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Symposium Reports |
1 Department of Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl– channel important in transepithelial salt and water transport. While there is a paucity of direct structural information on CFTR, much has been learned about the molecular determinants of the CFTR Cl– channel pore region and the mechanism of Cl– permeation through the pore from indirect structure–function studies. The first and sixth transmembrane regions of the CFTR protein play major roles in forming the channel pore and determining its functional properties by interacting with permeating Cl– ions. Positively charged amino acid side-chains are involved in attracting negatively charged Cl– ions into the pore region, where they interact briefly with a number of discrete sites on the pore walls. The pore appears able to accommodate more than one Cl– ion at a time, and Cl– ions bound inside the pore are probably sensitive to one another's presence. Repulsive interactions between Cl– ions bound concurrently within the pore may be important in ensuring rapid movement of Cl– ions through the pore. Chloride ion binding sites also interact with larger anions that can occlude the pore and block Cl– permeation, thus inhibiting CFTR function. Other ions besides Cl– are capable of passing through the pore, and specific amino acid residues that may be important in allowing the channel to discriminate between different anions have been identified. This brief review summarizes these mechanistic insights and tries to incorporate them into a simple cartoon model depicting the interactions between the channel and Cl– ions that are important for ion translocation.
(Received 11 August 2005;
accepted after revision 9 September 2005; first published online 12 September 2005)
Corresponding author P. Linsdell: Sir Charles Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia B3H 1X5, Canada. Email: paul.linsdell{at}dal.ca
Cystic fibrosis is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that functions as a Cl– channel in the apical membrane of many different epithelial cell types. While there is currently a paucity of direct structural information on CFTR, much has been learned about the mechanism by which this fascinating protein allows Cl– ions to move across the cell membrane. This brief review emphasizes the indirect structural information and mechanistic insights that have been gained from functional studies of CFTR.
Molecular determinants of the CFTR channel pore
The CFTR molecule is made up of two homologous repeats, each containing six transmembrane (TM) regions followed by an intracellular nucleotide binding domain (NBD; Fig. 1). These two halves are joined by an intracellular regulatory or R domain. Recently a low-resolution crystal structure of CFTR was obtained (Rosenberg et al. 2004), which showed membrane-spanning regions lining a central pore; the pathway through which Cl– ions cross the membrane. However, the identity of the TM regions forming the pore, or even the number of TMs that line the pore, cannot be identified in this structure. Nevertheless, homology with the structures of other ATP-binding cassette (ABC) proteins (Locher et al. 2002; Chang, 2003; Rosenberg et al. 2005) suggests that the CFTR pore is lined by multiple 
-helical TM regions in a reasonably parallel fashion. This overall pore architecture is common with ligand-gated Cl– channels (Unwin, 2003; Cascio, 2004) but in stark contrast with the seemingly haphazard arrangement of membrane-associated -helices observed in ClC Cl– channels (Dutzler et al. 2002).
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What about the C-terminal TMs? Historically these have received less experimental attention. It is tempting, based simply on the primary structure (Fig. 1), to suggest a symmetrical arrangement, and indeed TM12, like its counterpart TM6, has been proposed to play a role in forming the pore (McDonough et al. 1994; Vankeerberghen et al. 1998; Zhang et al. 2000). However, comparison of the functional effects of mutations in TMs 6 and 12 led to the proposal that the pore structure is asymmetric, with the N-terminal TMs (1–6) in fact being much more important in determining the permeation phenotype than their C-terminal partners (Gupta et al. 2001). This is consistent with earlier work using chimeric human–Xenopus CFTR channels that suggested that the main determinants of anion permeation were located in the N-terminal region (Price et al. 1996).
Positive charges attract Cl– ions into the pore
To ensure rapid movement of Cl– ions across the membrane, the inside of the channel pore must be an accomodating place for these ions, and recent work has emphasized the role played by positively charged amino acid side-chains in attracting Cl– into the pore. Careful analysis of the effects of site-directed mutagenesis on the shape of the current–voltage relationship suggests that a positively charged arginine (R334) in TM6 attracts Cl– ions into the pore from the extracellular solution, while another positive charge, on a lysine (K95) in TM1 pulls in Cl– from the intracellular side of the membrane (Smith et al. 2001; Linsdell, 2005). The apparent interactions of these two residues with Cl– ions on opposite sides of the membrane suggests that their fixed positive charges are located at the outer and inner entrances to a functionally important part of the pore (Fig. 2A). The importance of these electrostatic interactions in drawing Cl– ions into the pore is emphasized by the fact that removing either of these fixed positive charges dramatically reduces the rate at which Cl– ions pass through the channel (Ge et al. 2004; Gong & Linsdell, 2004). Indeed, this may contribute to the mechanism by which mutations at R334 cause cystic fibrosis (CF; Gong & Linsdell, 2004).
