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Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function.
The phenylalanine residue at position 508 in the first nucleotide-binding domain (NBD1) of cystic fibrosis transmembrane conductance regulator (CFTR) has been the focus of much attention because a mutation causing its deletion is the cause of most cystic fibrosis (Cheng et al. 1990). The mutation results in the biosynthetic arrest of the protein in the endoplasmic reticulum, where it is degraded rather than proceeding further through the secretory pathway to its functional site at the cell surface (Kopito, 1999). Recent structural studies (Lewis et al. 2005; Thibodeau et al. 2005) and experiments with intracellular CFTR folding (Du et al. 2005) suggest the deletion causes only a minor surface perturbation of NBD1 where it normally resides, and probably interferes with the normal domain assembly of the protein. When the protein is partially rescued by growth of cells at reduced temperature it has at least some chloride channel activity (Dalemans et al. 1991; Denning et al. 1992). The significance of the functional defects induced by Phe508 deletion is controversial (Schultz et al. 1999; Wang et al. 2000). When Phe508 is replaced by one of several other residues rather than being deleted, the protein is able to mature and be transported to the cell surface (Du et al. 2005). The fact that cysteine is one of the permissive residues at this position provides the opportunity to utilize a wide variety of sulfhydryl reactive reagents to investigate the role of this surface residue in CFTR ion channel function.
However, to specifically probe the 508 position with sulfhydryl reagents it is necessary to remove the 18 endogenous cysteines from CFTR. When this was done recently by Chen et al. (2004) they found that cysless CFTR did not mature and reach the surface of cells in which it was transiently expressed.
We now have generated BHK cell lines stably expressing cysless CFTR which does mature and function in cells cultured at reduced temperature. The cysless protein mediates cyclic AMP-stimulated 36Cl efflux from cells, binds and hydrolyses ATP and exhibits characteristic single-channel activity. Combining cysless CFTR single-channel recording with site-directed mutagenesis and cysteine chemical modification revealed that the side chain of Phe508 has a strong impact on CFTR function. This supports a direct role of the Phe508 side chain in CFTR channel gating and provides a tool with which to characterize the intramolecular interaction involved.
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These experiments have two main outcomes. First they describe an initial characterization of cysless CFTR, indicating that it is functional, although with an extended single-channel closed time. Second, modification of a single cysteine (508C) in the cysless background with methanethiosulphonate reagents provide evidence of involvement of an aromatic moiety at this position in channel gating.
Neither ATP binding, nor the rate of ATP hydrolysis, is strongly affected by complete cysteine substitution in the mature CFTR molecule (Fig. 2). However the turnover of bound 8N3[γ-32P]ATP at the second ATP binding site provides only a qualitative measure of hydrolytic ability. Quantitative comparisons will require purification and conventional catalytic rate measurements, as has been done with wild-type CFTR (Li et al. 1996; Aleksandrov et al. 2002).
We employed single-channel recording to compare functional properties of the cysless and wild-type CFTR ion channels in more detail. It is important to distinguish between the requirements for this channel function and so-called conformational maturation. Conformational maturation, a term widely used in connection with the biosynthetic processing and ER export of CFTR, is not equivalent to functional competence. Some mutants can meet the requirements for maturation but are not sufficient for the normal protein function.
Our approach also illustrates the advantage of being able to introduce modifications of the cysteine after assembly has occurred rather than by mutagenesis. In the latter case, it is not possible to determine whether an effect on function has resulted indirectly from perturbed assembly. With the cysteine modification it is possible to monitor the impact of the induced structural perturbation at the single-molecule level in real time (see control in Supplemental material).
Complete cysteine substitution does not affect ion channel mean open time. Moreover, the open state has the same ligand specificity for both cysless and wild-type CFTR (see Table 1). Based on these experimental data we can conclude that complete cysteine substitution does not affect the open state structure. Therefore, we could expect the same structural organization for both cysless and wild-type CFTR under experimental conditions where the open state is predominant. The E1371 mutation that locks the channel open when 8BrATP is used as a ligand provides an identical structural background for both cysless and wild-type CFTR (Fig. 6 upper trace, Fig. 7 upper trace). That is why our experiments with the F508C mutation and its chemical modification were done on this particular background.
