http://informahealthcare.com/mbc ISSN: 0968-7688 (print), 1464-5203 (electronic) Mol Membr Biol, 2014; 31(1): 1–16 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/09687688.2013.868055

REVIEW

Functional architecture of the CFTR chloride channel Paul Linsdell

Abstract

Keywords

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) family of membrane transport proteins. CFTR is unique among ABC proteins in that it functions not as an active transporter but as an ATP-gated Cl channel. As an ion channel, the function of the CFTR transmembrane channel pore that mediates Cl movement has been studied in great detail. On the other hand, only low resolution structural data is available on the transmembrane parts of the protein. The structure of the channel pore has, however, been modeled on the known structure of active transporter ABC proteins. Currently, significant barriers exist to building a unified view of CFTR pore structure and function. Reconciling functional data on the channel with indirect structural data based on other proteins with very different transport functions and substrates has proven problematic. This review summarizes current structural and functional models of the CFTR Cl channel pore, including a comprehensive review of previous electrophysiological investigations of channel structure and function. In addition, functional data on the threedimensional arrangement of pore-lining helices, as well as contemporary hypotheses concerning conformational changes in the pore that occur during channel opening and closing, are discussed. Important similarities and differences between different models of the pore highlight current gaps in our knowledge of CFTR structure and function. In order to fill these gaps, structural and functional models of the membrane-spanning pore need to become better integrated.

ABC protein, chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR)

Introduction Cystic fibrosis is caused by loss-of-function mutations in a single gene, that encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (Lubamba et al. 2012). CFTR is a member of a large family of membrane transport proteins, the ATP-binding cassette (ABC) transporters (Dean et al. 2001, Rees et al. 2009). All ABC proteins share a common minimal architecture, with two nucleotide-binding domains (NBDs) that dimerize in order to bind and hydrolyze ATP, and two membrane-spanning domains (MSDs) that come together to form a single transmembrane substrate transport pathway (Kos and Ford 2009, Locher 2009, Rees et al. 2009). This common modular structure is used by different ABC proteins to couple ATP hydrolysis to the transmembrane transport of a broad range of different transport substrates. In most ABC proteins the mechanism of transport is active, with the energy associated with ATP hydrolysis being used to power unidirectional movement of substrate across the membrane, potentially against a substrate concentration gradient. In contrast to this conserved transport mechanism, CFTR appears a unique exception within the ABC family, in Correspondence: Dr Paul Linsdell, PhD, Department of Physiology & Biophysics, Dalhousie University, PO Box 15000, Halifax, Nova Scotia B3H 4R2, Canada. Tel: +1 902 494 2265. Fax: +1 902 494 1685. E-mail: [email protected]

History Received 1 October 2013 Revised 14 November 2013 Accepted 18 November 2013 Published online 24 January 2014

that it functions as an ion channel that mediates the passive, electrodiffusional movement of Cl, HCO 3 and other small anions across the cell membrane (McCarty 2000, Liu et al. 2003, Linsdell 2006, Hwang and Kirk 2013). In CFTR, ATP binding and hydrolysis by the cytoplasmic NBD dimer is coupled to opening and closing of a single anion channel pore formed by the MSDs (Muallem and Vergani 2009, Kirk and Wang 2011, Jih and Hwang 2012). Channel activity is regulated by phosphorylation of the cytoplasmic regulatory domain (R domain), a region that is also a unique structural feature of CFTR (Chong et al. 2013, Hwang and Kirk 2013). CFTR is expressed in the apical membrane of many different epithelial cell types, and the symptoms of CF are thought to result directly from a loss of epithelial Cl and HCO 3 permeability (Frizzell and Hanrahan 2013, Lubamba et al. 2012). The ABC protein family encompasses 49 different genes in humans, divided into seven subfamilies (ABCA–ABCG) (Dean et al. 2001). CFTR (ABCC7) is one of 13 members of the ABCC subfamily, most members of which function as ATP-driven exporters of a diverse range of large organic substances. Presumably at some point in evolutionary history, CFTR diverged functionally (Chen and Hwang 2008, Jordan et al. 2008) and structurally (Rishishwar et al. 2012, Hunt et al. 2013, Sebastian et al. 2013) from its ABCC relatives, and its transport function changed from one of active

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Department of Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada

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Mol Membr Biol, 2014; 31(1): 1–16

transport to passive, electrodiffusional ion channel activity. This evolutionary relationship to active transporters has led to CFTR occasionally being referred to, somewhat pejoratively, as a ‘broken pump’ (Gadsby 2009, Mornon et al. 2009, Miller 2010). In fact, although text books tell us that active transporters and ion channels function by very different mechanisms, recent evidence has led to significant blurring of the lines between the two (Miller 2006, DeFelice and Goswami 2007, Chen and Hwang 2008, Gadsby 2009, Miller 2010). As active transporters, ABC proteins are thought to follow an ‘alternating access’ model of transport, in which conformational changes in the MSDs change the transport pathway between ‘inward facing’ and ‘outward facing’ states which allow bound substrate to be alternately exposed to the intracellular and extracellular solution respectively (Kos and Ford 2009, Locher 2009, Rees et al. 2009) (Figure 1A). Indeed, the crystal structures of ABC proteins have identified both inward facing (transmembrane transport pathway apparently accessible only from the inside) and outward facing (transmembrane transport pathway apparently accessible only from the outside) structures of the MSDs (see later section entitled Structural properties of the channel). It is assumed that the conformational change in the MSDs that effectively switches the substrate from one side of the membrane to the other is somehow coupled to ATP hydrolysis at the NBDs (Kos and Ford 2009, Locher 2009, Rees et al. 2009). Of course, such global conformational changes that underlie active transport are not necessary in ion channel mechanisms of transmembrane transport (Gadsby 2009). In some ion channel types, relatively localized (A) Pump (alternating access)

(B) Channel (one gate)

(C) Pump (two gates)

Figure 1. Simple mechanisms of pumps and channels. (A) Alternating access or ‘flip-flop’ model of pump function. A conformational change converts the MSDs between ‘outward facing’ and ‘inward facing’ orientations, alternatively exposing the substrate (red) to one side of the membrane or the other. (B) Single gate model of channel function. A localized conformational change allows the channel to exist in ‘closed’ (transport not allowed) or ‘open’ (transport allowed) states. (C) Two gate model of pump function. Two gates open or occlude the transport pathway. In order for active transport to occur, both gates are never open simultaneously, since this would create a passive, open ion channel-type transport pathway. As a result, it is presumed that a substrate occluded state (centre) must exist. This is a greatly simplified version; in order for