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Biophysical studies dating back to the 1950s suggested that ions bind to specific sites within channel pores (see Hille, 2001), and when ion channel crystal structures were finally obtained, one of their most striking features was the existence of multiple, discrete permeant ion binding sites (Doyle et al. 1998; Zhou et al. 2001; Dutzler et al. 2003). Ion binding is thought to be a key link between tight ionic selectivity and rapid ion transport in channel pores (MacKinnon, 2003; Sather & McCleskey, 2003).
While we do not yet have any direct structural evidence for Cl– binding sites in the CFTR pore, functional interactions between anions and the pore have been extensively studied since Tabcharani et al. (1993) first showed that a foreign anion, SCN–, could compete with Cl– for passage through the pore. Much has been learned by the use of surrogate anions, such as SCN–, that appear to bind inside the pore much more tightly than Cl– itself. Most useful in this respect have been the pseudohalide anions (such as Au(CN)2–, C(CN)3–, Pt(NO2)42– and Fe(CN)63–) that were introduced to the anion channel community by David Dawson (Smith et al. 1999). Physicochemical reasons why these ions are so ‘sticky’ inside the pore have been recently reviewed (Liu et al. 2003). But where do they stick? Identification of the sites within the pore at which permeant anions might bind has been based largely on the effects of site-directed mutagenesis within the TMs on Au(CN)2– inhibition of Cl– permeation. While the results of such studies have shown that anion binding is highly sensitive to mutagenesis in several TMs (Ge et al. 2004), three amino acid residues seem particularly important for tight Au(CN)2– binding: K95 in TM1 (Ge et al. 2004), R334 in TM6 (Gong & Linsdell, 2003b) and T338 in TM6 (Gong et al. 2002). Based on the relationship between anion physicochemical properties (in particular the energetic strength of anion interactions with water and other solvents) and binding to CFTR (Smith et al. 1999; Linsdell, 2001), it seems reasonable to assume that these sites also bind Cl– ions as they pass through the channel.
How many Cl– ion binding sites are there in the pore? The answer to this question may have important mechanistic implications. There is a wealth of biophysical evidence that the CFTR pore can accommodate more than one anion at the same time (Tabcharani et al. 1993; Linsdell et al. 1997a; Zhou et al. 2002; Gong & Linsdell, 2003a,c), implying the existence of more than one binding site. Interestingly, the crystal structure of a ClC Cl– channel suggested the existence of three distinct but closely adjacent Cl– ion binding sites inside the pore (Dutzler et al. 2003). Concurrent binding of multiple permeant ions within the pore is thought to be an important aspect of the permeation mechanism in a number of different ion channel types (Dutzler et al. 2003; MacKinnon, 2003; Sather & McCleskey, 2003). The functional importance of multiple anion binding in the CFTR pore is supported by the findings that: (i) movement of one anion inside the pore can be ‘coupled’ to the movement of another anion, suggesting that anions do not move through the pore independently of one another but instead influence each other's movement (Gong & Linsdell, 2003a); (ii) entry of an anion into the pore accelerates the exit of anions that are already bound within the pore (Gong & Linsdell, 2003c); and (iii) mutations that disrupt anion–anion interactions inside the pore result in a decrease in unitary Cl– conductance (Gong & Linsdell, 2004). Based on this evidence, I suggest that the existence of multiple binding sites inside the pore allows CFTR to accommodate multiple Cl– ions simultaneously, and that these Cl– ions then experience mutual repulsive effects, probably of an electrostatic nature, that accelerate their exit from the pore, thus ensuring high overall rates of Cl– transport through the pore.
Anion binding makes the pore susceptible to inhibitors
CFTR-mediated Cl– transport is inhibited by a broad range of substances that bind within the pore and physically occlude Cl– transport. These ‘open channel blockers’ are not only widely used experimentally, but are also of some clinical interest (reviewed by Schultz et al. 1999; Sheppard, 2004). It has been known for some time that many of these substances act preferentially or exclusively from the intracellular side of the membrane, leading to the suggestion that the CFTR pore has a wide inner vestibule that accomodates these large organic anions (Linsdell & Hanrahan, 1996; Sheppard & Robinson, 1997; Hwang & Sheppard, 1999). Recently I showed that mutagenesis of positively charged lysine residue K95 dramatically reduced the apparent affinity of five structurally unrelated open channel blockers: the sulphonylurea glibenclamide, the disulphonic stilbene 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS), the indazole lonidamine, the arylaminobenzoate 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and the conjugated bile salt taurolithocholate-3-sulphate (Linsdell, 2005). Thus the positive charge of K95 that is important for attracting intracellular Cl– ions into the pore (Fig. 2A) also attracts large organic anions into a wide inner pore vestibule (Fig. 2B) where they become lodged and prevent Cl– from passing.
