Mechanism of chloride permeation in the cystic fibrosis transmembrane by mrg10873


									Exp Physiol 91.1 pp 123–129                                                                                                                  123

                                            Experimental Physiology – Symposium Report

Mechanism of chloride permeation in the cystic fibrosis
transmembrane conductance regulator chloride channel
Paul Linsdell
Department of Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada

                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:

Cystic fibrosis is caused by mutations in the gene that                           intracellular regulatory or R domain. Recently a low-
encodes the cystic fibrosis transmembrane conductance                             resolution crystal structure of CFTR was obtained
regulator (CFTR), a protein that functions as a Cl− channel                      (Rosenberg et al. 2004), which showed membrane-
in the apical membrane of many different epithelial cell                         spanning regions lining a central pore; the pathway
types. While there is currently a paucity of direct structural                   through which Cl− ions cross the membrane. However,
information on CFTR, much has been learned about                                 the identity of the TM regions forming the pore, or
the mechanism by which this fascinating protein allows                           even the number of TMs that line the pore, cannot
Cl− ions to move across the cell membrane. This brief                            be identified in this structure. Nevertheless, homology
review emphasizes the indirect structural information and                        with the structures of other ATP-binding cassette (ABC)
mechanistic insights that have been gained from functional                       proteins (Locher et al. 2002; Chang, 2003; Rosenberg et al.
studies of CFTR.                                                                 2005) suggests that the CFTR pore is lined by multiple α-
                                                                                 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
Molecular determinants of the CFTR channel pore
                                                                                 with the seemingly haphazard arrangement of membrane-
The CFTR molecule is made up of two homologous                                   associated α-helices observed in ClC Cl− channels (Dutzler
repeats, each containing six transmembrane (TM) regions                          et al. 2002).
followed by an intracellular nucleotide binding domain                              Recently we presented a comparison of the functional
(NBD; Fig. 1). These two halves are joined by an                                 roles played by the first six TMs in the N-terminal ‘half’ of

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124                                                             P. Linsdell                                                Exp Physiol 91.1 pp 123–129

CFTR, based on alanine scanning mutagenesis (Ge et al.               et al. 2001; Linsdell, 2005). The apparent interactions of
2004). The results of this study strongly suggested that             these two residues with Cl− ions on opposite sides of the
TMs 1 and 6 were major players in forming the pore and               membrane suggests that their fixed positive charges are
determining its functional properties, with TM5 perhaps              located at the outer and inner entrances to a functionally
playing a lesser role. In contrast, there was no evidence to         important part of the pore (Fig. 2A). The importance
suggest a direct role for TMs 2, 3 or 4.                             of these electrostatic interactions in drawing Cl− ions
   What about the C-terminal TMs? Historically these                 into the pore is emphasized by the fact that removing
have received less experimental attention. It is tempting,           either of these fixed positive charges dramatically reduces
based simply on the primary structure (Fig. 1), to                   the rate at which Cl− ions pass through the channel
suggest a symmetrical arrangement, and indeed TM12,                  (Ge et al. 2004; Gong & Linsdell, 2004). Indeed, this
like its counterpart TM6, has been proposed to play                  may contribute to the mechanism by which mutations
a role in forming the pore (McDonough et al. 1994;                   at R334 cause cystic fibrosis (CF; Gong & Linsdell,
Vankeerberghen et al. 1998; Zhang et al. 2000). However,             2004).
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              Anions bind inside the pore
much more important in determining the permeation                    Biophysical studies dating back to the 1950s suggested
phenotype than their C-terminal partners (Gupta et al.               that ions bind to specific sites within channel pores (see
2001). This is consistent with earlier work using chimeric           Hille, 2001), and when ion channel crystal structures were
human–Xenopus CFTR channels that suggested that the                  finally obtained, one of their most striking features was
main determinants of anion permeation were located in                the existence of multiple, discrete permeant ion binding
the N-terminal region (Price et al. 1996).                           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
Positive charges attract Cl– ions into the pore
                                                                     pores (MacKinnon, 2003; Sather & McCleskey, 2003).
To ensure rapid movement of Cl− ions across the                         While we do not yet have any direct structural evidence
membrane, the inside of the channel pore must be an                  for Cl− binding sites in the CFTR pore, functional
accomodating place for these ions, and recent work has               interactions between anions and the pore have been
emphasized the role played by positively charged amino               extensively studied since Tabcharani et al. (1993) first
acid side-chains in attracting Cl− into the pore. Careful            showed that a foreign anion, SCN− , could compete with
analysis of the effects of site-directed mutagenesis on the          Cl− for passage through the pore. Much has been learned
shape of the current–voltage relationship suggests that              by the use of surrogate anions, such as SCN− , that
a positively charged arginine (R334) in TM6 attracts                 appear to bind inside the pore much more tightly
Cl− ions into the pore from the extracellular solution, while        than Cl− itself. Most useful in this respect have been
another positive charge, on a lysine (K95) in TM1 pulls in           the pseudohalide anions (such as Au(CN)2 − , C(CN)3 − ,
Cl− from the intracellular side of the membrane (Smith               Pt(NO2)4 2− and Fe(CN)6 3− ) that were introduced to the

