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Pflugers Arch
2023 Feb 01;4752:167-179. doi: 10.1007/s00424-022-02758-9.
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Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels.
Lin J
,
Gettings SM
,
Talbi K
,
Schreiber R
,
Taggart MJ
,
Preller M
,
Kunzelmann K
,
Althaus M
,
Gray MA
.
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The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.
Fig. 1. Time-dependent inhibition of SOCE by CFTRinh-172 in Calu-3 cells. A–D Representative Ca2+ fluorimetry traces tracking changes in [Ca2+]i following a Tg-activated, repeated Ca2+ addback protocol. Cells were exposed to the vehicle DMSO (A), or 20 µM CFTRinh-172 (CFinh172, B-D), in a nominally Ca2+ free solution for 3 (B), 10 (C), or 30 (D) min before the second SOCE was induced by adding back 1 mM Ca.2+ (black bars). The agents were removed from the perfusing solution for 15 (B, C) or 30 (D) min before the third SOCE was activated. (E) Box and whiskers summary of percentage change in SOCE peak amplitude and rate, from SOCE #1 to SOCE #2, following DMSO (control) or CFTRinh-172 treatment of different durations. One-way ANOVA with Holm-Sidak multiple comparisons tests was performed across the four groups. Boxes represent median ± 25th/75th percentiles, while whiskers represent minimum/maximum. * = p < 0.05 vs. control; # = p < 0.05 vs. 3 min. F Frequency of > 10% inhibition of SOCE amplitude and rate by DMSO or CFTRinh-172 treatment of different durations. Percentage inhibition was calculated as percentage of SOCE #2 amplitude/rate over that of SOCE #1, and the frequency of experiments with over and under 10% inhibition was tallied. A chi-square test was performed across the four groups. χ = p < 0.05 for chi-square test. n = 4–14
Fig. 2. Distinct CFTR inhibitors reduce SOCE in HEK293T cells. A, B HEK293T cells do not express CFTR. A Raw CT values for amplification of CFTR, and the housekeeping gene GAPDH, in Calu-3 and HEK293T cell samples. Each sample was run in duplicate. B Western blot image of Calu-3 and HEK293T samples probed for CFTR expression (Band C ~ 180 kDa, fully glycosylated CFTR). C–E Representative Ca2+ fluorimetry traces tracking changes in [Ca2+]i following a Tg-activated, repeated Ca.2+ addback protocol, with pre-treatment of DMSO (C), 20 µM CFTRinh-172 for 30 min (D) or 10 µM GlyH-101 for 10 min (E) before the second SOCE was activated. F Box and whiskers summary of percentage change in SOCE peak amplitude and rate, from SOCE #1 to SOCE #2, following treatment of DMSO (control) or the CFTR inhibitors. One-way ANOVA with Holm-Sidak multiple comparisons tests was performed across the three groups. Boxes represent median ± 25th/75th percentiles, while whiskers represent minimum/maximum. * = p < 0.05 vs. control. n = 5–6
Fig. 3. CFTRinh-172 and GlyH-101 inhibit Orai1/Stim1-mediated whole cell currents in HEK293T cells. A, B, D, E Current–voltage relationships for whole cell currents measured in cells pre-exposed to CPA (10 µM) to activate Orai1 channels, followed by CFTR inhibitors. The inhibitors were present for 3 min at each concentration tested. Cells were mock-transfected (black symbols) or overexpressed Orai1/Stim1 (red symbols). Increasing concentrations of CFTRinh-172 (CFinh172, 0.2, 2, 20 µM) or GlyH-101 (GlyH101, 0.5, 5, 50 µM) were applied in the continuous presence of CPA. Application of the highest concentration of CFTRinh-172 (20 µM) or GlyH-101 (50 µM) significantly inhibited the inward currents, but had no effect in mock-transfected cells. C, F Summary of the concentration-dependent inhibition of inward currents by CFTRinh-172 or GlyH-101. * = p < 0.05 vs. control (mock). Un-paired t-test
Fig. 4. CFTRinh-172 and GlyH-101 affect ENaC currents in Xenopus oocytes. A Left panel: representative current trace of a human αβγ-ENaC expressing oocyte. The application of amiloride (100 µM; ‘a’) is represented by the black bars and was used to determine amiloride-sensitive fractions of IM (ΔIami; right panel), before and after application of CFTRinh172 (20 µM; CFinh172). n = 9. B Similar experiments as shown in panel A, with human δβγ-ENaC expressing oocytes (n = 9). C/D Similar experiments as shown in panels A/B, where ΔIami were determined before and after application of GlyH-101 (GlyH101, 10 µM) n = 9 for both conditions. Student’s paired t-test was employed for all statistical analyses, with ** indicating p < 0.01 and *** indicating p < 0.001. E Left panel: Representative current trace of a water-injected control oocyte. The application of amiloride (100 µM; ‘a’) is represented by the black bars, the application of CFTR inhibitors by grey bars. Neither of the CFTR-inhibitors had any significant effect on transmembrane currents (IM). n = 6. Wilcoxon matched-pairs signed rank test was employed for statistical analyses
Fig. 5. Putative CFTR-inhibitor binding sites in human ENaC. Molecular docking experiments identified two potential binding pockets for CFTRinh-172 (yellow) and GlyH-101 (orange) in the human α-ENaC structure. α-ENaC is shown in blue, γ-ENaC in brown and β-ENaC in green. The structure of each ENaC subunit represents a clenched hand holding a ball of β-sheets [32]. One binding pocket is located at the ‘finger’/’ thumb’ domain interface, the second binding pocket is located between the ‘β-ball’ and ‘palm’ domains
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