**Fig. 3. **Each Ttyh1/2/3 isoform is sufficient for VRAC_{swell} activity. (A) Representative I_{Cl,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 I_{Cl,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 I_{Cl,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 I_{Cl,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 I_{Cl,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 I_{Cl,swell} recorded from naïve HEK293T cells in response to HOS (black) and HOS(NaCl) (pink). (I) Summary bar graph showing the maximal amplitude for I_{Cl,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 I_{Cl,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 I_{Cl,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 I_{Cl,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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