KCa3.1 potentiation stimulates Cl- secretion in F508del and G551D CFTR-corrected primary human bronchial epithelial cells

Abstract

We previously identified potentiators of KCa3.1 (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; DCEBIO) that stimulate Cl^- secretion across human bronchial epithelial cells (HBEs) expressing wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR). However, these compounds failed to stimulate Cl^- secretion in F508del CFTR HBEs. Drug discovery efforts identified CFTR potentiators (VX-770) and correctors (VX-445, VX-661) for cystic fibrosis (CF) disease-causing mutations, including F508del and G551D. Herein, we evaluated the effect of KCa3.1 potentiation on Cl^- equivalent current (I_Cl) across primary HBEs expressing WT, F508del, and G551D CFTR. Transepithelial impedance analysis was used to obtain estimates of apical (R_a) and basolateral membrane (BLM; R_b) resistances. In WT CFTR HBEs, DCEBIO stimulated I_Cl, which was increased by forskolin. Similarly, forskolin stimulated I_Cl, and this was increased by DCEBIO. The KCa3.1 blocker, TRAM-34 inhibited I_Cl. DCEBIO decreased R_b, whereas TRAM-34 increased R_b, consistent with BLM localization of KCa3.1. Following correction of F508del CFTR with VX-445 + VX-661, DCEBIO failed to stimulate I_Cl, although the subsequent addition of forskolin + VX-770 increased I_Cl. Importantly, following stimulation of I_Cl with forskolin + VX-770, DCEBIO induced a further significant increase in I_Cl. As above, DCEBIO reduced R_b, whereas TRAM-34 increased R_b, consistent with BLM localized KCa3.1. Finally, we assessed KCa3.1 potentiation on I_Cl in G551D/F508del CFTR HBEs in the absence or presence of VX-445 + VX-661. In both cases, DCEBIO failed to stimulate I_Cl. However, following stimulation with forskolin + VX-770, DCEBIO nearly doubled I_Cl. Our results demonstrate that following correction/potentiation of F508del and G551D CFTR, potentiation of KCa3.1 increases the Cl^- secretory response, suggesting this class of compounds may represent a novel means of further increasing Cl^- secretion across CF airway.

Keywords: CFTR; DCEBIO; KCa3.1; VX-445; VX-661; cystic fibrosis; rikafta.