Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice

Abstract

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green-fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K⁺ and Cl⁻ channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K(2P)) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K(2P) channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K⁺ effluxes. Additionally, the inhibition of Na⁺-K⁺-Cl⁻ co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K⁺ channels (Kir4.1), K(2P) channels (TREK-1 and TWIK-1) and Cl⁻ channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K(2P) channels.

Figure 1. Inhibitors of glutamate transporters and K⁺-Cl⁻ co-transporters reduce the OGD-induced swelling in both astrocytic subpopulations.

The effect of 100 µM DL-TBOA, an inhibitor of excitatory amino acid transporters (A), 100 µM DIOA, an inhibitor of K⁺-Cl⁻ co-transporter (B), and 100 µM bumetanide, an inhibitor of Na⁺-K⁺-Cl⁻ co-transporter (C). A–C top: Time course of volume changes in HR-astrocytes (filled circles) and LR-astrocytes (empty circles) during 40-minute OGD (control) and during 20-minute OGD followed by 20-minute OGD with the application of an inhibitor in HR-/LR-astrocytes (filled/empty triangles). A–C bottom: The effect of inhibitors was evaluated in each individual cell and expressed as the percent cell volume increase/decrease related to the maximal volume after 20-minute OGD, which was set as 0%. Note that the application of DL-TBOA and DIOA led to a swelling reduction in both HR- and LR- astrocytes, while bumetanide only affected the swelling in HR-astrocytes. Asterisks indicate significant differences from controls (p<0.05 (*, significant), p<0.01 (**, very significant), p<0.001 (***, extremely significant)).

Figure 2. Inhibitors of Cl⁻ channels differently affect OGD-induced swelling in HR-and LR-astrocytes.

The effect of 100 µM NPPB, a non-specific inhibitor of chloride channels (A), 30 µM Tamoxifen (B) and 30 µM DCPIB (C), inhibitors of volume regulated anion channels. A–C top: Time course of volume changes in HR-astrocytes (filled circles) and LR-astrocytes (empty circles) during 40-minute OGD (control) and during 20-minute OGD followed by 20-minute co-application of ACSF_OGD plus an inhibitor in HR-/LR-astrocytes (filled/empty triangles). A–C bottom: The effect of the inhibitors was evaluated in each individual cell and expressed as the percent cell volume increase/decrease related to the maximal volume after 20-minute OGD, which was set as 0%. Note that in HR-astrocytes the application of VRAC inhibitors (Tamoxifen and DCPIB) reduced the swelling induced by OGD, while in LR-astrocytes the application of DCPIB resulted in an additional swelling. Asterisks indicate significant differences from controls (p<0.05 (*, significant), p<0.01 (**, very significant), p<0.001 (***, extremely significant)).

Figure 3. Inhibitors of K⁺ channels have an opposite effect on HR-/LR-astrocytes when applied during OGD.

The effect of 100 µM BaCl₂ (A), an inhibitor of inwardly rectifying potassium channels, 1 mM BaCl₂ (B) and 200 µM Quinine (C), inhibitors of two-pore domain potassium channels. A–C top: Time course of volume changes in HR-astrocytes (filled circles) and LR-astrocytes (empty circles) during 40-minute OGD (control) and during 20-minute OGD followed by 20-minute co-application of ACSF_OGD plus an inhibitor in HR-/LR-astrocytes (filled/empty triangles). A–C bottom: The effect of inhibitors was evaluated in each individual cell and expressed as the percent cell volume increase/decrease related to the maximal volume after 20-minute OGD, which was set as 0%. Note that the application of 1 mM BaCl₂ or Quinine inhibited swelling in HR-astrocytes, while in LR-astrocytes it had the opposite effect. In contrast, the application of 100 µM BaCl₂ had the same effect in both groups of astrocytes. Asterisks indicate significant differences from controls (p<0.05 (*, significant), p<0.01 (**, very significant), p<0.001 (***, extremely significant)).

Figure 4. Experiment I: gene expression profiling of distinct astrocytic subpopulations.

A: Bar plot with SEM for all the expressed genes; significant differences are indicated with asterisks (p<0.05 (*), p<0.01 (**), p<0.001 (***). B: Principal component analysis. The identification of 2 astrocytic subpopulations is along the first principal component, which accounts for most of the variation in the measured data. C: Clustering of astrocytes using Kohonen SOMs. The expression levels of all genes were mean-centered. Each dot represents one cell. D: Dendrogram based on all astrocytic genes. The y-axis shows the distance between groups.

Figure 5. Experiment II: gene expression profiling of distinct astrocytic subpopulations.

A: Bar plot with SEM for all the expressed genes; significant differences are indicated with asterisks (p<0.05 (*), p<0.01 (**), p<0.001 (***). B: Principal component analysis. The identification of 2 astrocytic subpopulations is along the first principal component, which accounts for most of the variation in the measured data. C: Clustering of astrocytes using Kohonen SOMs. The expression levels of all genes were mean-centered. Each dot represents one cell. D: Dendrogram based on all astrocytic genes. The y-axis shows the distance between groups.

Figure 6. Two astrocytic subpopulations differing in the gene expression levels of K⁺/Cl⁻ channels.

We propose a correlation of subpopulation 1 with LR-astrocytes and subpopulation 2 with HR-astrocytes due to the diverse gene expression levels for ClC2 and TREK-1, which are responsible for K⁺ and Cl⁻ efflux and thus contribute to cell volume regulation. Ion channels outlined with dashed line indicate their low gene expression levels. Arrows indicate the proposed direction of ion/EAA fluxes through the channels/transporters during OGD based on the effect of the relevant inhibitors. AQP4 – aquaporin channel (subtype AQP4), ClC2 - chloride channel (subtype ClC2), EAAT – excitatory amino acid transporter (subtypes EAAT1 and EAAT2), HR – high response astrocyte, TWIK1 and TREK1 – two-pore domain potassium channels (subtypes TWIK-1, TREK-1), KCC1 – K⁺-Cl⁻ co-transporter (subtype KCC1), Kir4.1 – inwardly rectifying potassium channel, LR – low response astrocyte, NKCC1 – Na⁺-K⁺-Cl⁻ co-transporter (subtype NKCC1).

Figure 7. Proposed K⁺ movement from HR- to LR-astrocytes during OGD.

HR-astrocytes exposed to higher [K⁺]_o take up K⁺ (by K_2P channels or co-transporters), which is either released via Kir channels or redistributed through the astrocytic syncytium and then extruded by Kir and K_2P channels in LR-astrocytes. HR – high response astrocyte, LR – low response astrocyte, N – neuron, K2P – two-pore domain potassium channel, Kir – inwardly rectifying potassium channel, GJ – gap junction.