Weak Acids as Endogenous Inhibitors of the Proton-Activated Chloride Channel

Figures

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  Figure 1. Acetic acid inhibits endogenous PAC-mediated currents in HEK293T cells. (A) Representative PAC current from HEK293T cells evoked by pH 5.0 and concentration-dependent inhibition by acetic acid (HOAc) at pH 5.0 (n = 9). (B) Box diagrams with dot plots displaying the induced current reduction by increasing concentrations of HOAc at pH 5.0 (n = 9 for each concentration). (C) Membrane current in a HEK293T cell elicited by pH 5.0 during 500 ms long ramps from −100 mV through 100 mV with control solution (pH 7.4), pH 5.0, or the combination of pH 5.0 and 30 mM HOAc (n = 11). (D) Box diagram with dot plots demonstrating that PAC currents elicited by voltage ramps are inhibited by 30 mM HOAc (n = 11). (E–G) Voltage-dependent membrane currents in HEK293T cells evoked by 200 ms long, 20 mV voltage step pulses from −60 mV through 120 mV at pH 7.4 (E), pH 5.0 (F), or after application of 30 mM HOAc at pH 5.0 (G) (n = 10). (H) Normalized currents evoked by voltage step pulses and application of either control solution (black), pH 5.0 (red), or the combination of 30 mM acetic acid and pH 5.0 (blue) (n = 10). (I) Current inhibition caused by co-application of pH 5.0 and 30 mM acetic acid is…

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  Figure 2. Endogenous weak acids inhibit PAC-mediated currents in HEK293T cells. (A) HEK293T cells held at +60 mV generate a PAC-induced current which is inhibited by increasing concentrations of lactic acid at pH 5.0 (n = 8). (B) Box diagram with dots displaying the concentration-dependent current inhibition by lactic acid (unpaired t-test; *** denotes p < 0.001). (C) Representative PAC current and its inhibition by increasing concentrations of propionic acid (n = 8). (D) Box diagram with dots demonstrating the current inhibition by propionic acid (Mann–Whitney U test; ns denotes not significant). (E) PAC-mediated current inhibited by 30 mM itaconic acid at pH 5.0. Termination of itaconic acid and application of solely pH 5.0 induced a partial recovery of the PAC-invoked current (n = 8). (F) Box diagram with dots illustrating the concentration-dependent current inhibition by a combination of pH 5.0 and itaconic acid (Kruskal–Wallis ANOVA with Dunn–Bonferroni post hoc test, * denotes p < 0.05, and *** denotes p < 0.001, n = 4–8). (G) PAC-mediated current inhibition by increasing concentrations of β-hydroxybutyrate (BHB) (n = 8). (H) Box diagram with dots illustrating the…

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  Figure 3. Properties of acetic acid sensitivity of endogenous PAC channels. (A–D) Representative PAC currents from a HEK293T cells held at +60 mV. Currents were evoked by pH 5.4 ((A), n = 7), pH 5.2 ((B), n = 7), pH 5.0 ((C), n = 5), or pH 4.0 ((D), n = 6). (E) Box diagram with dot plots representing PAC current inhibition by 3 mM HOAc at different pH values (Kruskal–Wallis ANOVA with Dunn–Bonferroni post hoc test; ** denotes p < 0.01; *** denotes p < 0.001). (F) Concentration- and pH-dependency of the inhibition of PAC by HOAc (n = 7–9 cells for each concentration and pH value). (G,H) Current traces demonstrating inhibition of PAC-mediated currents by 30 mM HOAc at pH 5.0 using pipette solutions with pH 7.4 ((G), n = 8) or pH 5.0 ((H), n = 8). (I,J) Acidification of the cytoplasm with pH 5.0 neither had an influence on the current density of PAC-mediated currents (I), nor on the inhibitory effect of 30 mM HOAc (J) (unpaired t-test, ns denotes not significant). (K) PAC-mediated current induced by pH 5.0 with an extracellular solution containing 2 mM calcium (n = 4). (L) Box diagram with dots representing the current inhibition by 30 mM HOAc in calcium-containing and calcium-free…

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  Figure 4. Reduced inhibition by weak acids on hPAC–Arg93Ala. (A) Typical recordings on PAC-knockout HEK293 cells transfected with hPAC-WT, showing a representative PAC-mediated current in a cell held at +20 mV upon stimulation with pH 5.0 and inhibition by 30 mM HOAc (n = 9). (B) Current mediated by hPAC–Arg93Ala expressed in PAC-knockout HEK293 cells. Note that 30 mM HOAc failed to inhibit this mutant (n = 11). (C) Box diagram with dots displaying the current inhibition induced by 30 mM HOAc in (A,B) and further mutant hPAC constructs (Kruskal–Wallis ANOVA with Dunn–Bonferroni post hoc test; ns denotes not significant; **** denotes, p < 0.0001). (D) Box diagram with dots demonstrating the significantly reduced current inhibition by 30 mM lactic acid on hPAC–Arg93Ala (Mann–Whitney U test; * denotes p < 0.05; n = 12). (E) Box diagram with dots showing that inhibition of hPAC–Arg93Ala by 30 mM propionic acid was not significantly reduced (Mann–Whitney U test; n = 8). (F,G). Typical currents generated by hPAC-WT ((F), n = 12) and hPAC–Arg93Ala ((G), n = 11). While hPAC-WT was almost completely inhibited by 30 µM arachidonic acid (ARA) at pH 5.0, inhibition of hPAC–Arg93Ala was strongly…

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  Figure 5. hiPSC-derived cardiomyocytes generate PAC-like currents, which are inhibited by weak acids. (A,B) Membrane currents in hiPSC-derived cardiomyocytes challenged with pH 5.0 or the combination of pH 5.0 and (A) 100 µM pregnenolone sulfate (PS, n = 6), or (B) 500 µM pregnenolone sulfate (n = 7 cells). (C,D) Membrane currents in hiPSC-derived cardiomyocytes evoked by pH 5.0 or the combination of pH 5.0 and 10 or 30 mM lactic acid ((C), n = 8), or 30 mM HOAc ((D), n = 8). (E) Box diagram with dots representing the PAC current inhibition by different concentrations of lactic (n = 8) and acetic acid (n = 8). (F–H) Representative figures of HEK293 cells following treatment with pH 7.4 (F), pH 5.0 (G), or pH 5.0 and 10 mM lactic acid (H) for 2 h and subsequent incubation with standard medium for 4 h at 37 °C. Cells were double-stained with DAPI (blue) and PI (red). (I) Box diagrams with dots displaying the percentage of dead cells (PI-positive) over absolute (DAPI-positive) cell count for two experimental conditions (2 h 37 °C or 2 h 37 °C and subsequent 4 h standard cell culture incubation; each condition n = 5 with independent experiments).