The pore discriminates between anions
Ion channels are defined by their selectivity, the ability to allow certain ions to pass at a high rate while effectively excluding others. CFTR, like most other Cl– channels, does not show particularly stringent selectivity and allows most small anions to permeate to some extent (Linsdell & Hanrahan, 1998a). In fact, the permeation of anions other than Cl– through CFTR appears to be physiologically relevant (Poulsen et al. 1994; Linsdell & Hanrahan, 1998b; Wine, 2001). Loose selectivity in Cl– channels probably reflects a lack of necessity for strong discrimination; unlike cation channels that must discriminate between Na+, K+ and Ca2+ ions with extremely high fidelity, anion channels only have to choose between Cl– and large organic anions that the cell does not usually want to lose. Nevertheless, Cl– channels can tell the difference between different small monovalent anions and, interestingly, they tend to show similar patterns of discrimination: most Cl– channels that have been studied in detail show lyotropic anion selectivity patterns, with weakly hydrated anions (lyotropes) showing a higher permeability than those that bind water molecules more strongly (kosmotropes; e.g. Bormann et al. 1987; Halm & Frizzell, 1992; Kubo & Okada, 1992; Arreola et al. 1995; Verdon et al. 1995; Smith et al. 1999; Qu & Hartzell, 2000; Machaca et al. 2002; Thompson et al. 2002; Qu et al. 2004). This pattern is observed even when Cl– transport is not the primary function of the protein in question (Wadiche & Kavanaugh, 1998).
It is open to question whether a specific mechanism is required to achieve such a low level of selectivity. Indeed, the anion selectivity of CFTR and other Cl– channels can be reproduced by simple mathematical models in which the channel pore is a featureless tunnel with a different effective dielectric constant than the aqueous environment that exists on either side of the membrane (Smith et al. 1999; Liu et al. 2003). Put simply, this model postulates that the inside of the channel (as a whole) reproduces a water-like environment that accomodates permeating anions, but that small differences in the strength of interactions between anions and water and between anions and the channel result in the channel interior being a more or less accomodating place for different anions. This results in differences in the ability of different anions to enter the pore, resulting in the observed permeability ‘selectivity’ patterns. According to this model, there is no need for a ‘selectivity filter’ that provides discrimination; instead, selectivity is a ‘global’ feature of the pore. This model is attractive in its elegant simplicity and because it explains why different Cl– channels with their different structures would end up sharing more or less the same selectivity patterns. However, point mutations within the CFTR channel pore can significantly alter selectivity patterns and, interestingly, selectivity-altering mutations seem to cluster around the centre of the pore, raising the possibility that this part of the pore forms some kind of a ‘selectivity filter’. Thus, mutations of TM6 residues T338 and S341 significantly alter the relative permeability of different anions (Linsdell et al. 1998; McCarty & Zhang, 2001; Gupta & Linsdell, 2003). Even more interestingly, mutagenesis of the nearby F337 – and more specifically, mutations that reduce the side-chain volume at this position – greatly diminish the ability of CFTR to discriminate between different anions, resulting in a channel that no longer shows the familiar lyotropic anion selectivity pattern (Linsdell et al. 2000). The mechanism by which a bulky amino acid side-chain at this position is necessary for lyotropic anion selectivity has not been explained and, as such, the precise role of this phenylalanine residue remains unclear. Mutations outside of TM6 have not been associated with significant changes in anion selectivity (Gupta et al. 2001; McCarty & Zhang, 2001; Ge et al. 2004).
If this region of TM6 (residues F337–5341) contains the main determinants of anion relative permeability, is it appropriate to call this region the ‘selectivity filter’? Can a channel that shows such weak selectivity have a selectivity filter in the traditional sense of the word? There is evidence for permeant anion binding within this region (Linsdell, 2001; Gong et al. 2002; Ge et al. 2004), and it is also generally assumed that this is the narrowest part of the pore (Linsdell et al. 1997b; McCarty & Zhang, 2001; Gong & Linsdell, 2003c). In contrast, an anion binding site observed in the narrowest region of the ClC Cl– channel crystal structure was referred to as the ‘selectivity filter’ without any functional evidence that this region was involved in the determination of anion selectivity (Dutzler et al. 2002). Although it has the advantage of being familiar to most ion channel researchers, the shorthand notation ‘selectivity filter’ does carry implications of a highly localized mechanism of ionic selectivity that remain controversial in CFTR.
