Ca2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl– Channels

Figures

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  FIGURE 1. Activation of LRRC8A/C is Ca2+ dependent. (A) Coexpression of LRRC8A and LRRC8C by a bicistronic vector. Detection of LRRC8A by Western blotting. (B) Continuous recording of whole-cell currents activated by hypotonic cell swelling (Hypo, 200 mosm/l). Patch pipette [Ca2+ ] was 100 nM; extracellular Ringer [Ca2+ ] was 1.3 mM. (C) Whole-cell current overlays obtained with 100, 10, or 0 nM Ca2+ in the patch pipette filling solution. Clamp voltages ranged from −100 to +100 mV in steps of 20 mV. (D) Summary of Hypo-induced whole-cell current densities (Vc = +100 mV) obtained in mock-transfected and LRRC8A/C-expressing HEK293 cells (p < 0.001 and < 0.01, respectively). Mean ± SEM; n = 5–9 for each series). #Significant increase when compared to mock (unpaired t-tests). Blots were done as replicates.

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  FIGURE 2. TMEM16A is activated by Hypo and augments ICl,swell. (A) Continues recording of whole cell currents activated in HEK293 cells by hypotonic cell swelling (Hypo, 200 mosm/l). (B) Current/voltage relationships of Hypo-activated whole-cell currents. TMEM16A is activated by Hypo and further augments ICl,swell in cells coexpressing TMEM16A and LRRC8A/C. Mean ± SEM; n = 8–11 for each series). #Significant increase when compared to mock (p < 0.05–0.0001; unpaired t-tests).

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  FIGURE 3. TMEM16A overexpressed in HEK293 cells is spontaneously active. (A) Continuous current recordings of a mock-transfected and a TMEM16A-expressing HEK293 cell. In these experiments, the pipette filling solution and the bath solution contained CsCl (c.f. Methods). Cl– removal from the extracellular bath solution except of 5 mM (5Cl–) had very little effect on the mock-transfected cell but strongly depolarized the TMEM16A-expressing cell. (B) Corresponding I/V curves indicate small currents and little effect of 5Cl– in mock-transfected cells, while currents were large and strongly inhibited with a shift of the reversal potential in TMEM16A-overexpressing cells.

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  FIGURE 4. TMEM16A supports activation of LRRC8. (A) Hypo (200 mosm/l)-induced whole-cell current overlays obtained in HT29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, LRRC8A, or both. The free Ca2+ concentration in the patch pipette filling solution was 100 nM. (B) Corresponding current–voltage relationships. (C) Hypo-induced whole-cell current densities (Vc = +100 mV). (D) Time-dependent activation of whole-cell currents by Hypo. Mean ± SEM; n = 9–14 for each series). #Significant decrease when compared to mock (p < 0.01 for all; unpaired t-tests).

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  FIGURE 5. Hypotonicity activates TMEM16A-dependent Ca2+ increase. (A) Summary of Hypo (200 mosm/l)-induced current densities inhibited by DCPIB in HT29 cells (n = 9–15). The free Ca2+ concentration in the patch pipette filling solution was 100 nM. (B) Hypo-induced whole-cell current overlays in HT29 cells treated with scrambled RNA (scrbld), siRNA for TMEM16A, or both TMEM16F. (C) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM; p < 0.001) on Hypo-induced currents densities (Vc = +100 mV) (n = 9–11). (D) ATP (100 μM)-induced whole-cell current overlays in the absence or presence of siRNAs. (E) Corresponding current–voltage relationships (n = 9–11). (F) Effect of 2-APB (50 μM; p < 0.001) and dantrolene (50 μM) on ATP-induced current densities (Vc = +100 mV) (n = 7–9). (G) Effect of TMEM16A-siRNA on ATP-induced Ca2+-increase (n = 21–45; p < 0.000004 and p < 0.01, respectively). (H) Effect of TMEM16A-siRNA on Hypo-induced Ca2+-increase (n = 21–55; p < 0.003). Plateau Ca2+ was determined at the end of the plateau. Mean ± SEM; #significant decrease (unpaired t-test).

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  FIGURE 6. Hypo-induced anion permeability measured under non-voltage clamp conditions. (A) siRNA knockdown of LRRC8A detected by Western blotting. The knockdown efficiency was 72%. Knockdown of TMEM16A enhanced the expression of LRRC8A, corresponding to Benedetto et al. (2016). (B) Summary of time dependence for YFP quenching induced by 20 mM iodide in the presence of isotonic Ringer (Iso; 300 mosmol/l) or Hypo (200 mosm/l). (C) Summary of initial rate of quenching (arbitrary units/s) (n = 4; p < 0.006). (D) siRNA knockdown of TMEM16A detected by Western blotting. (E) Summary of time dependence for Hypo-induced YFP quenching in the absence or presence of siRNA. (F) Summary of initial rates of quenching (arbitrary units/s) (n = 19). p < 0.0005 (siTMEM16A), p < 2 × 10–11 (siLRRC8A), p < 2 × 10–11 (siLRRC8A/siTMEM16A). (G,H) Hypo-induced YFP quenching was absent in the presence of 0 mM extracellular iodide but was gradually increased with 5 mM (n = 4; p < 0.003), 20 mM (n = 4; p < 0.0009), and 50 mM (n = 4; p < 0.0005). Mean ± SEM; #significant increase or decrease, respectively (unpaired t-test). Blots were done as replicates.

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  FIGURE 7. Role of Ca2+ signaling for Hypo- and ATP-induced anion permeability. (A) Time-dependent YFP quenching induced by different concentrations of ATP (n = 3–5). (B) Concentration dependence of ATP-induced quenching (arbitrary units/s) and effects of knockdown of TMEM16A or LRRC8A (n = 4). (C) Summary of ATP-induced quenching in the absence (control) or presence of IP3R blockers xestospongin C (10 μM; p < 10–19), suramin (500 μM; p < 10–7), and probenecid (1 mM; p < 2 × 10–9) (n = 4–5). (D) Summary of Hypo (200 mosm/l)-induced quenching in the absence (control) or presence of DCPIB (30 μM; p < 0.02), Ani9 (10 μM), suramin (500 μM; p < 2 × 10–7), U73122 (10 μM; p < 0.01), 2-APB (50 μM; p < 10–17), dantrolene (50 μM; p < 0.02), probenecid (1 mM; p < 0.03), and NS3728 (10 μM; p < 0.03) (n = 4–5). (E,F) The contribution of Ca2+ influx on Hypo-induced quenching was examined by exposing the cells to a hypotonic solution in the presence of physiological (1.3 mM; n = 5) or low (1 μM; n = 4; p < 0.02) extracellular Ca2+ concentration. Mean ± SEM; #significant inhibition (unpaired t-test).