                                                                                                    Figure 1. Overall topology of CFTR
                                                                                                    CFTR comprises 12 TM regions (organized
                                                                                                    into two groups of six), two intracellular
                                                                                                    NBDs and the intracellular R domain.

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Exp Physiol 91.1 pp 123–129                             Cl− permeation in the CFTR Cl− channel                                              125

anion channel community by David Dawson (Smith et al.                            than one binding site. Interestingly, the crystal structure
1999). Physicochemical reasons why these ions are so                             of a ClC Cl− channel suggested the existence of three
‘sticky’ inside the pore have been recently reviewed (Liu                        distinct but closely adjacent Cl− ion binding sites inside
et al. 2003). But where do they stick? Identification of                          the pore (Dutzler et al. 2003). Concurrent binding of
the sites within the pore at which permeant anions might                         multiple permeant ions within the pore is thought to
bind has been based largely on the effects of site-directed                      be an important aspect of the permeation mechanism in
mutagenesis within the TMs on Au(CN)2 − inhibition of                            a number of different ion channel types (Dutzler et al.
Cl− permeation. While the results of such studies have                           2003; MacKinnon, 2003; Sather & McCleskey, 2003). The
shown that anion binding is highly sensitive to mutagenesis                      functional importance of multiple anion binding in the
in several TMs (Ge et al. 2004), three amino acid residues                       CFTR pore is supported by the findings that: (i) movement
seem particularly important for tight Au(CN)2 − binding:                         of one anion inside the pore can be ‘coupled’ to the
K95 in TM1 (Ge et al. 2004), R334 in TM6 (Gong &                                 movement of another anion, suggesting that anions do not
Linsdell, 2003b) and T338 in TM6 (Gong et al. 2002).                             move through the pore independently of one another but
Based on the relationship between anion physicochemical                          instead influence each other’s movement (Gong & Linsdell,
properties (in particular the energetic strength of anion                        2003a); (ii) entry of an anion into the pore accelerates the
interactions with water and other solvents) and binding                          exit of anions that are already bound within the pore (Gong
to CFTR (Smith et al. 1999; Linsdell, 2001), it seems                            & Linsdell, 2003c); and (iii) mutations that disrupt anion–
reasonable to assume that these sites also bind Cl− ions                         anion interactions inside the pore result in a decrease in
as they pass through the channel.                                                unitary Cl− conductance (Gong & Linsdell, 2004). Based
    How many Cl− ion binding sites are there in the                              on this evidence, I suggest that the existence of multiple
pore? The answer to this question may have important                             binding sites inside the pore allows CFTR to accommodate
mechanistic implications. There is a wealth of biophysical                       multiple Cl− ions simultaneously, and that these Cl− ions
evidence that the CFTR pore can accommodate more                                 then experience mutual repulsive effects, probably of an
than one anion at the same time (Tabcharani et al.                               electrostatic nature, that accelerate their exit from the pore,
1993; Linsdell et al. 1997a; Zhou et al. 2002; Gong                              thus ensuring high overall rates of Cl− transport through
& Linsdell, 2003a,c), implying the existence of more                             the pore.

Figure 2. Simple cartoon models of the
CFTR pore
A and B, positive charges attract anions into
the CFTR pore. A, chloride ions are
attracted into the pore from the
extracellular solution (by R334 in TM6) and
from the intracellular solution (by K95 in
TM1). B, large organic anions are similarly
attracted into a wide inner vestibule of the
pore by K95; should these anions become
lodged within the inner vestibule, they will
physically occlude Cl− permeation and
thereby act as open channel blockers.
C, the central, narrow region of the pore is
lined by TM6 residues F337 and T338; this
narrow region is flanked by the important
positively charged amino acid side-chains of
K95 (TM1) and R334 (TM6). D, these three
sites form Cl− ion binding sites in the pore;
loading of these sites by Cl− ions leads to
mutual electrostatic repulsion and Cl− exit
from the pore, resulting in Cl− permeation
across the membrane.

