Fig. 5. Each Ttyh1/2/3 shares the same pharmacological properties (sensitive to both genistein and PD98059) as astrocytic VRACswell. (A) Representative traces of ICl,swell recording from cultured astrocytes and Ttyh1/2/3 with AQP4 co-expressing HEK293T cells during treatment HOS (black bar) in the presence or absence of Genistein (50 µM, sky blue bar). Right upper insets represent cultured astrocytes and Ttyh1/2/3 (red, blue and green, respectively) with AQP4 (grey) over-expressing HEK293T cells. (B) Averaged I~V curves for ICl,swell in control (grey) and genistein-treated group (sky blue) recorded from cultured astrocytes. (C, D) Summary bar graph showing the maximal amplitude of ICl,swell from +100 mV to −100 mV recorded from cultured astrocytes and Ttyh1/2/3 with AQP4 co-expressing HEK293T cells, respectively. Data are represented as mean±SEM (n=8 for both control and genistein treatment in astrocytes; **<0.01, Wilcoxon test; n=10 for both control and genistein treatment in TTYH1 with AQP4 co-expressing HEK293T cells, n=12 and 9 for control and genistein treatment in TTYH2 with AQP4 co-expressing HEK293T cells, and n=10 and 15 for control and genistein treatment in TTYH2 with AQP4 co-expressing HEK293T cells; **<0.01, ***<0.001, ****<0.0001, 2way ANOVA). (E) Representative traces of ICl,swell from cultured astrocytes during treatment hypo-osmotic solution (black bar) and then PD98059 (50 µM, pink bar). Inset: schematic diagram of whole-cell patch-clamp recording in primary cultured astrocyte. (F) Averaged I~V curves for HOS induced current responses in control (black) and PD980959 (pink) recorded at cultured astrocytes. (G) Summary bar graph showing the maximal amplitude of HOS induced current density from −100mV to 100mV. Data are represented as mean±SEM (n=7 and 9 for control and PD98059; ***p<0.001; Mann-Whitney test). (H) Representative traces of ICl,swell from TTYH1 with AQP4 co-expressing CHO cells during treatment HOS and then PD98059 (50 µM). Inset: schematic diagram of whole-cell patch-clamp recording in TTYH1 (red) with AQP4 (grey) co-expressing CHO cells. (I) Averaged I~V curves for HOS induced current responses in control (red) and PD98059 (pink) recorded at TTYH1 with AQP4-co-expressing CHO cells. (J) Summary bar graph showing the maximal amplitude of ICl,swell from −100mV to 100mV. Data are represented as mean±SEM (n=7 and 9 for control and PD98059; **p<0.01; Mann-Whitney test). (K) Surface expression of TTYH1 in response to HOS(Tris-Cl) in cultured astrocytes, which were prevented by either TK and MAPK inhibitor. (L, M) Representative traces and summary bar graph of ICl,swell recording from naïve HEK293T in the absence of drug (black bar) or in the presence of genistein (TK inhibitor, light purple) or PD98059 (MAPK inhibitor, green) induced by HOS(NaCl, Pink line). For G, n=10, 4, 7 for in the absence or presence of drug, genistein or PD98059 in HEK293T cells, **<0.01, one-way ANOVA). (N) Summary bar graph showing block % of kinase inhibitor in cultured astrocytes (black, induced by HOS(Tris-Cl)), TTYH1+AQP4 overexpressed HEK293T cells (red, induced by HOS(Tris-Cl)), and naïve HEK293T cells (pink, induced by HOS(NaCl)). (n= 8, 10, and 4 for recorded from astrocyte, TTYH+AQP4 overexpressed, and naïve HEK293T cells after genistein treatment, and n= 9, 9, and 6 for recorded from astrocyte, TTYH+AQP4 overexpressed HEK293T cells, and naïve HEK293T cells after PD98059 treatment, **<0.01, Two-way ANOVA). (O) Schematic diagram for experimental conditions to record glutamate permeability of ICl,swell. HOS (black) is standard 83mM Cl− containing the hypo-osmotic solution. gHOS (orange) is 70mM glutamate and 13mM Cl− containing the hypo-osmotic solution. (P) Representative trace for glutamate permeability recording in cultured astrocytes. gHOS (orange) was treated at the peak of ICl,swell induced by standard HOS (black). (Q) Representative I~V curves for ICl,swell were recorded under whole-cell patch-clamp in cultured astrocytes firstly in HOS (83 mM Cl−, black) and switched to gHOS (70 mM glutamate and 13 mM Cl−, orange) in the same cell. (R) Representative I~V curve for ICl,swell in AQP4 (grey), TTYH1 (red), TTYH2 (blue), TTYH3 (green) with AQP4 co-expressing HEK293T cells, with standard HOS (black) changed to gHOS (orange) in the same cell. (S) Glutamate permeability ratio relative to Cl− (PGlu/PCl) of ICl,swell are summarized. PGlu/PCl was calculated from the Goldman-Hodgkin-Katz equation (see in methods). Data are represented as mean±SEM (n=6 for astrocytes and n=8, 10 and 8 for TTYH1, TTYH2 and TTYH3 with AQP4 co-expressing HEK293T cells; NS>0.05, *<0.05, Kruskal-Wallis test).
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