell. The pH dependent fluorescent signal was rapidly attenuated in response to a change of the extracellular pH from 7.4 to 6.5 (curves below images). Reversal of the pH restored the fluorescent signal, indicating that transfected Na,K-ATPase α1 and α2 were inserted in the plasma membrane. Scale bar 10 µm.
Figure 3
Figure 3. Transient changes in [Na+]i following exposure to glutamate 200 µM in astrocytes expressing predominantly Na,K-ATPase α1 or α2 isoforms.
A. Astrocyte [Na+]i (mean) in Na,K-ATPase α1 and Na,K-ATPase α2 expressing cells exposed to glutamate 200 µM for 10 min (indicated by horizontal white bar). B. Maximum [Na+]i after 10 min exposure to glutamate 200 µM in α1 (white) and α2 (grey) expressing astrocytes (One-way ANOVA, N = 18 cells, *P<0.05). C. Residual [Na+]i measured at 10 min (indicated with an arrow in Fig. 3A) after discontinuation of glutamate treatment in α1 (white) and α2 (grey) expressing astrocytes (One-way ANOVA, N = 18 cells, *P<0.05).
Figure 4
Figure 4. Na,K-ATPase α1 and α2 subunit interaction with astrocyte glutamate transporters GLT-1 and GLAST.
A and B. GLAST and GLT-1 were co-immunoprecipitated with Na,K-ATPase α1 and α2 from adult rat brain lysate. GLAST and GLT-1 interact with full length Na,K-ATPase α1 (A) and Na,K-ATPase α2 (B). C–H: Pull-down assays of GST and GST fused to the Na,K-ATPase α N-terminus (NT), cytosolic domains corresponding to first and second intracellular loops (CD2 and CD3) and C-terminus (CT), respectively, incubated with adult rat brain lysate after immobilization on GST-sepharose. The pull-down probes were subjected to SDS-PAGE and Western blot and probed with antibodies against GLAST or GLT-1. C and D. GLAST interacts with the CD2 domain of Na,K-ATPase α2 (C) and Na,K-ATPase α1 (D) but not with NT, CD3 or CT domains of either α1 or α2. E and F. GLT-1 interacts with the CD2 domain of Na,K-ATPase α2 (E) and Na,K-ATPase α1 (F) but not with NT, CD3 or CT domains of either α1 or α2. G and H. Comparison of the CD2 α1 and α2 interaction between GLT-1 and GLAST. Bar diagram shows mean integrated densities for GLAST (G) and GLT-1 (H) immunostaining in GST pull-down assays of CD2 α1 and CD2 α2, respectively, in percent of CD2 α2 staining density. (One-way ANOVA, N = 3 experiments, *P<0.05). I. Co-expression of GLAST and Na,K-ATPase α subunits in primary astrocytes. Primary astrocytes transfected with GLAST (magenta) and Na,K-ATPase α1 or α2 (green). Note co-expression (white) in plasma membrane compartments, including astrocyte processes, indicated by arrows. Scale bar 10 µm.
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Grants and funding

This study has been supported by grants from the Swedish Research Council (AA), the Erling-Persson Family Foundation, the Regional Agreement on Medical Training and Clinical Research (ALF) between Stockholm County Council and Karolinska Institutet (EG) and Sällskapet Barnavård. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.