The Functional Interaction of KATP and BK Channels with Aquaporin-4 in the U87 Glioblastoma Cell

Abstract

K⁺ channels do play a role in cell shape changes observed during cell proliferation and apoptosis. Research suggested that the dynamics of the aggregation of Aquaporin-4 (AQP4) into AQP4-OAP isoforms can trigger cell shape changes in malignant glioma cells. Here, we investigated the relationship between AQP4 and some K⁺ channels in the malignant glioma U87 line. The U87 cells transfected with the human M1-AQP4 and M23-AQP4 isoforms were investigated for morphology, the gene expression of KCNJ8, KCNJ11, ABCC8, ABCC9, KCNMA1, and Cyclin genes by RT-PCR, recording the whole-cell K⁺ ion currents by patch-clamp experiments. AQP4 aggregation into OAPs increases the plasma membrane functional expression of the Kir6.2 and SUR2 subunits of the KATP channels and of the KCNMA1 of the BK channels in U87 cells leading to a large increase in inward and outward K⁺ ion currents. These changes were associated with changes in morphology, with a decrease in cell volume in the U87 cells and an increase in the ER density. These U87 cells accumulate in the mitotic and G2 cell cycle. The KATP channel blocker zoledronic acid reduced cell proliferation in both M23 AQP4-OAP and M1 AQP4-tetramer-transfected cells, leading to early and late apoptosis, respectively. The BK channel sustains the efflux of K⁺ ions associated with the M23 AQP4-OAP expression in the U87 cells, but it is downregulated in the M1 AQP4-tetramer cells. The KATP channels are effective in the M1 AQP4-tetramer and M23 AQP4-OAP cells. Zoledronic acid can be effective in targeting pathogenic M1 AQP4-tetramer cell phenotypes inhibiting KATP channels and inducing early apoptosis.

Keywords: BK channels; KATP channels; M1-AQP4 (AQP4-tetramers forming isoform); M23-AQP4 (AQP4-OAPs forming isoform); aquaporin-4; cell cycle; glioblastoma.

Figure 1

Expression of AQP4 in U87 cells. (A) Epifluorescence images of U87wt and U87 expressing M1 AQP4-tetramers or M23 AQP4-OAPs. AQP4 staining is shown in red and DAPI in blue. Scale bar 20 μm. (B) Immunoblot detection of AQP4 expression levels in U87 cells transfected with M1-AQP4 (AQP4-tetramers) and M23-AQP4 (AQP4-OAPs). GFAP and GAPDH were used to normalize for equal loading.

Figure 2

Characterization of inward and outward macroscopic K⁺ ion currents recorded in U87wt cells. The currents were recorded using a whole cell configuration under physiological concentration of K⁺ ions in the bath and pipette and were obtained in response to voltage pulses from −120 to +120 mV in increments of 20 mV, starting at HP = −60 mV (Vm) and intracellular-free Ca²⁺ ions (1.6 × 10⁻⁶ M). (A) TEA (5 × 10⁻³ M) suppressed the outward K⁺ ion currents, and TEA/Ba²⁺ (5 × 10⁻³ M) fully suppressed also the inward K⁺ ion currents. (B) The selective BK channel blocker IbTX (4 × 10⁻⁷ M) reduced the outward K⁺ ion currents that were fully reduced by TEA (5 × 10⁻³ M). (C) KATP currents recorded in U87wt cells in low intracellular ATP 1 × 10⁻³ M. Glibenclamide (5 × 10⁻⁸ M) suppressed the inward currents, suggesting the presence of KATP channels in the cells. Cells of the same size were selected for patch-clamp experiments. Each point represented the mean ± SEM (N patches = 10^–15). (D) Percentage of blocking of K⁺ ion currents in the presence of the antagonists with respect to the control condition at 60 mV and −60 mV (Vm).

Figure 3

Effects of antagonists and agonists of the K⁺ channels on the proliferation of U87wt cells. * p < 0.05 and ** p < 0.01 when compared to the experimental group not treated after 48 h of incubation. Each point represented the mean ± SEM.