conformational changes lead to the opening and closing of a ‘gate’ within the transmembrane pore (Jiang et al. 2002, Dutzler et al. 2003, Piechotta et al. 2011, Faure et al. 2012, Hattori and Gouaux 2012, McCusker et al. 2012, Auerbach 2013), resulting in opening and closing of the channel (Figure 1B). Active transport mechanisms can also be conceptualized in terms of ‘gates’ that open or close the transport pathway. However for active transport to take place there must be at least two gates, and these gates cannot all be open at the same time (Figure 1C). In this formulism, an open inner gate corresponds conceptually to the ‘inward facing’ conformation, and an open outer gate corresponds to the ‘outward facing’ conformation (Figure 1). In order that the gates never be open simultaneously, both must be closed at some point with the transport substrate occluded between the two gates in the transport pathway. In order to understand the change in transport pathway structure and function that has allowed CFTR to evolve from an active transporter to an ion channel, it has been suggested that one of the ‘gates’ has become atrophied or uncoupled, so that only one functional gate remains coupled to NBD function (Chen and Hwang 2008, Gadsby 2009, Miller 2010). Topics such as the structure of the NBDs and their role in the gating (opening and closing) of the CFTR channel (Muallem and Vergani 2009, Jih and Hwang 2012, Hunt et al. 2013, Hwang and Kirk 2013), structure of the R domain and its role in the regulation of channel activity (Bozoky et al. 2013, Chong et al. 2013, Hunt et al. 2013), folding and misfolding of wild type and mutant CFTR protein (Cheung and Deber 2008, Lukacs and Verkman 2012, Okiyoneda and Lukacs 2012) and the potential pharmacological manipulation of CFTR activity in the treatment of cystic fibrosis (Lubamba et al. 2012, Hanrahan et al. 2013, Rowe and Verkman 2013) and other diseases (Li and Sheppard 2009, Thiagarajah and Verkman 2012) have been reviewed in detail recently. This review addresses the structural and functional properties of the CFTR anion channel pore formed by the MSDs. The structural properties of the MSDs are relatively poorly defined in terms of direct experimental evidence, but several atomic level models of MSD/pore structure have been proposed based on homology with other ABC proteins (Figures 2 and 3). Conversely, since CFTR is unique amongst ABC proteins in its function as an ion channel, its transport function can be and has been studied in far greater detail than other ABC proteins using high resolution electrophysiological techniques. Unfortunately, significant barriers exist in bringing together these different lines of evidence to develop a unified view of channel structure and function. I will review what is known about the structure of the CFTR channel pore, what has been proposed based on homology models (Figure 3), and what has been proposed based on electrophysiology-based structure-function investigations (Figures 4 and 5), before trying to find some common ground by which structural and functional models might become better integrated.

Structural properties of the channel Direct, high resolution structural information on full-length CFTR is lacking. The structures of isolated, cytoplasmic

CFTR chloride channel pore

DOI: 10.3109/09687688.2013.868055

(A) Sav1866 (outward facing)

(B)

P-glycoprotein (inward facing)

(C)

3

(D)

TM287/288 (inward facing)

ABCB10 (inward facing)

MSDs

NBDs

Figure 2. Structures of example ABC proteins. Published structures of Sav1866 (NBDs associated, MSDs outward facing), P-glycoprotein (NBDs dissociated, MSDs inward facing), TM287/288 (NBDs partially associated, MSDs inward facing) and ABCB10 (NBDs associated, MSDs inward

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(A)

(B)

(C)

(A) TM8 TM6

TM12 TM2

Figure 3. Examples of CFTR homology models based on ABC protein templates. (A) Outward facing model, based on Sav1866 template, published by Serohijos et al. (2008); a very similar model was also published by Mornon et al. (2008). (B) Inward facing model based on MsbA template, published by Mornon et al. (2009). (C) ‘Channel-like’ configuration, based on Sav1866 template, published by Dalton et al. (2012). In each case, the N-terminal ‘half’ of CFTR (TMs 1-6, NBD2) is coloured blue, and the C-terminal half (TMs 7-12, NBD2) orange. The R domain in (A) and (B) is coloured gray; the R domain was not included in the model shown in (C). Each panel was visualized with PyMol using coordinates provided in the described publications.

TM9 TM11 TM1

TM7

TM10

TM5 TM3

(B) TM12

TM1

TM3 TM4 TM5

TM2

TM8

TM11 TM10

NBDs have been solved at residue-level resolution, while the cytoplasmic R domain is unstructured (Patrick and Thomas 2012, Bozoky et al. 2013, Chong et al. 2013, Hunt et al. 2013). In contrast, the structure of the membrane-spanning parts of the protein have been observed directly only at low resolution (Rosenberg et al. 2004, 2011, Mio et al. 2008, Zhang et al. 2009, Hunt et al. 2013). The MSDs appear to be in an outward facing conformation in the presumed absence of nucleotide (Rosenberg et al. 2011). In other ion channel types, tremendous new insights into the mechanisms of gating and permeation have been obtained from high resolution crystal structures (Gouaux and MacKinnon 2005, Miller 2006, Hilf and Dutzler 2009, Grigoryan et al. 2011, Catterall 2012, Baconguis et al. 2013, Kumar and Meyer 2013). In the case of ClC proteins, the same crystal structures serve as templates for both Cl channels and Cl/H+ exchangers (Dutzler et al. 2002, 2003, Miller 2006, Jayaram et al. 2008), emphasizing that minor structural changes may separate channels from active transporters. However, these crystal structures were solved initially for bacterial homologs of mammalian ion channels (Doyle et al. 1998, Dutzler et al. 2002, Jiang et al. 2002,

TM4

(C) I1112 (TM11)

TM6 TM9

TM7 K95 (TM1)

I344 (TM6) S1141

Figure 4. Proposed arrangement of TMs in CFTR-MSDs. (A, B) Symmetrical arrangement of TMs in the ‘channel-like’ homology model presented by Dalton et al. (2012), viewed from the side (A) or from the extracellular side of the membrane (B). Symmetrical ‘pairs’ of TMs from the N- and C-terminal parts of the protein are indicated in the same colour, namely green (TMs 1, 7), brown (TMs 2, 8), yellow (TMs 3, 9), gray (TMs 4, 10), blue (TMs 5, 11) and red (TMs 6, 12). (C) Asymmetrical arrangement of pore-lining TMs 1, 6, 11 and 12. The apparent location of the pore lumen is indicated by the indicated side chains of pore-lining residues from approximately the same level in each of these four TMs, namely K95 (TM1), I344 (TM6), I1112 (TM11) and S1141 (TM12) (see also Figure 5).

