Revisiting CFTR inhibition: a comparative study of CFTRinh -172 and GlyH-101 inhibitors

Abstract

Background and purpose: For decades, inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been used as tools to investigate the role and function of CFTR conductance in cystic fibrosis research. In the early 2000s, two new and potent inhibitors of CFTR, CFTRinh -172 and GlyH-101, were described and are now widely used to inhibit specifically CFTR. However, despite some evidence, the effects of both drugs on other types of Cl(-) -conductance have been overlooked. In this context, we explore the specificity and the cellular toxicity of both inhibitors in CFTR-expressing and non-CFTR-expressing cells.

Experimental approach: Using patch-clamp technique, we tested the effects of CFTRinh -172 and GlyH-101 inhibitors on three distinct types of Cl(-) currents: the CFTR-like conductance, the volume-sensitive outwardly rectifying Cl(-) conductance (VSORC) and finally the Ca(2+) -dependent Cl(-) conductance (CaCC). We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines.

Key results: We confirmed that these two compounds were potent inhibitors of the CFTR-mediated Cl(-) conductance. However,GlyH-101 also inhibited the VSORC conductance and the CaCC at concentrations used to inhibit CFTR. The CFTRinh -172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM. Neither inhibitor (20 µM; 24 h exposure) affected cell viability, but both were cytotoxic at higher concentrations.

Conclusions and implications: Both inhibitors affected Cl(-) conductances apart from CFTR. Our results provided insights into their use in mouse models.

Keywords: CFTR; CaCC; VSORC; chloride channel; chloride conductance; cystic fibrosis; inhibitors; patch-clamp; whole-cell.

Figure 1

Effect of CFTR_inh-172 and GlyH-101 inhibitors on cellular toxicity. (A and B). Representative fluorescent dye staining (A) and related quantification of cell death (B) in CFTR-expressing cells (confluent kidney PCT cell monolayers) exposed for 24 h to increasing concentrations of CFTR_inh-172 or GlyH-101 (concentrations ranging from 0.5 to 50 µM). Live cells labelled with calcein-AM appeared green while dead cells labelled with homodimeric propidium iodide appeared red. Scale bars represents 80 µm. Values were normalized to the 100% of live cells in control experiments and were means (±SEM) of three to six individual experiments. (C) MTT assay performed on CFTR-expressing cells (kidney PCT cells) exposed as in (A) to increasing concentrations of both CFTR inhibitors. Values were normalized to control experiments and represent means (±SEM) of eight individual experiments. (D) MTT assay performed on non–CFTR-expressing cells (PS120) exposed to increasing concentrations of both inhibitors. Values were normalized to vehicle experiments and represent means (SEM) of eight individual experiments. *P < 0.05, Tukey's HSD test.

Figure 2

Inhibition of forskolin-activated CFTR-like conductance by CFTR_inh-172 and GlyH-101 inhibitors in CFTR-expressing cells. (A and B) Whole-cell current traces recorded in CFTR-expressing cells (kidney, DCT cells) under control condition and after forskolin exposure (FK, 1–10 µM). Once the Cl⁻ conductance is fully developed (3–4 min), CFTR_inh-172 or GlyH-101 were perfused at increasing concentrations (0.5, 1, 5, 10 µM). The membrane potential was held at −40 mV and currents were elicited by a train of 11 voltage steps (400 ms duration) between −100 and +100 mV in +20 mV increment. The zero current level is indicated by a dashed line. (C) Mean current/voltage relationships measured at 350 ms after the onset pulse corresponding to experiments performed (A and B) under control condition, after FK exposure and finally in the presence of CFTR_inh-172 (10 µM) or GlyH-101 (10 µM). Values are means (±SEM) of six to eight individual cells. (D and E) Histograms illustrating the concentration-dependent inhibition of the CFTR conductance obtained with increasing concentrations of CFTR_inh-172 (D) or GlyH-101 (E). Concentrations of both inhibitors vary from 0.5 up to 10 µM as indicated. Values were individually normalized for each concentration of inhibitors to the maximal current slope (recorded after FK stimulation) calculated between −100 and −60 mV and between +60 and +100 mV. Values are means (±SEM) of five to eight individual cells. *P < 0.05, Tukey's HSD test. The insets show the logarithmic dose–response curves corresponding to each inhibitor.