Figure 4

Characterization of macroscopic inward and outward K⁺ ion currents recorded in U87wt cells and after transfection with M1 AQP4-tetramer or M23 AQP4-OAP. Whole cell currents were recorded under physiological concentration of K⁺ ions in the bath and pipette and were obtained in response to voltage pulses from −120 to +120 mV in 20 mV increments, starting at HP = −60 mV (Vm). (A) Macroscopic K⁺ ion currents recorded in AQP4-OAP-transfected U87 cells. The presence of AQP4-OAPs caused a significant increase in the inward and outward currents that were reduced by TEA and BaCl₂. (B) Macroscopic K⁺ ion currents recorded in AQP4-tetramer-transfected U87 cells. The presence of M1 AQP4-tetramers caused a significant increase in the inward currents; however, the outward K⁺ ion currents were reduced in the amplitude. (C) The presence of M23 AQP4-OAPs caused a large increase in the currents at negative and positive membrane potentials vs. not transfected cell. Instead, the presence of AQP4-tetramers led to an increase in the currents at negative membrane potentials; conversely, the outward K⁺ ion currents decreased at positive membrane potentials. Data were pooled from N patches = 10–12. (D) Percentage of reduction in the K⁺ ion currents in the presence of the antagonist TEA with respect to the control condition at 60 mV and −60 mV (Vm). Each point represents the mean ± SEM.

Figure 5

Expression profile of KCNMA1, KCNJ11, KCNJ8, ABCC8, ABCC9, and TRPV1 genes in U87 glioma cells in the presence of the malignant M1 AQP4-tetramer and M23 AQP4-OAP aggregation vs. wt condition. * p < 0.05 when compared to the experimental group not treated (WT) after 48 h of incubation. Each point represented the mean ± SEM.

Figure 6

Effect of pharmacological inhibition of the Kir6.2-SUR2 channel activity with zoledronic acid on AQP4s expressing U87 cells. (A) Epifluorescence images of U87 cells expressing AQP4-tetramers or AQP4-OAPs treated with diazoxide or zoledronic acid. AQP4 staining is shown in red, and DAPI for nuclear staining is in blue. Phalloidin (in green) was used to visualize F-actin. The white arrowheads indicate the round-shaped cells, the arrows indicate irregular-shaped cells, and the blue arrowheads indicate the apoptotic beads. Scale bar 100 μm. ((B) top) Drawing/diagram showing the morphological change of U87 cells expressing AQP4-OAPs after pharmacological treatment with zoledronic acid according Coffin hypothesis and relative epifluorescence images. ((B) bottom) Epifluorescence images of U87 expressing AQP4-OAPs treated with zoledronic, showing the irregular-shaped cell, the round-shaped cell, and the apoptosis. AQP4 staining is shown in red. Phalloidin (in green) was used to visualize F-actin. Scale bar 10 µm. A histogram was created to display the percentage of round-shaped cells per field in U87 cells and those expressing AQP4-OAPs after treatment under different conditions. The conditions include treatment with zoledronic acid (an inhibitor), control conditions (vehicle), and diazoxide (an agonist). The histogram compares these percentages across the different treatment groups, highlighting the effects of the inhibitor and agonist on cell morphology. Values are expressed as mean ± SEM of percentage of cells with altered cell morphology out of the total number of transfected cells per field. ** p < 0.005; ((C) top) Representative image of expressing AQP4-OAPs in control condition or after treatment with zoledronic or diazoxide as indicated and stained with DAPI to visualize nuclei. Scale bar 50 µm. ((C) bottom) Dot plot showing the analysis of % of condensed nuclei for field and the nuclear area of images in A. Values are expressed in µm² and represent mean ± SEM. * p < 0.05, *** p < 0.001, n = 3, two-way ANOVA/Tukey’s tests.

Figure 7

RT PCR expression of the cyclin genes (E, A, and B1) in the U87 cells following M23 AQP4-OAP transfection. * p < 0.05 when compared to the experimental group not treated (WT) after 48 h of incubation. • represent the average of each single experiment. Each point represented the mean of at least three experiments± SEM.