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Mol Membr Biol, 2014; 31(1): 1–16

(A)

(B) Outer vestibule

+ R334 + K335 T337

R334 K335 F337 T338 S341 I344 V345 M348 A349

Narrow region

T338

K95 + Inner vestibule

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(C)

TM12

TM6

R334 K335 I336 F337 T338 T339 I340

Outside

I1131 I1132

S341

I1139

L346 R347

M1140 S1141 T1142 Inside

M348 A349 V350 T351

L102 L101 L100

L1149 Q1144 W1145

L1120 I1119 S1118 I1117 F1116

Both sides P99 Q98

T1115 V1114 A1113

V97 A96

I1112 K95

T94 V93

Inside

F1111 F1110 I1109

E92 A1146

R352 Q353

Outside

T1122 T1121

G103

N1138 I344 V345

TM11

R104 L1133 T1134 L1135 A1136 M1137

Both sides

F342 C343

TM1

V1147 N1148 S1149

F354 P355

G91 L90 Y89 L88 F87 I86 G85 Y84

Figure 5. Working model of different regions of the open channel pore. (A) Functional cartoon model of the pore, summarizing key features described in the text: A narrow central pore region lined by TM6 residues F337 and T338 (red), a shallow outer vestibule including positively charged TM6 residues R334 and K335 (green), and a deeper, wide inner vestibule including positively charged TM1 residue K95 (blue). (B) Approximate location of pore-lining TM6 side chains proposed to contribute to these three functionally separable pore regions, according to the same colour scheme. The image shows a cross-section through the MSD region of the model presented by Dalton et al. (2012) (see also Figure 4). (C) Pore-lining side chains identified in structurally symmetrical TMs 6 and 12 (left) and structurally asymmetric TMs 1 and 11 (right). Based on SCAM work from the author’s laboratory (El Hiani and Linsdell, 2010, Qian et al. 2011, Wang et al. 2011, 2014), residues that (when mutated to cysteine) are accessible to large cysteinereactive from the outside are coloured green, those accessible from the inside are coloured blue, and those accessible from both sides of the membrane are coloured red, a colour scheme that is deliberately common with that used in (A) and (B). Apparently non-pore-lining side chains are in black. Broadly similar results were obtained by other groups (Alexander et al. 2009, Bai et al. 2010, 2011, Gao et al. 2013), although importantly these other groups did not find that any residues were accessible from both sides of the membrane. Nevertheless, the ‘cut-off’ region that prevents permeation of large cysteine-reactive reagents from one side of the membrane to the other, which most likely corresponds to the narrowest part of the open channel pore, is consistently observed (see text).

Kuo et al. 2003, Hilf and Dutlzer 2008, Payandeh et al. 2011), while there is no bacterial ion channel homolog of CFTR. Those ABC proteins that are somewhat related to CFTR and for which high resolution structures are available include the bacterial multidrug efflux protein Sav1866 (Dawson and Locher 2006, 2007), the bacterial lipid translocase MsbA (Ward et al. 2007), mammalian and Caenorhabditis elegans homologs of the multidrug exporter P-glycoprotein (ABCB1) (Aller et al. 2009, Jin et al. 2012), the bacterial multidrug exporter TM287/288 (Hohl et al. 2012) and mammalian mitochondrial inner membrane protein ABCB10

(Shintre et al. 2013). Initially those proteins crystallized with bound nucleotides and dimerized NBDs showed outward facing conformations of the MSDs (Dawson and Locher 2006, 2007, Ward et al. 2007), whereas those crystallized in the absence of nucleotides showed well-separated NBDs and inward facing MSDs (Ward et al. 2007, Aller et al. 2009, Jin et al. 2012) (Figure 2A, 2B). This is consistent with the idea that ATP binding and NBD dimerization drives the conformational change of the MSDs from inward facing to outward facing (Kos and Ford 2009, Locher 2009, Rees et al. 2009). However, more recently solved structures have somewhat

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DOI: 10.3109/09687688.2013.868055

blurred this picture. First, the heterodimeric bacterial ABC protein TM287/288, which is thought to have an overall NBD arrangement more similar to that of CFTR, was crystallized in an inward-facing conformation with its NBDs bound to nucleotide and only partially separated (Hohl et al. 2012) (Figure 2C). More recently, the mammalian ABC protein ABCB10 was shown to present an inward-facing structure both with and without nucleotide bound to the NBDs (Shintre et al. 2013) (Figure 2D). Atomic homology models have been produced for CFTR, based on the solved outward-facing structure of Sav1866 (Mornon et al. 2008, Serohijos et al. 2008, Alexander et al. 2009, Mornon et al. 2009, Dalton et al. 2012, Norimatsu et al. 2012) (Figure 3A) and the inward-facing structure of MsbA (Mornon et al. 2009) (Figure 3B). Based on the relationship between nucleotide binding/NBD dimerization and MSD configuration in these two templates, it was proposed that the outward-facing structure would represent the ATP-bound, open state of the channel, and the inward-facing structure the nucleotide-free, closed state of the channel (Mornon et al. 2008, Serohijos et al. 2008, Gadsby 2009, Mornon et al. 2009). In spite of their visual clarity and detail, the sophisticated homology modeling procedures used to produce these models, and overall agreement with the observed lowresolution structure of CFTR (Rosenberg et al. 2011), there are some obvious caveats associated with these homology models. These include low functional similarity between CFTR and these templates (Cl channel versus active transporter of large organic substances), the presence of unique protein regions such as the R domain, and low sequence homology. While these problems might be relatively minor for domains such as the NBDs that are already structurally relatively well characterized (and relatively well conserved between different ABC proteins), they are likely to be most significant when modeling MSD structure and function. In fact, the MSDs represent the region of lowest sequence identity between CFTR and those ABC proteins on which its structure has been modeled (10–15%). Furthermore, since CFTR is the only ABC protein that functions as an ion channel, the transmembrane transport pathway formed by the MSDs is presumably a region of strong functional divergence between CFTR and its ABC protein relatives, raising important questions concerning the suitability of active transporter ABC proteins as templates for the ion channel CFTR. As an ion channel, CFTR must exist in an open state with a continuous transport pathway across the entire membrane, whereas in other, active transporter ABC proteins the transport pathway must be closed at one end to prevent passive movement of substrate. However, as described above, work with ClC proteins that are either Cl channels or Cl/ H+ exchangers illustrate that small structural changes may be sufficient to distinguish between ion channel and active transport mechanisms (Miller 2006, Chen and Hwang 2008, Gadsby 2009). Furthermore, CFTR has no high-affinity substrate which binds within the MSDs and whose transport is coupled directly to NBD function. In active transporter ABC proteins, binding of substrate, ATP binding and hydrolysis, and interconversion of inward facing and outward facing arrangements of the MSDs are thought to be intrinsically linked (Kos and Ford 2009, Locher 2009,

CFTR chloride channel pore

5

Rees et al. 2009, Shintre et al. 2013). Indeed, a number of problems or apparent inconsistencies between the structure of CFTR as shown in homology models and the presumed architecture of the open channel pore based on longstanding functional investigation (see Structure-function relationships: Transmembrane regions forming the channel pore) have already been noted (Bai et al. 2010, 2011, Wang et al. 2011, Dalton et al. 2012, Wang and Linsdell 2012a, Hunt et al. 2013). More recently, more ‘channel-like’ configurations of the MSDs have been presented within the overall framework of Sav1866-based models (Dalton et al. 2012, Norimatsu et al. 2012) (Figure 3C) that address some of the starkest discrepancies. Nevertheless, in spite of the problems associated with a simplistic correlation between functional states (channel open, channel closed) and presumed structural conformations (MSDs outward facing, inward facing), the hypothesis that the open state of the channel is structurally ‘outward facing’, and the closed state ‘inward facing’, now appears to be generally accepted (Gadsby 2009, Muallem and Vergani 2009, Miller 2010, Kirk and Wang 2011, Patrick and Thomas 2012, Chong et al. 2013, Hunt et al. 2013).

