Hernandez A et al. (2021), Positive modulation of the TMEM16B mediated cur...

XB-ART-58753

Biochem Biophys Rep 2021 Dec 02;28:101180. doi: 10.1016/j.bbrep.2021.101180.

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Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist.

Hernandez A , Alaniz-Palacios A , Contreras-Vite JA , Martínez-Torres A .

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Calcium-activated chloride channels (CaCCs) play important roles in many physiological processes and their malfunction is implicated in diverse pathologies such as cancer, asthma, and hypertension. TMEM16A and TMEM16B proteins are the structural components of the CaCCs. Recent studies in cell cultures and animal models have demonstrated that pharmacological inhibition of CaCCs could be helpful in the treatment of some diseases, however, there are few specific modulators of these channels. CaCCs and Transient Receptor Potential Vanilloid-4 (TRPV4) channels are co-expressed in some tissues where they functionally interact. TRPV4 is activated by different stimuli and forms a calcium permeable channel that is activated by GSK1016790A and antagonized by GSK2193874. Here we report that GSK2193874 enhances the chloride currents mediated by TMEM16B expressed in HEK cells at nanomolar concentrations and that GSK1016790A enhances native CaCCs of Xenopus oocytes. Thus, these compounds may be used as a tool for the study of CaCCs, TRPV4 and their interactions.

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Genes referenced: ano1 ano2 trpv4

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	Fig. 1. Transient outward chloride current and TRPV4 modulation in Xenopus oocytes. (A) Representative current activation traces from Xenopus oocytes at different voltages (10, 30 and 50 mV) shows the absence of the transient outward current (Tout) in normal Ringer's solution (upper panel), the presence in Ringer's containing 10 mM calcium and enhancement in presence of calcium 10 mM plus GSK1016790A (+GSKa) (lower panel). (B) Summary of the current-voltage relation constructed from the maximal peak of the current amplitude measured from the initial activation and normalized respect to the peak amplitude at 10 mV. The effect of GSKa was statistically significant only in 10 mM calcium at positive voltages after 30 mV without effect in normal Ringer. p < 0.05, *p < 0.01, vs. Normal Ringer's; &p < 0.05 vs Calcium 10 mM; Unpaired student t-test; n = 7 Normal ringer, n = 12 calcium 10 mM, n = 15 +GSKa. empty circles (Normal Ringer), filled circles (Normal Ringer + GSKa), empty squares (calcium 10 mM), filled squares (calcium 10 mM + GSKa). Symbols show mean ± S.E.M.
	Fig. 2. Niflumic acid on TMEM16B currents in HEK293 cells. (A) Representative current traces induced by square voltage steps from −60 mV to +120 mV. Typical exponential activation and deactivation is observed in control conditions and niflumic acid (N–A). Activation and deactivation time course is shown on scaled traces for control (black) and 30 μM N-A (gray, pointed by arrow) at the right side of the panel. (B) Summary of the current-voltage relation (current density) constructed by measuring the current amplitude at the steady state shows similar voltage activation in all conditions. (C) Summary of the time constant of activation and deactivation currents taken from 40 mV to 120 mV. Activation and deactivation time constants were statistically increased in presence of N-A compared to control in concentration dependent relation. **p < 0.0001, p < 0.001; N-A 30 μM vs. control, *p < 0.01, N-A 10 μM vs. control; one-way ANOVA with multiple comparisons correction, Tukey’s post-hoc. (D) Summary of the normalized current voltage relation from tail currents shows a shift to positive voltages (dashed lines) in presence of N-A fitted by the Boltzmann function (black lines). Symbols show mean ± S.E.M.
	Fig. 3. GSK1016790A on TMEM16B currents in HEK293 cells. (A) Representative current traces induced by square voltage steps recorded in different GSK1016790A (GSKa) concentrations (1 and 10 μM). The right side of the panel shows the scaled traces superimposed and compared to the control (black) and GSKa 10 μM (gray, pointed by arrow). (B) Summary of the current-voltage relation at the steady state expressed as current density. The plot shows similar voltage activation in control and two different concentrations of GSKa. (C) Summary of the time constant of activation shows a significant increase at all voltages in presence of GSKa 1 μM (p < 0.05, vs. control) and 10 μM (p < 0.001, vs. control), similarly the deactivation time constant increased statistically significant at all voltages only for 1 μM GSKa (*p < 0.001, vs. control). (D) Summary of the normalized current voltage relation shows non-significant changes (dashed lines) on the curves fitted by the Boltzmann function (black lines). One-way ANOVA with multiple comparisons correction, Tukey’s post-hoc. Symbols show mean ± S.E.M.
	Fig. 4. GSK2193874 on TMEM16B currents in HEK293 cells. (A) Representative current traces induced by 20 mV square voltage steps in different GSK2193874 (GSKb) concentrations and scaled traces superimposed from control (black) and 1.5 μM GSKb (gray, pointed by arrow) are shown at the right side of the panel. (B) Summary of the current-voltage relation expressed as current density shows a clear increase on the current density in concentration dependent relation. (C) Summary of the time constant of activation shows a significant increase at all voltages explored in presence of GSKb compared to control without follow a concentration dependent relation (***p < 0.0001, GSKb 1.5 nM; p < 0.05, GSKb 150 nM; p < 0.05, GSKb 1.5 μM), except for 15 nM. Similarly, the deactivation time constant increases statistically significant at all voltages without follow a concentration dependent relation compared to control (p < 0.01, GSKb 1.5 nM; p < 0.001, GSKb 150 nM; p < 0.01, GSKb 1.5 μM) except for 15 nM. (D) Concentration-response curve shows the percent of stimulation response respect to control at 40 (empty circles) and 120 mV (filled circles). Curve fitted indicates an EC50 at the nanomolar concentrations (black lines). One-way ANOVA with multiple comparisons correction, Tukey’s post-hoc. Symbols show mean ± S.E.M.

