Fig. 4. Each Ttyh1/2/3 isoform shares the same biophysical (voltage-dependent inactivation, outward rectification), biochemical (Ca²⁺-independency) and pharmacological properties (sensitivity to DCPIB) as astrocytic VRAC_swell. (A) Representative current traces with voltage step protocol from −100 mV to +140 mV, which is recorded in cultured astrocytes (black), TTYH1+AQP4 (blue), TTYH1+AQP4 with m/h Lrrc8a shRNA (green) expressing HEK293T cells, and naïve HEK293T cells (purple and red). The major constituent of HOS is indicated in each traces, (HOS(Tris-Cl) or HOS(NaCl). Bottom left, the schematic diagram of voltage step protocol and calculation of inactivation % (Inactivation % is calculated by a/b^*100, the ‘a’ indicates the initial 10% of current amplitude at +140 mV, and the ‘b’ indicates the last 90% of current amplitude at +140 mV). (B) Summary bar graph showing inactivation % at +140 mV. Data are represented as mean±SEM (n=8 for astrocytes by treating HOS(Tris-Cl), n=11 for TTYH1+AQP4 expressing HEK293T by treating HOS(Tris-Cl), n=9 for TTYH1+AQP4 with m/h Lrrc8a shRNA by treating HOS(NaCl), n=8 for naïve HEK293T cells by treating HOS(Tris-Cl), and n=14 for naïve HEK293T cells by treating HOS(NaCl), ^****<0.0001, one-way ANOVA). (C) Summary bar graph showing rectification index from cultured astrocyte and naïve HEK293T by treating HOS(Tris-Cl) (black and blue) and from naïve HEK293T cells by treating HOS(NaCl) (red). (n=8 for astrocyte by treating HOS(Tris-Cl), n=11 for TTYH1+AQP4 expressing HEK293T cells by treating HOS(Tris-Cl), n=9 for naïve HEK293T cells by treating HOS(NaCl), ns>0.05, one-way ANOVA). (D) Summary scatter plot showing the relative permeability of various anions such as SCN⁻, I⁻, Br⁻, and F- recorded from naïve HEK293T cells by treating Na⁺ containing HOS (grey circle) and recorded from TTYH1+AQP4 with m/h Lrrc8a shRNA expressing HEK3293T cells by treating Na⁺ containing HOS (skyblue square). (n=9, 8, 8, and 7 for naïve HEK293T cells for SCN⁻, I⁻, Br⁻, and F⁻, respectively, and n=8, 8, 8, and 8 for TTYH1+AQP4 with m/h Lrrc8a shRNA expressing HEK293T cells for SCN⁻, I⁻, Br⁻, and F⁻, respectively). (E) Summary bar graph showing rectification index (|I_Cl,swell at −100 mV|/I_Cl,swell at +100 mV) from whole-cell patch-clamp recording of cultured astrocyte and Ttyh1/2/3 with AQP4 co-expressing HEK293T cells. Data are represented as mean±SEM (n=24 for astrocytes and n=21, 23 and 13 for TTYH1, TTYH2 and TTYH3 with AQP4 co-expressing HEK293T cells; NS>0.05, Kruskal-Wallis test). (F) Representative I_Cl,swell from TTYH1, 2 and 3 with AQP4 overexpressing CHO-K1 cells using 0.15 mM external Ca2+ and 20 mM BAPTA containing internal solution. Inset: schematic diagram of whole-cell patch-clamp recordings in TTYH1 (red), TTYH2 (blue) and TTYH3 (green) with AQP4 (grey) co-expressing CHO-K1 cells. (G) Averaged I~V curves for I_Cl,swell recorded at TTYH1 (red), TTYH2 (blue) and TTYH3 (green) with AQP4 (grey) co-expressing CHO-K1 cells. (H) Summary bar graph showing the maximal amplitude of I_Cl,swell from −100mV to 100mV. Data are represented as mean±SEM (n=10, 8 and 10 for TTYH1, TTYH2 and TTYH3 with AQP4 co-expressing CHO-K1 cells; NS>0.05, Kruskal-Wallis test). (I, L) Representative traces of I_Cl,swell recording from cultured astrocytes and Ttyh1/2/3 with AQP4 co-expressing HEK293T cells during treatment HOS (black bar) and DCPIB (10 µM, sky blue bar). (J) Averaged I~V curves for I_Cl,swell from cultured astrocytes in control (black) and DCPIB (sky blue). (K, M) Summary bar graph showing the maximal amplitude for I_Cl,swell from +100 mV to −100 mV in cultured astrocytes and Ttyh1/2/3 with AQP4 co-expressing HEK293T cells, respectively. Data are represented as mean±SEM (n=10 and 8 for control or DCPIB treatment in astrocytes; ^****<0.0001, Mann-Whitney test; n=8, 15 and 8 for control or DCPIB treatment in each TTYH1, TTYH2 and TTYH3 with AQP4 co-expressing HEK293T cells; ^****<0.0001, 2way ANOVA).