Extracellular phosphate enhances the function of F508del-CFTR rescued by CFTR correctors

Abstract

Background: The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors.

Methods: With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature.

Results: Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl^- currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl^- currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate.

Conclusions: Extracellular phosphate modulates the magnitude of CFTR-mediated Cl^- currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors.

Keywords: Airway epithelia; CFTR correctors; Elexacaftor-tezacaftor-ivacaftor (Trikafta); F508del-CFTR; Phosphate; SLC34A2.

Fig. 1

Messenger RNA expression of the phosphate transporter SLC34A2 in hAECs and FRT cells. (A and C) Agarose gel electrophoresis (2% agarose) of PCR amplified products using specific primer pairs for (A) SLC34A2 and 18S rRNA with bands at 155 and 209 bp, respectively, and (C) slc34a2 and 18s rRNA with bands at 109 and 208 bp, respectively. (B and D) Relative quantity (RQ) of mRNA for (B) SLC34A2 in primary cultures of hAECs from CF and non-CF (NCF) donors and (D) slc34a2 in F508del- and wild-type CFTR FRT cells. Symbols represent individual values and lines are means ± SD (B, n = 8 from 3 donors each; P = 0.33; D, n = 3; P = 0.1; Mann-Whitney rank sum test).

Fig. 2

Phosphate increases lumacaftor-rescued F508del-CFTR-mediated Cl⁻ currents in FRT epithelia. (A and B) Representative I_sc recordings of lumacaftor-rescued F508del-CFTR in the presence (A) and absence (B) of phosphate (1.24 mM K₂HPO₄ and 2.4 mM KH₂PO₄) in the Krebs Ringer Buffer (KRB). Prior to study, F508del-CFTR-expressing FRT epithelia were treated with lumacaftor (VX-809; 3 μM) or DMSO (0.1% v·v⁻¹) for 48 h at 37°C. At the indicated times, F508del-CFTR-mediated Cl⁻ currents were activated with forskolin (Fsk; 10 μM), potentiated with P5 (10 μM) and inhibited with CFTR_inh-172 (I172; 20 μM); continuous lines indicate the presence of compounds in the apical solution only or the apical and basolateral solutions (forskolin) during I_sc recordings. Data were normalised by subtraction of the baseline current prior to F508del-CFTR activation by forskolin. (C – E) Summary data show the magnitude of baseline I_sc, R_t before forskolin addition and the change in I_sc (ΔI_sc) for the indicated conditions. Symbols represent individual values and lines are means ± SD (VX-809: +phosphate, n = 8; –phosphate, n = 6; DMSO: n = 4); *, P < 0.05; **, P < 0.01 (Two-way ANOVA with Tukey's multiple comparison test).

Fig. 3

Phosphate enhances lumacaftor-rescued F508del-CFTR-mediated Cl⁻ currents in hAEC epithelia. (A and B) Representative I_sc recordings of lumacaftor-rescued F508del-CFTR in the presence (A) and absence (B) of phosphate (1.24 mM K₂HPO₄ and 2.4 mM KH₂PO₄) in the KRB. Prior to study, hAEC epithelia (genotype: F508del/F508del) were treated with lumacaftor (VX-809; 3 μM) or DMSO (0.1% v·v⁻¹) for 48 h at 37°C. At the indicated times, F508del-CFTR-mediated Cl⁻ currents were activated with forskolin (Fsk; 10 μM), potentiated with P5 (10 μM) and inhibited with CFTR_inh-172 (I172; 20 μM); continuous lines indicate the presence of compounds in the apical solution only or the apical and basolateral solutions (forskolin) during I_sc recordings. Experiments were performed in the presence of amiloride (10 μM) in the apical solution. Data were normalised by subtraction of the steady-state current after amiloride addition prior to F508del-CFTR activation by forskolin. (C – E) Summary data show the magnitude of baseline I_sc and R_t before amiloride addition and the change in I_sc (ΔI_sc) for the indicated conditions. Symbols represent individual values and lines are means ± SD (VX-809: +phosphate, n = 9; –phosphate, n = 11; DMSO: +phosphate, n = 10; –phosphate, n = 11); *, P < 0.05; **, P < 0.01; ^†††, P < 0.001 (Repeated Measure two-way ANOVA with Sidak's multiple comparison test).

Fig. 4

Phosphate enhances elexacaftor-tezacaftor-ivacaftor-rescued F508del-CFTR-mediated Cl⁻ currents in hAEC epithelia. (A and B) Representative I_sc recordings of elexacaftor-tezacaftor-ivacaftor (ETI)-rescued F508del-CFTR in the presence (A) and absence (B) of phosphate (1.24 mM K₂HPO₄ and 2.4 mM KH₂PO₄) in the KRB. Prior to study, hAEC epithelia (genotype: F508del/F508del) were treated with VX-445 (2 µM), VX-661 (3 µM) and VX-770 (1 µM) or DMSO (0.06% v·v⁻¹) for 24 h at 37°C. At the indicated times, F508del-CFTR-mediated Cl⁻ currents were activated with forskolin (Fsk; 10 μM) and inhibited with CFTR_inh-172 (I172; 20 μM); continuous lines indicate the presence of compounds in the apical solution only, or the apical and basolateral solutions (forskolin) during I_sc recordings. Experiments were performed in the presence of amiloride (10 μM) in the apical solution. Data were normalised by subtraction of the steady-state current after amiloride addition prior to F508del-CFTR activation by forskolin. (C – E) Summary data show the magnitude of baseline I_sc and R_t before amiloride addition and the change in I_sc (ΔI_sc) for the indicated conditions. Symbols represent individual values and lines are means ± SD (elexacaftor-tezacaftor-ivacaftor (ETI): +phosphate, n = 9; –phosphate, n = 9; DMSO: +phosphate, n = 9; –phosphate, n = 9); * P < 0.05; ***, P < 0.001; ^†, P < 0.05 vs. –phosphate (Two-way ANOVA with Sidak's multiple comparison test).

Fig. 5

The enhancement by phosphate of F508del-CFTR function in hAEC epithelia after rescue by elexacaftor-tezacaftor-ivacaftor is dependent on external sodium. (A) Representative I_sc recordings of elexacaftor-tezacaftor-ivacaftor (ETI)-rescued F508del-CFTR in the absence or presence of phosphate (1.24 mM K₂HPO₄ and 2.4 mM KH₂PO₄) using a sodium-free KRB. Prior to study, hAEC epithelia (genotype: F508del/F508del) were treated with VX-445 (2 µM), VX-661 (3 µM) and VX-770 (1 µM) or DMSO (0.06% v·v⁻¹) for 24 h at 37°C. At the indicated times, F508del-CFTR-mediated Cl⁻ currents were activated with forskolin (Fsk; 10 μM) and inhibited with CFTR_inh-172 (I172; 20 μM); continuous lines indicate the presence of compounds in the apical solution only, or the apical and basolateral solutions (forskolin) during I_sc recordings. Experiments were performed in the presence of amiloride (10 μM) in the apical solution. Data were normalised by subtraction of the steady-state current after amiloride addition prior to F508del-CFTR activation by forskolin. (B – D) Summary data show the magnitude of baseline I_sc and R_t before amiloride addition and the change in I_sc (ΔI_sc) for the indicated conditions. Symbols represent individual values and lines are means ± SD (elexacaftor-tezacaftor-ivacaftor (ETI): +phosphate, n = 9; –phosphate, n = 9).