Functional properties of the channel Because CFTR is an ion channel its functional properties have been studied in great detail and high temporal resolution for over 20 years (Anderson et al. 1991, Berger et al. 1991, Dalemans et al. 1991, Tabcharani et al. 1991). Indeed, functional models of the CFTR pore were proposed long before structural information was available concerning the MSDs of any ABC protein (Dawson et al. 1999, Hwang and Sheppard 1999, Sheppard and Welsh 1999, Akabas 2000, McCarty 2000, Liu et al. 2003, Linsdell 2006) (see section entitled Functionally separable regions of the channel pore). The overall functional properties of the CFTR Cl channel pore have been reviewed in detail previously (Dawson et al. 1999, McCarty 2000, Liu et al. 2003, Linsdell 2006) and will be summarized only briefly here. Under high [Cl] conditions, the channel has a unitary conductance of 8–10 pS and a linear current-voltage relationship. While the channel selects strongly for Cl over small cations such as Na+, selectivity between anions is quite low, with most monovalent anions that have diameters below ˚ being able to pass through the channel to some around 5.3 A extent (Linsdell et al. 1997, Linsdell and Hanrahan 1998). In fact, in addition to Cl itself, other anions including HCO 3 (Bridges 2013), glutathione (Frizzell and Hanrahan 2013), SCN (Frizzell and Hanrahan 2013) and I (Fong 2011) might be physiologically relevant substrates for CFTRmediated transport. Amongst anions that are small enough to pass, the channel shows a so-called ‘lyotropic’ anion permeability sequence, meaning that anions with a relatively low free energy of hydration (lyotropes; those anions that can lose their waters of hydration relatively easily) tend to show a higher permeability (they are more likely to pass through the channel) than anions with a higher free energy of hydration (kosmotropes; those anions that retain their waters of hydration relatively tightly). Chloride itself is mildly lyotropic, while HCO 3 is kosmotropic. Highly lyotropic anions such as SCN and AuðCNÞ 2 show a much higher

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permeability than Cl itself (Linsdell and Hanrahan 1998, Smith et al. 1999, Gong et al. 2002, Liu et al. 2003), while small kosmotropes such as F show very low permeability (Linsdell and Hanrahan 1998). This lyotropic selectivity pattern is consistent with the idea that anions lose at least some of their waters of hydration as they pass through the channel pore (Liu et al. 2003, Linsdell 2006), which is in line with current models of ion permeation in most channel types (Hille 2001, Gouaux and MacKinnon 2005, Armstrong 2007, Andersen 2011). Lyotropic anion selectivity also suggests that the energy required to remove the waters of hydration is a major factor in determining whether an anion will be permeant in the CFTR channel. These features of moderate lyotropic anion selectivity are, in fact, common to almost all Cl channels that have been studied in detail (Liu et al. 2003, Linsdell 2006), perhaps hinting at some similarities in the mechanisms of anion permeation. A combination of moderate lyotropic selectivity and size selectivity is sufficient to allow Cl permeation while limiting the permeation of large organic anions that cells must retain within their cytoplasm, which is probably the most important level of discrimination between anions that a plasma membrane Cl channel is required to carry out. Lyotropic anions with high permeability (preferred substrates for transport) – such as SCN and AuðCNÞ 2 mentioned above – also tend to show relatively low conductance in CFTR (they pass through the channel more slowly) (Smith et al. 1999, Linsdell 2001a, Liu et al. 2003). Furthermore, low concentrations of these ions are able to ‘block’ Cl permeation through the channel, as if the slow passage of these anions physically prevents the passage of the higher conductance Cl ions (Smith et al. 1999, Linsdell 2001a, Gong et al. 2002, Liu et al. 2003). Both of these properties have been interpreted as being consistent with tight binding of lyotropic anions inside the pore, although the existence of specific permeant anion binding sites within the pore is debatable (Liu et al. 2003, Linsdell 2006). Nevertheless, permeant anion binding sites are observed in the crystal structures of other Cl channels (Dutzler et al. 2002, 2003, Hibbs and Gouaux 2011). Furthermore, although both anion selectivity and anion binding appear to follow approximate lyotropic selectivities, there is a lack of evidence supporting a strong functional relationship between binding and permeability (Linsdell 2001b, McCarty and Zhang 2001). These functional parameters of the CFTR channel – its single channel conductance, relative permeability of different anions (both lyotropic selectivity and size selectivity), and evidence for anion binding (such as block of permeation of one anion by a relatively low concentration of another anion) – represent ‘fingerprints’ of pore function that can be assayed by electrophysiological investigation. Where there is a change in one or more of these properties (for example, in a channel mutant), there is a change in pore function. Understanding the ways in which pore function can be altered by changes in the physiocochemical properties of individual amino acid side chains can then provide important insight into the interactions between anions and the channel protein, suggest key structural features of the channel pore that underlie its function, and provide some insight into the mechanism of anion permeation itself.

Mol Membr Biol, 2014; 31(1): 1–16

Structure-function relationships: Transmembrane regions forming the channel pore The two MSDs of CFTR are made up of six transmembrane helices (TMs) each, and appear symmetrical in overall arrangement (Figures 3–5). The structure of the membranespanning parts of CFTR has been observed only at low resolution (Rosenberg et al. 2011, Hunt et al. 2013) and appears to show a central pore surrounded and lined by multiple TMs. Consistent with this, homology models show a central pore lined by a symmetrical arrangement of TMs (Mornon et al. 2008, 2009, Serohijos et al. 2008, Dalton et al. 2012, Norimatsu et al. 2012) (Figure 3), as observed directly in other ABC proteins (Dawson and Locher 2006, 2007, Ward et al. 2007, Aller et al. 2009, Hohl et al. 2012, Jin et al. 2012, Shintre et al. 2013) (Figure 2). Based on the overall domainswapped architecture of their ABC templates, these models show the MSDs organized into two symmetrical ‘wings’, one made up of TMs 1, 2, 9, 10, 11 and 12, and the other containing TMs 3, 4, 5, 6, 7 and 8 (Figure 4A, 4B). It is not known how many of CFTR’s 12 TMs contribute to the lining of the channel pore. Longstanding functional evidence suggests that TM6 contributes to all parts of the pore and plays a conspicuously large role in determining channel functional properties (McCarty 2000, Ge et al. 2004, Linsdell 2006, Hwang and Kirk 2013) (Figure 4). This is supported by substantial substituted cysteine accessibility mutagenesis (SCAM) evidence showing that residues from throughout TM6 are accessible to intracellular and/or extracellular cysteine reactive reagents (Cheung and Akabas 1996, Beck et al. 2008, Alexander et al. 2009, Bai et al. 2010, El Hiani and Linsdell 2010) (see also Functionally separable regions of the channel pore, and Figure 5). Consistent with a symmetrical architecture, SCAM indicates that TM12 also lines the pore (Bai et al. 2011, Qian et al. 2011, Norimatsu et al. 2012) (Figures 4 and 5). However, it has consistently been noted that the functional effects of mutagenesis on pore functional properties is much less in TM12 than in TM6 (Gupta et al. 2001, McCarty and Zhang 2001, Fatehi and Linsdell 2009, Qian et al. 2011, Cui et al. 2012). In fact, comparison of the functional effects of mutagenesis within these two symmetrical TMs led to the first suggestion of an asymmetric channel pore – at least in terms of determination of channel function (Gupta et al. 2001). An important poreforming role for TM1 is suggested by functional (Ge et al. 2004, Linsdell 2005) and SCAM (Akabas et al, 1994, Wang et al. 2011, Gao et al. 2013) experiments. Similarly, TM11 has been ascribed as pore-lining based on both functional and SCAM results (Fatehi and Linsdell 2009, Wang et al. 2014). However, a recent SCAM investigation failed to identify porelining residues in either TM5 or TM7 (Wang et al. 2014). This result is consistent with a lack of strong functional evidence demonstrating a role for either of these TMs in contributing to channel permeation properties, however, it is surprising in terms of a symmetrical arrangement of TMs (Wang et al. 2014) (Figure 4). Based on a strictly symmetrical TM arrangement, one would expect TMs 1 and 7 to play analogous structural roles, as would also be predicted for TMs 5 and 11 (Figure 4). Recent SCAM evidence also suggests that TMs 3 and 9 contribute to the cytoplasmic