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Anion binding makes the pore susceptible                             2004). This pattern is observed even when Cl− transport
to inhibitors                                                        is not the primary function of the protein in question
                                                                     (Wadiche & Kavanaugh, 1998).
CFTR-mediated Cl− transport is inhibited by a broad
                                                                         It is open to question whether a specific mechanism is
range of substances that bind within the pore and
                                                                     required to achieve such a low level of selectivity. Indeed,
physically occlude Cl− transport. These ‘open channel
                                                                     the anion selectivity of CFTR and other Cl− channels can
blockers’ are not only widely used experimentally, but
                                                                     be reproduced by simple mathematical models in which
are also of some clinical interest (reviewed by Schultz
                                                                     the channel pore is a featureless tunnel with a different
et al. 1999; Sheppard, 2004). It has been known for some
                                                                     effective dielectric constant than the aqueous environment
time that many of these substances act preferentially or
                                                                     that exists on either side of the membrane (Smith et al.
exclusively from the intracellular side of the membrane,
                                                                     1999; Liu et al. 2003). Put simply, this model postulates that
leading to the suggestion that the CFTR pore has
                                                                     the inside of the channel (as a whole) reproduces a water-
a wide inner vestibule that accomodates these large
                                                                     like environment that accomodates permeating anions,
organic anions (Linsdell & Hanrahan, 1996; Sheppard &
                                                                     but that small differences in the strength of interactions
Robinson, 1997; Hwang & Sheppard, 1999). Recently I
                                                                     between anions and water and between anions and the
showed that mutagenesis of positively charged lysine
                                                                     channel result in the channel interior being a more or less
residue K95 dramatically reduced the apparent affinity
                                                                     accomodating place for different anions. This results in
of five structurally unrelated open channel blockers: the
                                                                     differences in the ability of different anions to enter the
sulphonylurea glibenclamide, the disulphonic stilbene
                                                                     pore, resulting in the observed permeability ‘selectivity’
4,4 -dinitrostilbene-2,2 -disulphonic acid (DNDS), the
                                                                     patterns. According to this model, there is no need for
indazole lonidamine, the arylaminobenzoate 5-nitro-
                                                                     a ‘selectivity filter’ that provides discrimination; instead,
2-(3-phenylpropylamino)benzoic acid (NPPB) and
                                                                     selectivity is a ‘global’ feature of the pore. This model is
the conjugated bile salt taurolithocholate-3-sulphate
                                                                     attractive in its elegant simplicity and because it explains
(Linsdell, 2005). Thus the positive charge of K95 that is
                                                                     why different Cl− channels with their different structures
important for attracting intracellular Cl− ions into the
                                                                     would end up sharing more or less the same selectivity
pore (Fig. 2A) also attracts large organic anions into a
                                                                     patterns. However, point mutations within the CFTR
wide inner pore vestibule (Fig. 2B) where they become
                                                                     channel pore can significantly alter selectivity patterns and,
lodged and prevent Cl− from passing.
                                                                     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
The pore discriminates between anions
                                                                     filter’. Thus, mutations of TM6 residues T338 and S341
Ion channels are defined by their selectivity, the ability to         significantly alter the relative permeability of different
allow certain ions to pass at a high rate while effectively          anions (Linsdell et al. 1998; McCarty & Zhang, 2001; Gupta
excluding others. CFTR, like most other Cl− channels,                & Linsdell, 2003). Even more interestingly, mutagenesis of
does not show particularly stringent selectivity and allows          the nearby F337 – and more specifically, mutations that
most small anions to permeate to some extent (Linsdell               reduce the side-chain volume at this position – greatly
& Hanrahan, 1998a). In fact, the permeation of anions                diminish the ability of CFTR to discriminate between
other than Cl− through CFTR appears to be physiologically            different anions, resulting in a channel that no longer
relevant (Poulsen et al. 1994; Linsdell & Hanrahan, 1998b;           shows the familiar lyotropic anion selectivity pattern
Wine, 2001). Loose selectivity in Cl− channels probably              (Linsdell et al. 2000). The mechanism by which a bulky
reflects a lack of necessity for strong discrimination; unlike        amino acid side-chain at this position is necessary for
cation channels that must discriminate between Na+ , K+              lyotropic anion selectivity has not been explained and,
and Ca2+ ions with extremely high fidelity, anion channels            as such, the precise role of this phenylalanine residue
only have to choose between Cl− and large organic anions             remains unclear. Mutations outside of TM6 have not been
that the cell does not usually want to lose. Nevertheless,           associated with significant changes in anion selectivity
Cl− channels can tell the difference between different small         (Gupta et al. 2001; McCarty & Zhang, 2001; Ge et al. 2004).
monovalent anions and, interestingly, they tend to show                  If this region of TM6 (residues F337–5341) contains
similar patterns of discrimination: most Cl− channels that           the main determinants of anion relative permeability, is
have been studied in detail show lyotropic anion selectivity         it appropriate to call this region the ‘selectivity filter’?
patterns, with weakly hydrated anions (lyotropes) showing            Can a channel that shows such weak selectivity have a
a higher permeability than those that bind water molecules           selectivity filter in the traditional sense of the word? There
more strongly (kosmotropes; e.g. Bormann et al. 1987;                is evidence for permeant anion binding within this region
Halm & Frizzell, 1992; Kubo & Okada, 1992; Arreola et al.            (Linsdell, 2001; Gong et al. 2002; Ge et al. 2004), and it
1995; Verdon et al. 1995; Smith et al. 1999; Qu & Hartzell,          is also generally assumed that this is the narrowest part of
2000; Machaca et al. 2002; Thompson et al. 2002; Qu et al.           the pore (Linsdell et al. 1997b; McCarty & Zhang, 2001;

