Fig. 3. Each Ttyh1/2/3 isoform is sufficient for VRACswell activity. (A) Representative ICl,swell from Naïve, TTYH2 only, AQP4 only or TTYH1, TTYH2 and TTYH3 with AQP4 co-expressing HEK293T cells. Inset: schematic diagram of whole-cell patch-clamp recording in AQP4 (grey), TTYH1 (red), TTYH2 (blue), TTYH3 (green) expressing HEK293T cells. (B) Averaged I–V curves for ICl,swell in Naïve (black), TTYH2 only (skyblue), AQP4 only (grey), and TTYH1 (red), TTYH2 (blue), TTYH3 (green) with AQP4 co-expressing HEK293T cells. (C) Summary bar graph showing the maximal amplitude of ICl,swell from +100 mV to −100 mV. Data are represented as mean±SEM (n=18 for Naïve HEK293T cells, n=5 for TTYH2 over-expressing HEK293T cells, n=22 for AQP4 over-expressing HEK293T cells, n=21, 23 and 13 for TTYH1, TTYH2 and TTYH3 with AQP4 co-expressing HEK293T cells; *<0.05, **<0.01, ****<0.0001 and NS>0.05, Kruskal-Wallis test). (D~F) Representative traces, averaged I~V curves and summary bar graphs of maximal ICl,swell currents from either TTYH1 or LRRC8A with AQP4 co-expressed CHO-K1 cells (n=15 for TTYH1+AQP4, n=10 for LRRC8A+AQP4; ****p<0.0001, Mann-Whitney test). (G) Representative traces of ICl,swell recording from naïve HEK293T cells during HOS (black bar) and NaCl−containing HOS (HOS(NaCl)) treatment, respectively (pink bar). (H) Averaged I~V curves for maximal ICl,swell recorded from naïve HEK293T cells in response to HOS (black) and HOS(NaCl) (pink). (I) Summary bar graph showing the maximal amplitude for ICl,swell from +100 mV to −100 mV recorded from naïve HEK293T cells in response to HOS (black) and HOS(NaCl) (pink). Data are represented as mean±SEM (n=8 and 10 for HOS or HOS(NaCl); ****<0.0001, unpaired t-test). (J~L) Representative traces, averaged I~V curves, and summary bar graph of ICl,swell recorded from control shRNA (black), m/h Lrrc8a shRNA (orange)–expressing and TTYH1 (red), AQP4 (grey) with m/h Lrrc8a shRNA (orange)-expressing HEK293T cells in response to HOS(NaCl) (pink). Data are represented as mean±SEM (n=10, 11, and 11 for Control shRNA, m/h Lrrc8a shRNA, and TTYH1+AQP4 with m/h Lrrc8a; ***<0.001, one-way ANOVA). (M~O) Representative traces, averaged I~V curves and summary bar graphs of maximal ICl,swell currents from control shRNA or m/h Lrrc8a shRNA expressing condition in cultured astrocytes (n=9 for control shRNA, n=9 for m/h Lrrc8a shRNA; NS: p>0.05, unpaired t-test). (P~R) Representative traces, averaged I~V curves and summary bar graphs of maximal ICl,swell currents from control shRNA expressing condition (n=10) or m/h Lrrc8a shRNA expressing condition (n=13) in HEK293T cells with expression of TTYH1+AQP4. Statistical analysis was performed with (NS: p>0.05, Mann-Whitney test) Data are represented as mean±SEM for all averaged I~V curves and summary bar graphs.
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