VX770 Modulated Bicarbonate Conductance in CFTR‐expressing FRT Cell (2022)

The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in epithelia and other tissues, is compromised by genetic mutations that cause cystic fibrosis (CF). Although CFTR is reportedly permeant to both Cl¯ and HCO3¯, high throughput screening studies identified drug scaffolds that enhanced Cl¯ conductance of mutated CFTR by ‘correcting’ processing deficiets and/or ‘potentiating’ aggregate conductance of channels in epithelial cell membranes while being agnostic to HCO3‐. Ongoing work seeks to determine whether mutated forms of CFTR exhibit typical HCO3¯ conductance and to access clinically‐used drugs for their effects on HCO3¯ conductance by wild‐type and mutated CFTR. Fisher rat thyroid (FRT) cells, which form electrically tight epithelial monolayers, express no functionally detectable CFTR and which traffick exogenously expressed CFTR to both the apical and basolateral membranes, were employed to assess transepithelial anion conductance in modified Ussing‐style flux chambers. Initial experiments employing FRT cells expressing human wtCFTR validated that forskolin‐stimulated conductance, which was inhibited by selective CFTR blockers, was proportional to extracellular Cl¯ concentration over the range of ~70‐140 mEq/l. Further, a clear increment in forskolin‐stimulated conductance was observed when HCO3¯ (25 mEq/l) was incorporated into the media. VX770 potentiated forskolin‐stimulated conductance, but had no effect in the absence of forskolin. Foskolin‐stimulated and VX770‐potentiated conductance was compared in base medium (100 mEq/l Cl‐) supplemented with 25 mEq/l HCO3‐ or Cl‐ and bicarbonate (B) conductance was quantified relative to Cl‐ (C) conductance (i.e., gB/gC). Following forskolin exposure the gB/gC ratio for wtCFTR was 0.62±0.20, a value that decreased to 0.11±0.10 following exposure to VX770, suggesting that VX770 may negatively impact gB. Forskolin‐ and VX770‐stimulated conductance across cells expressing F508del CFTR was nearly undetectable unless cells were pretreated with CFTR correctors. Following corrector exposure, forskolin stimulated and VX770 potentiated gC, but gB was virtually undetectable, suggesting that this variant may lack gB. Alternatively, forskolin stimulated little conductance across cells expressing G551D CFTR, but a substantial potentiation with VX770 was observed. Notably, the gB/gC ratio approached 1.0 for both foskolin stimulation and VX770 potentiation. Taken together, these results suggest that CFTR HCO3‐ conductance differs by disease‐associated variant and that gB of some variants are sensitive to VX770. Ongoing experiments are designed to determine the optimal forskolin concentration to maximize VX770 potentiation. Initial outcomes suggest that there is interaction between the CFTR variant and forskolin concentration dependence such that VX770‐potentiated conductance is maximized at different forskolin concentrations. Taken together, results from this investigation suggest that additional or alternative interventions may be required to optimize bicarbonate secretion and pH regulation in CF patients expressing alternative CFTR variants.