Using a cysteine-less mutant to provide insight into the structure and mechanism of CFTR.

Abstract

Cystic fibrosis (CF) is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator protein (CFTR) (Riordan et al. 1989). CFTR is a member of the ABC (ATP-binding cassette) family of transporters and acts as a cAMP-regulated chloride channel at the apical surface of epithelial cells. The most common mutation in CF is deletion of phenylalanine 508 in CFTR (ΔF508 CFTR) that causes the protein to be incompletely folded and retained in the endoplasmic reticulum (processing mutant). A fraction of the mutant ΔF508 CFTR protein can fold and be trafficked to the cell surface in a functional form, however, when it is expressed at low temperature (Denning et al. 1992). A potential therapy for CF would be to use specific pharmacological chaperones to induce proper folding of misfolded CFTR (Loo et al. 2005; Pedemonte et al. 2005). There are problems, however, with ‘rescued’ΔF508 CFTR at the cell surface. The rescued ΔF508 CFTR protein shows more than a threefold reduction in open probability after cAMP stimulation. There also appears to be two types of rescued ΔF508 CFTRs at the cell surface – a forskolin-resistant form (FR-CFTR) and a forskolin-insensitive form (FS-CFTR). We have observed that channel activation of ΔF508 CFTR rescued at low temperature required the presence of forskolin and a channel opener such as genistein (FR-CFTR). By contrast, ΔF508 CFTR rescued with some pharmacological chaperones required only forskolin to be activated (FS-CFTR) (authors' unpublished observations). Therefore, these subtle differences between the rescued ΔF508 CFTRs would probably be technically difficult to study by crystallography. Another approach to probe for subtle changes in CFTR structure is to introduce cysteine residues into CFTR and to study the effects of thiol-modifying compounds on its structure and function. Such an approach was used to show that the structures of mature and immature CFTR processing mutants were quite different (Loo et al. 2006). Initial studies with cysteine-less (Cys-less) CFTR (all cysteines mutated to serine) were not successful because the mutant protein did not mature (Chen et al. 2004) and could not be rescued by expression at low temperature (authors' unpublished observations). In this issue of The Journal of Physiology, Cui et al. (2006) constructed a better Cys-less CFTR by changing 16 of the cysteines to alanine and two to leucine. Leucines were introduced at positions Cys590 and Cys592 because it had been reported that hydrophobic amino acids at these positions promoted delivery of the Cys-less CFTR to the cell surface in oocytes (Mense et al. 2002). Although this Cys-less CFTR did not mature at 37°C, it matured into a functional form at 27°C. The authors then replaced Phe508 of Cys-less CFTR with a cysteine followed by thiol modification to study the effects on channel gating. Their results suggest that the side chain of Phe508 plays a direct role in determining the residency time in the closed state and help explain why rescued ΔF508 CFTR shows defective open probability. The study by Cui et al. (2006) clearly shows the usefulness of the Cys-less CFTR and its potential use will be limited only by one's imagination. The Cys-less CFTR could be used in cysteine-modification studies for mechanistic studies such as comparing the FS and FR forms of rescued ΔF508 CFTR to wild-type CFTR, or for cross-linking studies that provide insight into the structure of CFTR. A wide variety of studies are possible as shown with the Cys-less mutant of P-glycoprotein (Cys-less P-gp), a sister protein of CFTR (Loo & Clarke, 2005). For example, the Cys-less P-gp has been used to study the ligand binding sites (Loo et al. 2003b) and nucleotide binding by ESR spectroscopy (Delannoy et al. 2005), to determine packing of the transmembrane domains (Loo et al. 2004) and to follow conformational changes between the transmembrane and nucleotide-binding domains (Loo et al. 2003a). Cysteine mutagenesis and thiol modification techniques have also been used extensively to study other non-ABC membrane proteins such as lactose permease (Frillingos et al. 1998). It must be kept in mind, however, that work with a Cys-less mutant does have potential problems. The Cys-less CFTR developed by Cui et al. (2006) did not mature at 37°C. One wonders whether changing other cysteines to leucine may overcome this problem. Also, introduction of new cysteine residues into the Cys-less CFTR could potentially compound the folding problem. A recent study by Liu et al. (2006) showed that another potential problem with introducing cysteines into Cys-less CFTR is that the thiol group could exist in three different chemical states with different reactivities to thiol-modifying compounds. Despite these drawbacks, use of Cys-less CFTR should provide important new insights into the structure and mechanism of CFTR.