Figure 3

Effects of CFTR_inh-172 on the VSORC measured in CFTR-expressing cells (kidney) and in non–CFTR-expressing cells (PS120). (A and B) Whole-cell currents recorded in CFTR-expressing cells (A) and in non–CFTR-expressing cells (B). Cl⁻ currents were recorded in control conditions and after replacing the hypertonic bath by a hypotonic solution (hypo). Once the Cl⁻ conductance is fully developed (3–4 min), CFTR_inh-172 was perfused (5, 10 µM as indicated). Normal bath solution was made hypertonic (340 mOsmol including 30–40 mOsm of Mannitol), and the hypotonic one was adjusted by removing mannitol from the normal bath solution (290 mOsm). NPPB (100 µM) completely inhibited swelling-activated Cl⁻ current. The membrane potential was held at −40 mV and currents were elicited by a train of 11 voltage steps (400 ms duration) between −100 and +100 mV in +20 mV increment. (C and D) Mean current/voltage relationships measured in CFTR-expressing cells (C) and in non–CFTR-expressing cells (D) recorded in control condition, after the stabilization of the VSORC Cl⁻ current (hypo) and in the presence of CFTR_inh-172 (10 µM). Current values were measured 5 ms after the onset pulse. Values are means (±SEM) of five to six individual cells. (E and F) Histogram illustrating the remaining fraction of the VSORC conductance for increasing concentrations of CFTR_inh-172 (1, 5, 10 µM) measured in CFTR-expressing cells and in non–CFTR-expressing cells. Values were individually normalized for each concentration of CFTR_inh-172 to the maximal current slope (recorded after hypotonic solution exposure) calculated between −100 and −60 mV (E) and between +60 and +100 mV (F). Values are means (±SEM) of five to six individual cells. *P < 0.05, Tukey's honestly significant difference test. The insets show the logarithmic dose–response curves calculated between −100 and −60 mV (E) and between +60 and +100 mV (F).

Figure 4

Effects of GlyH-101 on VSORC conductance measured in CFTR-expressing cells (kidney) and in non–CFTR-expressing cells (PS120). (A and C) Whole-cell current traces recorded in CFTR-expressing cells (A) and in non–CFTR-expressing cells (C) in control condition and after replacing the hypertonic bath by a hypotonic solution (hypo). Once the Cl⁻ conductance is fully developed, GlyH-101 was perfused at increasing concentrations (0.5, 1, 5, 10 µM) as indicated. The membrane potential was held at −40 mV and currents were elicited by a train of 11 voltage steps (400 ms duration) between −100 and +100 mV in +20 mV increments. (B and D) Means current/voltage relationships measured in CFTR-expressing cells (B) and in non–CFTR-expressing cells (D) recorded in control conditions, after the stabilization of the VSORC Cl⁻ current (hypo) and in the presence of GlyH-101 (10 µM). Currents values were measured 5 ms after the onset pulse. Values are means (±SEM) of five to six individual cells. (E and F) Histogram illustrating the remaining fraction of the VSORC conductance for increasing concentrations of GlyH-101 (0.5, 1, 5, 10 µM) measured in CFTR-expressing cells and in non–CFTR-expressing cells. Values were individually normalized for each concentration of GlyH-101 to the maximal current slope (recorded after hypotonic solution exposure) calculated between −100 and −60 mV (E) and between +60 and +100 mV (F). Values are means (±SEM) of five to six individual cells. *P < 0.05, Tukey's HSD test. The insets show the logarithmic dose–response curves calculated between −100 and −60 mV (E) and between +60 and +100 mV (F).

Figure 5

Effects of CFTR_inh-172 and GlyH-101 inhibitors on the CaCC. (A and B) Whole-cell current traces recorded in CFTR-expressing cells (kidney) in control conditions and after ionomycin treatment (iono, 2 µM). Once the CaCC is fully developed (<5 min), CFTR_inh-172 (A) or GlyH-101 (B) were perfused at increasing concentrations (1, 5, 10 µM). The membrane potential was held at −40 mV and currents were elicited by a train of 12 voltage steps (400-ms duration) between −100 and +120 mV in +20 mV increments. (C) Mean current/voltage relationships measured at 350 ms after the onset pulse corresponding to experiments performed as in (A) and (B) under control conditions, after ionomycin exposure and finally in the presence of GlyH-101 (10 µM) or CFTR_inh-172 (10 µM). Values are means (±SEM) of five to six individual cells. (D) Histogram illustrating the remaining fraction of the CaCC conductance as a function of GlyH-101 or CFTR_inh-172 concentrations (1, 5, 10 µM). Values measured for each concentration were individually normalized to the maximal current slope measured between +60 and +120 mV in the absence of any inhibitor. Values are means (±SEM) of five to six individual cells. *P < 0.05, Tukey's HSD test. The insets show the logarithmic dose–response curve for GlyH-101 and calculated between −100 and −60 mV (E) and between +60 and +120 mV (F).

Figure 6

Schematic representation of the specificity of CFTR_inh-172 and GlyH-101 towards Cl⁻ conductances, depending on the concentration used.