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	Fig. 1. Transient outward chloride current and TRPV4 modulation in Xenopus oocytes. (A) Representative current activation traces from Xenopus oocytes at different voltages (10, 30 and 50 mV) shows the absence of the transient outward current (Tout) in normal Ringer's solution (upper panel), the presence in Ringer's containing 10 mM calcium and enhancement in presence of calcium 10 mM plus GSK1016790A (+GSKa) (lower panel). (B) Summary of the current-voltage relation constructed from the maximal peak of the current amplitude measured from the initial activation and normalized respect to the peak amplitude at 10 mV. The effect of GSKa was statistically significant only in 10 mM calcium at positive voltages after 30 mV without effect in normal Ringer. p < 0.05, *p < 0.01, vs. Normal Ringer's; &p < 0.05 vs Calcium 10 mM; Unpaired student t-test; n = 7 Normal ringer, n = 12 calcium 10 mM, n = 15 +GSKa. empty circles (Normal Ringer), filled circles (Normal Ringer + GSKa), empty squares (calcium 10 mM), filled squares (calcium 10 mM + GSKa). Symbols show mean ± S.E.M.
	Fig. 2. Niflumic acid on TMEM16B currents in HEK293 cells. (A) Representative current traces induced by square voltage steps from −60 mV to +120 mV. Typical exponential activation and deactivation is observed in control conditions and niflumic acid (N–A). Activation and deactivation time course is shown on scaled traces for control (black) and 30 μM N-A (gray, pointed by arrow) at the right side of the panel. (B) Summary of the current-voltage relation (current density) constructed by measuring the current amplitude at the steady state shows similar voltage activation in all conditions. (C) Summary of the time constant of activation and deactivation currents taken from 40 mV to 120 mV. Activation and deactivation time constants were statistically increased in presence of N-A compared to control in concentration dependent relation. **p < 0.0001, p < 0.001; N-A 30 μM vs. control, *p < 0.01, N-A 10 μM vs. control; one-way ANOVA with multiple comparisons correction, Tukey’s post-hoc. (D) Summary of the normalized current voltage relation from tail currents shows a shift to positive voltages (dashed lines) in presence of N-A fitted by the Boltzmann function (black lines). Symbols show mean ± S.E.M.
	Fig. 3. GSK1016790A on TMEM16B currents in HEK293 cells. (A) Representative current traces induced by square voltage steps recorded in different GSK1016790A (GSKa) concentrations (1 and 10 μM). The right side of the panel shows the scaled traces superimposed and compared to the control (black) and GSKa 10 μM (gray, pointed by arrow). (B) Summary of the current-voltage relation at the steady state expressed as current density. The plot shows similar voltage activation in control and two different concentrations of GSKa. (C) Summary of the time constant of activation shows a significant increase at all voltages in presence of GSKa 1 μM (p < 0.05, vs. control) and 10 μM (p < 0.001, vs. control), similarly the deactivation time constant increased statistically significant at all voltages only for 1 μM GSKa (*p < 0.001, vs. control). (D) Summary of the normalized current voltage relation shows non-significant changes (dashed lines) on the curves fitted by the Boltzmann function (black lines). One-way ANOVA with multiple comparisons correction, Tukey’s post-hoc. Symbols show mean ± S.E.M.
	Fig. 4. GSK2193874 on TMEM16B currents in HEK293 cells. (A) Representative current traces induced by 20 mV square voltage steps in different GSK2193874 (GSKb) concentrations and scaled traces superimposed from control (black) and 1.5 μM GSKb (gray, pointed by arrow) are shown at the right side of the panel. (B) Summary of the current-voltage relation expressed as current density shows a clear increase on the current density in concentration dependent relation. (C) Summary of the time constant of activation shows a significant increase at all voltages explored in presence of GSKb compared to control without follow a concentration dependent relation (***p < 0.0001, GSKb 1.5 nM; p < 0.05, GSKb 150 nM; p < 0.05, GSKb 1.5 μM), except for 15 nM. Similarly, the deactivation time constant increases statistically significant at all voltages without follow a concentration dependent relation compared to control (p < 0.01, GSKb 1.5 nM; p < 0.001, GSKb 150 nM; p < 0.01, GSKb 1.5 μM) except for 15 nM. (D) Concentration-response curve shows the percent of stimulation response respect to control at 40 (empty circles) and 120 mV (filled circles). Curve fitted indicates an EC50 at the nanomolar concentrations (black lines). One-way ANOVA with multiple comparisons correction, Tukey’s post-hoc. Symbols show mean ± S.E.M.