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DOI: 10.3109/09687688.2013.868055

aspect of the pore (Norimatsu et al. 2012); however, these TMs were found to make little contribution to the more extracellular parts of the pore. Again, there is a lack of functional evidence regarding the contribution of these TMs to channel properties. Furthermore, cysteine side chains introduced into these two TMs have been shown to react only with externally-applied, channel-permeant cysteine reactive reagents (Norimatsu et al. 2012), whereas TMs 1, 5, 6, 7, 11 and 12 have all been studied using intracellular application of larger cysteine-reactive methanethiosulfonate (MTS) reagents (Bai et al. 2010, El Hiani and Linsdell 2010, Bai et al. 2011, Qian et al. 2011, Wang et al. 2011, Gao et al. 2013, Wang et al. 2014) as well as by reaction with extracellular substances (Akabas et al. 1994, Cheung and Akabas 1996, Beck et al. 2008, Alexander et al. 2009, Fatehi and Linsdell 2009, Wang et al. 2011, Norimatsu et al. 2012, Wang and Linsdell, 2012a, 2012b). In summary, functional evidence suggests that the pore is lined by TMs 1, 6, 11 and 12, with its cytoplasmic part perhaps also lined by TMs 3 and 9. This arrangement of TMs suggests that the channel pore is not, in fact, symmetrical (Figure 4C). As pointed out previously (Wang et al. 2014), whereas the functionally most important TM (TM6) is donated from one ‘wing’ of TMs, other supporting TMs (1, 11, 12) come from the other wing (Figure 4). This apparent functional asymmetry (Figure 4) is one major discrepancy from highly symmetrical MSD models (Figure 3) based on other, structurally symmetrical ABC transporters (Figure 2).

Functionally separable regions of the channel pore Functional characterization of CFTR by electrophysiological investigation – as discussed in detail in the following sections – has led to the development of a working model (Figure 5A). The pore has a relatively narrow central region flanked by wider regions termed the inner and outer vestibules, respectively. The functional effects of mutations on pore properties – together with SCAM experiments that probe the accessibility of individual amino acid side chains from the intracellular and extracellular solutions (Figure 5B, 5C) – can be used to ascribe amino acids lining the CFTR pore to these different pore regions. While the boundaries between these regions of the pore are not discrete and may be somewhat arbitrary, this distinction does allow the pore to be divided into different parts that play somewhat different functional roles. I will therefore separately address the function of each of these regions – referred to here as the narrow pore region, inner vestibule and outer vestibule – independently of what has been inferred from homology models. In particular, the central functional role apparently played by TM6 – and the wealth of experimental detail that follows from structurefunction experiments focused on TM6 – allows this TM segment to be used as a guide (Figure 5B, 5C), to which residues from other TMs can be compared. Narrow pore region As described in the previous section entitled Functional properties of the channel, only anions with diameters less ˚ are able to pass through the CFTR pore at than 5.3 A

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functionally measurable rates. This suggests that, at its narrowest part, the pore must adopt dimensions close to this cut-off. SCAM evidence using channel-impermeant MTS reagents applied to either side of the membrane suggest that the narrowest part of the pore lies somewhere between TM6 residues F337 and S341 (Beck et al. 2008, Alexander et al. 2009, Bai et al. 2010, El Hiani and Linsdell 2010) (Figure 5). This is consistent with functional evidence that pore-lining residues F337, T338 and S341 each make important interactions with permeating Cl ions. Mutagenesis of F337 (Figure 5) has dramatic effects on the conductance (Linsdell 2001b) and selectivity (Linsdell et al. 2000) of the channel. Specifically, mutations that reduce the size of the amino acid side chain (F337A, F337S) led to a dramatic decrease in conductance to 525% of wild type values (Linsdell 2001b). These same two mutations also caused an almost complete loss of lyotropic anion selectivity (Linsdell et al., 2000). These effects were not observed when larger amino acids were substituted (in F337L, F337Y or F337W). In fact, the relationship between the relative permeabilities of different anions and the volume of the amino acid side chain present at position 337 suggested that while lyotropic anions (Br, I, SCN) preferred a large side chain, permeability of kosmotropic F ion was favoured by smaller side chains (Linsdell et al. 2000). While the reasons for the need for a relatively large amino acid side chain at this position for normal channel conductance and lyotropic anion selectivity are not clear, the apparent size-dependent effects of mutations at F337 are consistent with this residue being located in a physically restricted part of the pore where changes in amino acid side chain volume can have a large impact on anion permeation. The dramatic effects of the F337A and F337S mutations on anion selectivity – effects that have not been observed following mutagenesis of any other residue in CFTR – led to the suggestion that F337 might contribute to a highly localized lyotropic ‘selectivity filter’ located within the narrow pore region (Linsdell et al., 2000) (see below). Mutation of T338 (Figure 5) also has major effects on channel properties that are correlated with the size of the amino acid side chain substituted. Thus while replacement with an amino acid with a bulkier side chain (T338I, T338N, T338V) led to a dramatic (490%) decrease in conductance, smaller substituents (T338A, T338S) actually increased conductance by 25% (Linsdell et al. 1998). Several mutations at T338 also strongly affect permeability of different anions (Linsdell et al. 1998, McCarty and Zhang 2001), although unlike F337A and F337S these mutations do not disrupt overall selectivity for lyotropic over kosmotropic anions. T338 mutations were described as altering selectivity between different lyotropic anions without affecting selectivity for lyotropes over kosmotropes (Linsdell et al. 1998, Gong et al. 2002). While the strongly size-dependent effects of mutations on channel conductance are consistent with T338 being located near the narrow part of the pore, the effects of mutations on selectivity are not obviously correlated with the size (or other properties) of the side chain substituted (Linsdell et al. 1998). Furthermore, while some changes in the permeability of organic anions (which are likely limited by steric factors) were observed in T338 mutants, overall

