CFTR function, pathology and pharmacology at single-molecule resolution

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes1. Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure2,3. Electrophysiological properties of CFTR have been analysed for decades4–6. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.

Structural basis for CFTR inhibition by CFTRinh-172

“…Conformational isomerizations of individual CFTR molecules were monitored as transitions between a low FRET efficiency (0.25 ± 0.01) NBD-separated state and a high FRET efficiency (0.49 ± 0.02) NBD-dimerized state. As we have previously reported (38), at a saturating (3 mM) ATP concentration, WT CFTR predominantly occupies NBD-dimerized conformations with brief excursions to the NBD-separated state (Figure 4C). The presence of 10 µM CFTRinh-172 did not significantly affect the probability of NBD dimerization or the dwell time of the NBD-dimerized state for WT CFTR (Figure 4D-E).…”

“…Potentiators Ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while the NBDs are dimerized. Additionally, the potentiators increase ATP turnover (38). In comparing the structure of CFTR (E1371Q) bound to GLPG1837 with that bound to CFTRinh-172, we observe that the CFTRinh-172 site is located along the pore-lining side of the TM8 hinge region, in direct juxtaposition to the potentiator binding site (Figure 4F).…”

“…The Michaelis-Menten constant (Km) for ATP was not significantly changed (Figure 4A). To assess the mechanism by which ATP hydrolysis is inhibited by CFTRinh-172, we used a recently established smFRET assay, which reports on the conformational state of CFTR's NBDs (38). In this assay, position 388 in NBD1 and position 1435 in NBD2 were labeled with donor and acceptor fluorophores (Figure 4B).…”

“…Substantial effort has been devoted to understanding CFTR in the context of cystic fibrosis. Disease-causing mutations have been extensively characterized, and small molecule drugs that potentiate gating or correct folding of CFTR have been successfully developed for clinical use (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). By contrast, relatively few studies have addressed CFTR hyperactivation.…”

Placing steroid hormones within the human ABCC3 transporter reveals a compatible amphiphilic substrate‐binding pocket

“…In fact, a combined administration of three drugs targeting ABCC7 has been applied to the treatment of cystic fibrosis (Middleton et al, 2019). Recently, a couple of drug-bound ABCC7 structures also provide more insights for the rational optimization of these clinical drugs (Fiedorczuk & Chen, 2022a,b;Levring et al, 2023).…”