In contrast, there must be substantial differences between cysless and wild-type CFTR ion channels with respect to the structural organization within the closed state (see Supplemental material). These differences must account for the large increment in the mean closed time which persists even with ATP analogues that normally shorten the closed time of the wild-type CFTR ion channel (see Table 1). Mean closed time for wild-type CFTR over the range of nucleotide concentration close to saturation is ligand specific, as is the mean open time (see Table 1). However, cysless CFTR demonstrate the appearance of the additional ligand-insensitive component in the total mean closed time whereas mean open time for both mutants remains ligand specific. Within the open state configuration, cysless CFTR binding site(s) still recognize minor differences in the ligand chemical structure with the same efficiency as the wild-type CFTR indicating that the affinity of binding sites should be unchanged. At the same time, cysless CFTR cannot support the relevant difference in the closed times. This may be explained by less efficient coupling between channel gating and the hydrolytic cycle for the cysless protein compared with the wild-type CFTR. Mechanistically this means that not every hydrolytic cycle will induce ion channel gating, and nucleotide binding is not a rate-limiting step for cysless CFTR ion channel gating. In the framework of this speculation, the apparent contradiction between the strong difference in channel gating and hydrolytic activity for cysless and wild-type CFTR is resolved.
Thus, although its gating is not identical to wild-type, cysless CFTR provides a means of gaining further insight into the role of specific features of the molecule such as the Phe508 side chain. Previous studies have focused on the rescued ΔPhe508 channel from which Phe508 is simply absent (Kopito, 1999; Riordan, 1999). More recent studies examined the effects of replacement of Phe508 by all other amino acids on the structure of isolated NBD1 (Thibodeau et al. 2005) and on the maturation of the whole CFTR protein (Du et al. 2005). Although the F508C variant appeared to mature similarly as wild-type and mediated iodide efflux (Du et al. 2005), we show that at the single-channel level it greatly increased the channel mean closed time in both the wild-type and cysless background, suggesting that the phenylalanine side chain plays a role in channel gating. This could be confirmed using a site II ATPase-inhibited mutant (E1371S) which is locked open in both the wild-type and cysless backgrounds, while the F508C version of cysless E1371S was unable to maintain the locked open state. Neither total cysteine removal, nor F508C substitution, affects open state. On the contrary, both increase residency time in the closed state (Figs 3, 4 and 5). It should be remembered that brief closing events still occur in the locked-open state. In the restricted bandwidth of our single-channel recording they are visible as incomplete transitions and reflected in the asymmetric shape of the peak for the open state in the all-points histogram (Fig. 7, upper panel). We speculate that aromatic ring removal from position 508 could impede channel reopening and by doing so, transform some of these brief closings to long-lasting ones.
The ability of F508C version of cysless E1371S to maintain the locked open state was fully restored on modificaton of Cys508 with MTSBn. Thus either the aromatic ring of MTSBn or the native phenylalanine can support locking open, whereas the unmodified thiol cannot. In contrast, modification with the charged MTSET was functionally disruptive, completely ablating gating. This latter effect on channel function parallels the effects of mutagenic substitutions of Phe508 with large charged residues on CFTR maturation. Thus whereas the small cysteine side chain is compatible with maturation but not adequate for normal channel function, large charged groups are strongly detrimental to both.
Beyond the provision of a new tool for studies of CFTR structure and function, and demonstrating a role of the Phe508 aromatic side chain in the process responsible for the residency time in the closed state, mechanistic interpretations of the results remain largely speculative at this time.
Thus, just as removal of all endogenous cysteines apparently alters the gating response to ATP binding rather than binding itself, this also appears to be the case for the further increment in mean closed time caused by F508C. We postulate that a specific interaction of the wild-type aromatic phenylalanine side chain at this position is necessary for the normal more rapid exit from the closed state, and future experiments will attempt to identify the site of this interaction.