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there was little change in apparent functional pore diameter when either larger or smaller side chains were substituted (Linsdell et al. 1998). Based on the effects of the T338A mutation on the movement of permeant lyotropic AuðCNÞ 2 ions between different sites inside the pore, it was proposed that T338 forms a major ‘barrier’ limiting the movement of permeant anions within the pore (Fatehi et al., 2007). The concept that T338 contributes to a high-resistance region inside the pore is consistent with the increased conductance observed in T338A and T338S (Linsdell et al. 1998, Linsdell 2001b, Fatehi et al. 2007). Somewhat similar effects are seen following mutation of S341 (Figure 5). Strongly decreased conductance has been observed in S341A (McDonough et al. 1994) and S341K (Zhou et al. 2010). Mutants S341A, S341E and S341T significantly affected the permeabilities of different anions without changing the overall anion selectivity sequence (McCarty and Zhang 2001, Gupta and Linsdell 2003). Mutations at this site also affect interactions with intracellular open channel blockers (McDonough et al. 1994, Zhang et al. 2000b, Zhou et al. 2010, Cui et al. 2012), a pore property more usually associated with the inner vestibule (see below). While S341 has been suggested to be located in the narrow region of the pore (McCarty and Zhang 2001), amino acid side chain size-dependence of the effects of S341 mutations have not been reported. In contrast to the major effects of these three important pore-lining residues, mutagenesis of nearby non pore-lining residues (I336, T339, I340) have been shown to have negligible effects on channel conductance or selectivity (Linsdell et al. 1998, McCarty and Zhang 2001, Gupta and Linsdell 2003, Ge et al. 2004). This implies that the effects of mutations at F337, T338 and S341 are likely the result of altered interactions between their pore-exposed side chains and ions inside the narrow pore region (Figure 5). If the functional effects of mutations within the narrow pore region – as exemplified by work on F337, T338 and S341 – are major changes in conductance and relative permeabilities of different anions, then very little functional evidence supports a role for residues from other TMs in this narrow region. Based on the symmetrical locations of TM6 and TM12, it has long been thought that TM12 should also line the narrow pore (McCarty 2000, Norimatsu et al. 2012); however, evidence for an important role for this TM in determining the functional properties of the narrow pore is scant. Functional studies have found little to no effect on either conductance or anion permeability following mutagenesis of I1132 (Fatehi and Linsdell 2009), T1134 (McDonough et al. 1994, McCarty and Zhang 2001, Gupta et al. 2001, Cui et al. 2012), M1137 (Gupta et al. 2001), N1138 (Gupta et al. 2001, Bai et al. 2011) or S1141 (Zhou et al. 2010, Bai et al. 2011, Cui et al. 2012). When compared to the major effects of mutations at F337, T338 and S341, these results suggest that TMs 6 and 12, in spite of their supposed structural symmetry, make highly asymmetric contributions to the functional properties of the narrow pore region (Gupta et al. 2001, Qian et al. 2011). In TM1, mutations at Q98 (Ge et al. 2004, El Hiani and Linsdell 2012, Gao et al. 2013) and P99 (Sheppard et al. 1996, Ge et al. 2004) significantly reduce conductance but have been found to have little effect

Mol Membr Biol, 2014; 31(1): 1–16

on selectivity. Evidence from SCAM experiments also places L102 close to the narrow region (Wang et al. 2011, Wang and Linsdell 2012a); however, functional effects of mutations at this site have not been reported. Based on SCAM results TM11 residues S1115 and T1118 have been proposed to reside within the narrow pore (Wang et al. 2014); however, mutations at these sites also have very minor effects on pore function (Zhang et al. 2000a, Fatehi and Linsdell 2009, Wang et al. 2014). Mutations of T1121 and T1122 have small effects on conductance and selectivity, however, this was proposed to locate these residues in the outer vestibule of the pore rather than the narrow region (Fatehi and Linsdell 2009; see below). Given that the CFTR pore is a three-dimensional structure and the pore must be surrounded by multiple TM a-helices along its entire length (Figure 4), it is unclear why TM6 apparently plays such a dominant role in determining the functional properties of the narrow pore region (Figure 5). One possibility is that the process of anion permeation through this narrow region is a mechanistically very simple one with limited opportunities for functionally meaningful interactions between permeating anions and pore-lining amino acid side chains. It is also possible that, for some reason, permeating anions approach particularly close to side chains originating from TM6, perhaps hinting at some structural asymmetry in this part of the open channel pore. The narrow region is described as the main determinant of anion relative permeability, with evidence that F337 in particular contributes to some kind of lyotropic anion ‘selectivity filter’ in the pore (Figure 5). However, whether a relatively non-selective channel such as CFTR has either the structural basis or the need for a stringent selectivity filter is questionable (Liu et al. 2003, Linsdell 2006, Hwang and Kirk 2013). In fact it has been proposed that the lyotropic permeability sequence and apparent strong lyotropic anion binding in CFTR results not from specific interactions between permeant anions and the pore, but instead from anion stabilization inside a polarizable ‘dielectric tunnel’ (Smith et al. 1999, Liu et al. 2003). In this model, the interior of the pore is reduced to a polarizable space with an effective dielectric constant of about 19. This simple functional model has the advantages that not only does it explain quantitatively the salient features of anion selectivity and binding in CFTR, but also in other Cl channel types and even man-made anion-selective membranes (Smith et al. 1999, Liu et al. 2003). However, it is apparent that even the low-grade selectivity of CFTR occurs via a process that can be disrupted by point mutations, since the relationship between anion permeability and anion free energy of hydration that characterizes wild type CFTR is effectively lost in F337A and F337S (Linsdell et al. 2000). One possible reconciliation of the ‘dielectric tunnel’ and ‘selectivity filter’ models of CFTR anion selectivity is that the effective dielectric constant of the pore is determined only over a very short part of the pore – the narrow region where permeating anions approach close to the pore walls (Figure 5) – such that even point mutations within the pore may be sufficient to significantly affect the effective dielectric constant of the pore. Alternatively, the narrow region may form a lyotropic anion binding site (or sites) that allows selectivity by selective ion binding, as in cationselective ion channel selectivity filters (Hille 2001, Sather and

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McCleskey 2003, Roux 2005, Armstrong 2007). There is some evidence for anion binding within the narrow pore region (Linsdell 2001b, McCarty and Zhang 2001, Liu et al. 2003, Linsdell 2006), although the importance of this binding in the anion selectivity process has been questioned (Linsdell 2001b). In Cl-selective ion channel crystal structures, anion binding is observed at narrow parts of the channel pore that are referred to as the ‘selectivity filter’ (Dutzler et al. 2002, 2003, Miller 2006, Jayaram et al. 2008, Hibbs and Gouaux 2011). As pointed out previously, given the lack of evolutionary pressure to develop or maintain strong selectivity for Cl over other anions, the narrow region of CFTR may be organized to optimize Cl conductance rather than Cl selectivity (Linsdell 2001b, Linsdell 2006).

