(682) CFTR variants in FRT cells exhibit distinct bicarbonate to chloride conductance ratios that are affected by forskolin and ivacaftor

Background: The absence of bicarbonate (HCO₃^-) secretion or inappropriate extracellular pH is associated in cystic fibrosis (CF) with tissue and organ damage that leads to morbidity and mortality. Thus, in CF treatment, we speculate that recovery of HCO₃^- secretion will play a critical role in ongoing patient health. CFTR is reportedly permeant to both Cl^- and HCO₃^- although cell models coupling CFTR to Cl^-: HCO₃^- exchangers have been proposed to account for CFTR-associated HCO₃^- secretion. Importantly, a survey of HCO₃^- transport in tissues expressing CFTR variants has not been reported.

Methods: Fisher rat thyroid (FRT) cells, which form electrically tight monolayers that do not exhibit forskolin-stimulated conductance (g) or forskolin-stimulated electrogenic ion transport (I_sc), traffic exogenously expressed CFTR to both the apical and basolateral membranes, making this an ideal system to assess CFTR-associated anion conductance.

Results: Previously, we reported that forskolin-stimulated and GlyH101-inhibited g across FRT cells expressing wtCFTR was proportional to extracellular Cl^- concentration (g_Cl-). Further, we reported an increment in g when Cl^--containing buffer was supplemented with HCO₃^- (i.e., g_HCO3-). Most importantly, we showed that ivacaftor potentiated forskolin-stimulated g_Cl- but reduced g_HCO3- (i.e., reduced the g_HCO3-:g_Cl- ratio). More recent studies assessed forskolin concentration dependence of the g_HCO3-:g_Cl- ratio for wtCFTR. At low forskolin concentration (0.3 μM) the ratio ranged from 2 to 9 although overall g was low (< 2 mS cm^-2). Higher forskolin concentrations were associated with lower conductance ratios until at 10 μM forskolin, the conductance ratio was always < 1. At all concentrations of forskolin tested, subsequent exposure to ivacaftor increased g, but reduced g_HCO3-:g_Cl- to < 1. Ongoing experiments seek to determine optimal concentrations of forskolin and ivacaftor to maximize total g and the g_HCO3-:g_Cl- ratio in CFTR variants F508del and G551D following exposure to elexacaftor and/or tezacaftor (3 μM). Maximal g_Cl- was observed with 1 μM ivacaftor following 3 μM forskolin for F508del and 10 μM forskolin for G551D. Importantly, as we reported previously using correctors C17 and C18, little or no g_HCO3- could be detected in cells expressing F508del CFTR that were corrected with elexacaftor and/or tezacaftor. Cells expressing G551D CFTR consistently showed a HCO₃^--associated increment in ivacaftor potentiated g that was enhanced by elexacaftor and/or tezacaftor.

Conclusions: Taken together, these results extend previous observations by suggesting that CFTR variants exhibit different sensitivities to activators (e.g., forskolin), that the degree of potentiation by ivacaftor is sensitive to activation level and that ivacaftor can reduce g_HCO3-:g_Cl- ratio. More importantly, the results suggest that the g_HCO3-:g_Cl- ratio is unique to each CFTR variant and that the ratio may be modulated (reduced) by ivacaftor. The outcomes suggest that current therapies that greatly enhance the health and quality of life for many CF patients, may not correct bicarbonate secretion, which may reveal new pH-associated challenges to patient health.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation [grant number SCHULT21XX0].

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