Although several anions show a higher permeability than Cl– in CFTR, studies at both the macroscopic (McCarty & Zhang, 2001) and single channel levels (Linsdell, 2001) have shown that Cl– has the highest conductance amongst anions, i.e. it passes through the pore more quickly than any other anion tested. Since the function of CFTR is to allow Cl– to cross the membrane quickly, conductance may be a more pertinent physiological parameter than permeation selectivity.
A minimal permeation mechanism
Our current working model of the CFTR pore is summarized simply in Fig. 2C and D. The pore has a relatively wide inner vestibule and a shorter, narrower extracellular entrance. TMs 1 and 6 make major contributions to the pore walls and interact with permeant ions to determine the functional properties of the channel. Several permeant anion binding sites exist: a central site involving TM6 residues F337 and T338, located at the narrowest part of the pore, is a primary determinant of anion selectivity, and this site is flanked by others formed by the positively charged residues K95 (TM1) and R334 (TM6). These positive charges play an important role in drawing intracellular and extracellular Cl– ions, respectively, into the narrow central pore region; a side-effect of this mechanism is that K95 also draws larger anions into the centre of the pore where they may become lodged and block Cl– permeation, leading to a decrease in channel function. Simultaneous occupancy of anion binding sites results in repulsion between bound Cl– ions, leading to accelerated Cl– exit from the pore.
As it stands, this simple cartoon encompasses much of what we have learned, but much still remains to be added. For example, K95 is thought to be located in the outer half of TM1; what parts of CFTR form the inner part of the pore, between K95 and the intracellular solution, and what are the size, shape and extent of the pore in this extended inner vestibule? Is there symmetry between TMs 1–6 and TMs 7–12, and what is the contribution of the C-terminal TMs to the pore? Where is the ‘gate’ that opens and closes the pore, and how is this gate linked to the intracellular machinery that controls channel activity? How many anion binding sites are there, where are they located, and what is the extent of interactions between Cl– ions bound to these sites? By what mechanism does this arrangement of amino acids and anions result in the observed selectivity between different anions?
Beyond CFTR: lessons learned and lessons to share
The simple model of the CFTR pore shown in Fig. 2C and D – derived entirely from functional work – shows clear parallels to the structural model of the ClC Cl– channel pore, derived from the crystal structure (Dutzler et al. 2002, 2003; Dutzler, 2004). Both feature binding of multiple Cl– ions in a central, narrow pore region, and both suggest that concurrently bound Cl– ions will repel one another out of the pore. Furthermore, in both cases the central Cl– ion binding site is in some way associated with a ‘selectivity filter’.
Should these parallels imply that structurally diverse Cl– channels work in basically the same way? This is an attractive thought, since different Cl– channel types share many things in common: most exhibit lyotropic anion selectivity, open channel block by lyotropic and hydrophobic anions, different manifestations of multi-ion pore behaviour, similar minimal pore diameters, and frustratingly overlapping pharmacology (in terms of the low specificity of most known Cl– channel blockers). A narrow pore flanked by positively charged amino acid side-chains that attract Cl– and also larger organic anions, as shown in Fig. 2, represents a simple structural motif, and it may be that different types of anion transporting proteins have found different ways of constructing such a motif.
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Acknowledgements
The author's participation in the Physiology of Anion Transport meeting was supported by a travel grant from the Canadian Cystic Fibrosis Foundation (CCFF). Work in the author's laboratory is supported by CCFF, the Canadian Institutes of Health Research, and the Nova Scotia Health Research Foundation.
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 M. Fatehi, C. N. St. Aubin, and P. Linsdell On the Origin of Asymmetric Interactions between Permeant Anions and the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore Biophys. J., February 15, 2007; 92(4): 1241 - 1253. [Abstract] [Full Text] [PDF]
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C. N. St. Aubin and P. Linsdell Positive Charges at the Intracellular Mouth of the Pore Regulate Anion Conduction in the CFTR Chloride Channel J. Gen. Physiol., November 1, 2006; 128(5): 535 - 545. [Abstract] [Full Text] [PDF]
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 D. N Sheppard, T.-C. Hwang, and M. A Gray The physiology of anion transport: tales of the bizarre and unexpected Exp Physiol, January 1, 2006; 91(1): 121 - 122. [Full Text] [PDF]
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