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Inner vestibule CFTR was originally described as having a wide, deep inner vestibule between the cytoplasm and the narrow pore region (Figure 5A) based on its susceptibility to open channel block by large organic anions acting from the cytoplasmic side of the membrane (McDonough et al. 1994, Linsdell and Hanrahan 1996, Hwang and Sheppard 1999, Linsdell 2006). Modification by membrane-impermeant MTS reagents applied to the cytoplasmic side of the membrane confirm that this inner vestibule is lined by the intracellular ends of TMs 1 (Wang et al. 2011, Gao et al. 2013), 6 (Bai et al. 2010, El Hiani and Linsdell 2010), 11 (Wang et al. 2014) and 12 (Bai et al. 2011, Qian et al. 2011) (Figure 5C). The residue that has received the most experimental attention – and which may play a uniquely important role in the function of the inner vestibule – comes not from TM6, but from TM1. Mutations that remove the positively charged side chain of lysine residue K95 (Figure 5A) cause outward rectification of the current-voltage relationship (suggesting this charge is involved in attraction of cytoplasmic Cl ions to the pore) (Linsdell 2005), dramatically reduce single channel conductance by 85% (Ge et al. 2004, Zhou et al. 2010, El Hiani and Linsdell 2012), and weaken the blocking effects of many different organic anions that act as open channel blockers (Linsdell 2005, Zhou et al. 2010, Li et al. 2011). Mutagenesis of all of the positively charged lysine and arginine residues within the TMs (St Aubin and Linsdell 2006, Zhou et al. 2008) suggests that K95 plays a unique role within the inner vestibule in attracting both permeant and blocking ions from the cytoplasm to the pore. Furthermore, it has been shown that one is the optimal number of positive charges in this region of the pore to maximize channel function – when reduced to zero (when K95 is mutated to a neutral glutamine or serine), single channel conductance is greatly reduced, but when it is increased to two (by introduction of a second lysine at nearby pore lining sites) open channel block is strengthened without any further increase in conductance, leading to an overall decrease in CFTR channel current (Zhou et al. 2010, El Hiani and Linsdell 2012). In addition, the effect of toggling the number of supposedly functionally equivalent positive charges between zero and two by mutagenesis and/or by altering cytoplasmic pH supports the idea that the role of K95 in attracting Cl ions and maximizing channel conductance is

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predominantly electrostatic in nature (Zhou et al. 2010, El Hiani and Linsdell 2012). Mutation of pore-lining residues in the intracellular part of TM6 have not been reported to cause large changes in channel properties. For example, mutations at I344, V345, M348 and A349 (Figure 5B, 5C) have been shown to have small to negligible effects on single channel conductance (Bai et al. 2010, Cui et al. 2012, El Hiani and Linsdell 2012) and selectivity (Linsdell et al. 2000), although chemical modifications that alter the charge at some of these sites can have a small effect on conductance (Bai et al. 2010). Introduction of a positive charge into this region of TM6 (by the mutations I344K, V345K, M348K and A349K) can ‘rescue’ the functional effects of mutations that remove the endogenous charge at K95, leading to wild type-like channel conductance and blocker sensitivity (El Hiani and Linsdell 2012), which suggests that these residues in TMs 1 and 6 are somewhat interchangeable functionally. In particular, I344 and V345 appeared well situated to host this important positive charge, which was taken as evidence that the side chains of these residues may lie close to that of K95 in the inner vestibule (El Hiani and Linsdell 2012) (Figure 5B, 5C). Close to the cytoplasmic end of TM6 are two positively charged arginine residues, R347 and R352 (Figure 5C), the most important roles of which appear to be to interact with negatively charged residues in other TMs. Thus, R347 forms a salt bridge with D924 in TM8 (Cotten and Welsh 1999), and R352 forms a salt bridge with D993 in TM9 (Cui et al. 2008). More recently, it was shown that R347 can also form a salt bridge with D993, suggesting a three-way interaction between R347, D924 and D993 (Cui et al. 2013). It was proposed that the pattern of salt bridge formation between these four charged amino acid side chains (R347, R352, D924, D993) changes during channel gating, resulting in the stabilization of different channel pore conformations (Cui et al. 2013). Given the minor functional effects of mutations of TM6 residues predicted to line the inner vestibule, it is perhaps not surprising that mutations in the cytoplasmic parts of other TMs have also not been reported to dramatically change pore function. In fact, the effects of mutations have predominantly been shown to result in: (i) Changes in interactions with cytoplasmic blockers that are thought to bind within the inner vestibule, and (ii) charge-dependent effects on conductance following neutralization of the endogenous positive charge at K95. Mutations at many positions in TM12 (N1138, M1140, T1142, V1147, N1148, S1149, S1150, I1151, D1152) (Figure 5C) alter block by the sulfonylurea glibenclamide (Gupta and Linsdell 2003, Cui et al. 2012). Furthermore, following the suggestion that S1141 might also interact with open channel blockers (McDonough et al. 1994), it was shown that a positive charge introduced at this site (using a S1141K mutation) could support wild type-like blocker interactions that are lost following neutralization of K95 (Zhou et al. 2010). Given that the K95S/S1141K mutation also restored single channel conductance to close to wild type values, it was concluded that K95 in TM1 and S1141 in TM12 are functionally interchangeable within the inner vestibule (Zhou et al. 2010).

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Outer vestibule

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Positively charged residues also attract extracellular Cl ions to the pore, and these positive charges are thought to be located in a shallower outer vestibule that surrounds the extracellular entrance to the pore (Figure 5A). Mutations or covalent modifications that alter the charge at R334 or K335 (Figure 5A, 5B) cause rectification of the current-voltage relationship (suggesting an electrostatic attraction of extracellular Cl ions) and alter single channel conductance (Smith et al. 2001, Gong and Linsdell 2003, 2004, Zhang et al. 2005, Zhou et al. 2008). These positive charges may also attract small negatively charged blocking ions to the outer mouth of the pore (Zhou et al. 2007, 2008). Interpretation of the effects of mutagenesis at R334 in particular requires caution since it has been shown to affect the properties of distant parts of the pore (Zhou et al. 2007), which was proposed to represent a mutation-induced propagated change in channel conformation. On the extracellular side of R334 and K335, SCAM evidence suggests that I331 and L333 (Figure 5C) may also line the outer vestibule (Beck et al. 2008, Alexander et al. 2009). Other TMs, as well as the short extracellular loops (ECLs) that connect different TMs, also contribute to the outer vestibule. Positive charges from other parts of the protein also act to attract extracellular anions to the outer pore mouth, namely R104 (TM1/ECL1) and R117 (ECL1/TM2) (Zhou et al. 2008). Based on functional effects, it was proposed that these two charged residues were positioned at a more superficial location in the outer vestibule, with R334 and K335 being situated more deeply into the pore from the outside (Zhou et al. 2008). SCAM evidence suggests that uncharged amino acid side chains in TM1/ECL1 (I106, A107, Y109) may also contribute to the outer vestibule (Gao et al. 2013). The effects of charge deposition at various sites in TM11-ECL6-TM12 loop (T1121, T1122, G1127, V1129, I1131, I1132) was used to suggest that this loop also contributes to the outer vestibule, however, the functional effects of mutations at these positions were modest (Fatehi and Linsdell 2009). No functionally important charged amino acid side chains were identified in this loop.