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Exp Physiol 91.1 pp 123–129                             Cl− permeation in the CFTR Cl− channel                                              127

Gong & Linsdell, 2003c). In contrast, an anion binding                           sites? By what mechanism does this arrangement of amino
site observed in the narrowest region of the ClC Cl−                             acids and anions result in the observed selectivity between
channel crystal structure was referred to as the ‘selectivity                    different anions?
filter’ without any functional evidence that this region
was involved in the determination of anion selectivity                           Beyond CFTR: lessons learned and lessons to share
(Dutzler et al. 2002). Although it has the advantage of being
familiar to most ion channel researchers, the shorthand                          The simple model of the CFTR pore shown in Fig. 2C and
notation ‘selectivity filter’ does carry implications of a                        D – derived entirely from functional work – shows clear
highly localized mechanism of ionic selectivity that remain                      parallels to the structural model of the ClC Cl− channel
controversial in CFTR.                                                           pore, derived from the crystal structure (Dutzler et al. 2002,
   Although several anions show a higher permeability                            2003; Dutzler, 2004). Both feature binding of multiple Cl−
than Cl− in CFTR, studies at both the macroscopic                                ions in a central, narrow pore region, and both suggest that
(McCarty & Zhang, 2001) and single channel levels                                concurrently bound Cl− ions will repel one another out of
(Linsdell, 2001) have shown that Cl− has the highest                             the pore. Furthermore, in both cases the central Cl− ion
conductance amongst anions, i.e. it passes through the                           binding site is in some way associated with a ‘selectivity
pore more quickly than any other anion tested. Since                             filter’.
the function of CFTR is to allow Cl− to cross the                                   Should these parallels imply that structurally diverse
membrane quickly, conductance may be a more pertinent                            Cl− channels work in basically the same way? This is
physiological parameter than permeation selectivity.                             an attractive thought, since different Cl− channel types
                                                                                 share many things in common: most exhibit lyotropic
                                                                                 anion selectivity, open channel block by lyotropic and
A minimal permeation mechanism                                                   hydrophobic anions, different manifestations of multi-
Our current working model of the CFTR pore is                                    ion pore behaviour, similar minimal pore diameters, and
summarized simply in Fig. 2C and D. The pore                                     frustratingly overlapping pharmacology (in terms of the
has a relatively wide inner vestibule and a shorter,                             low specificity of most known Cl− channel blockers). A
narrower extracellular entrance. TMs 1 and 6 make major                          narrow pore flanked by positively charged amino acid side-
contributions to the pore walls and interact with permeant                       chains that attract Cl− and also larger organic anions, as
ions to determine the functional properties of the channel.                      shown in Fig. 2, represents a simple structural motif, and it
Several permeant anion binding sites exist: a central site                       may be that different types of anion transporting proteins
involving TM6 residues F337 and T338, located at the                             have found different ways of constructing such a motif.
narrowest part of the pore, is a primary determinant
of anion selectivity, and this site is flanked by others                          References
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