Three-dimensional organization of TMs The preceding section addresses CFTR pore function in a two dimensional, residue-by-residue fashion (Figure 5A, 5C). An attractive advantage of homology models of the pore is that they represent the pore as a three-dimensional entity (Figure 3). The overall arrangement of the TMs in these models (Figures 3 and 4), being based on transporters with homologous overall TM architecture, are presumably correct; however, how accurate are the fine details of three-dimensional structure? This question is particularly important when we begin to think about three-dimensional changes in the structure of the pore during channel gating (see Conformational changes of the pore during channel gating). Experimental investigation is now beginning to provide some evidence for three-dimensional pore architecture that provides important validation and constraints upon the models. This approach is based on cross-linking of cysteine side chains introduced into different parts of the protein. Initially,

Western blotting was used to demonstrate that long cysteine ˚ in length) could cause crosscross-linking molecules (49 A link formation between the intracellular ends of TM6 and TM12 (M348C/T1142C, T351C/T1142C, W356C/W1145C) (Chen et al. 2004) and also between TM6 and TM7 (I340C/ S877C) (Wang et al. 2007). More recently, functional investigation has been used to demonstrate oxidant-induced disulfide bond formation between cysteine side chains introduced into different TMs. In order to form disulfide bonds, it is generally assumed that the b-carbon to b-carbon distance of the two cysteine side chains must be in the range ˚ , and also subject to strict angular constraints of 4–8 A (Careaga and Falke 1992, Bass et al. 2007). Using this approach, cross-link formation has been demonstrated between pore-accessible cysteine side chains in the inner vestibule of the pore, between TM1 and TM12 (K95C/ S1141C) (Zhou et al. 2010) and between TM1 and TM6 (K95C/I344C, Q98C/I344C) (Wang et al. 2011) (Figure 5C). Furthermore, in the outer vestibule/narrow pore region, crosslinks can be formed between accessible side chains in TM6 and TM11 (R344C/T1122C; R344C/G1127C; T338C/ S1118C) (Wang and Linsdell 2012b) (Figure 5C). This kind of functional evidence for physical proximity of individual amino acids from different parts of the protein places important constraints on structural models of the MSDs (Dalton et al. 2012). Similar functional approaches have been used to demonstrate physical proximity between other parts of the CFTR protein, for example between the NBDs and MSDs (Mense et al. 2006, Loo et al. 2008, Serohijos et al. 2008).

Conformational changes of the pore during channel gating Functional data on the ion channel pore, derived from electrophysiological recordings of ions moving through the channel, by definition gives information only on the structure and function of the open state of the channel. What happens to the pore when the channel closes? Understanding the structure of the closed channel is central to understanding the presumed conformational changes that the protein undergoes during ATP-dependent channel gating. Differences in the structure of open and closed CFTR channels have been inferred from functional experiments taking advantage of substituted cysteine chemistry. The types of conformational changes that have been investigated in this way are both twodimensional (changes in the accessibility of individual side chains associated with channel opening and closing) and three-dimensional (changes in the apparent proximity of pairs of cysteine side chains). The first demonstration of state-dependent access to the pore was the finding that R334C, in the outer vestibule (Figure 5), was accessible to extracellular MTS reagents only in the closed state (Zhang et al. 2005). This result is somewhat surprising since the positively charged arginine side chain at this position normally interacts with extracellular anions in open channels (see section entitled Outer vestibule), suggesting it should be accessible to the extracellular solution when the channel is open. Nevertheless, there is also evidence that access from the extracellular solution to other sites in the pore – for example L333, K335 and T338 – is reduced in open

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channels (Beck et al. 2008, Wang and Linsdell 2012a, 2012b). Access of intracellular cysteine reactive reagents to sites in the inner vestibule has also been shown to be altered by channel gating, at cysteines introduced into TM1 (Wang et al. 2011, Wang and Linsdell, 2012a, 2012c, Gao et al. 2013), TM6 (Bai et al. 2010, El Hiani and Linsdell 2010, Wang and Linsdell 2012a, 2012c), TM11 (Wang et al. 2014) and TM12 (Bai et al. 2011, Qian et al. 2011). Both increases and decreases in access to different sites within the inner vestibule are observed in open channels, likely resulting from different kinds of conformational changes occurring during the closed-open transition. Changes in accessibility to the pore during channel gating have been interpreted in different ways in terms of the types of conformational changes that the pore undergoes. Decreased access from the extracellular solution to the pore in the open channel has been proposed to reflect a constriction in the outer vestibule/narrow pore region that occurs during channel opening (Wang and Linsdell 2012a, 2012b, Wang et al. 2014). Increased access from the intracellular solution in the open channel has been used to suggest the existence of a channel ‘gate’ near the boundary between the pore inner vestibule and narrow region (El Hiani and Linsdell 2010, Wang and Linsdell 2012c). This gate is proposed to exist in a region of the pore that dilates and constricts during opening and closing, respectively (Wang and Linsdell 2012c). However, these results are based on work using large MTS reagents, and the relevance of this ‘gate’ to the turning on and off of Cl permeation during channel opening and closing is uncertain. Work from T-C Hwang’s group has emphasized rotational movements of individual TMs during channel gating, suggesting that both TM6 (Bai et al. 2010) and TM12 (Bai et al. 2011) undergo significant (up to 100 ) rotations, exposing and concealing different amino acid side chains within the inner vestibule as the channel opens and closes. At the three-dimensional level, it has also been shown that the apparent proximity of side chains from TM6 and TM11 in the outer vestibule/narrow pore region changes during channel gating. It was proposed that amino acid pairs R334/ T1122 and T338/S1118 are closer together in open channels, with the R334/G1127 pair being closer together when the channel is closed (Wang and Linsdell 2012b) (Figure 5C). These results were interpreted as supporting a relative translational movement of different TMs during channel gating, with TM6 moving toward the cytoplasm (relative to TM11) during channel opening (Wang and Linsdell 2012b) (Figure 5C). While different kinds of conformational changes – constrictional/dilational, rotational, and translational – have been proposed, the importance of these different kinds of rearrangements in the transition between open and closed channels is not known. As described previously (Wang and Linsdell 2012b), these different kinds of conformational rearrangements are not mutually exclusive. Different TMs may move translationally, as well as tilt and rotate, in response to ATP actions at the NBDs, and this may lead to local constrictions or dilations at different points in the permeation pathway. While different kinds of TM movements have been suggested, it has not yet been shown that preventing any particular movement can ‘lock’ the channel pore in either

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the open or the closed state. Such a finding would not only identify essential conformational changes that occur during gating, but could potentially also point the way toward being able to control pore structure and function directly in order to manipulate CFTR function in disease states associated with insufficient or excessive CFTR activity. While most work has focused on the open-closed channel conformational change that is presumed to be driven by ATP binding and hydrolysis, there is also limited functional evidence that the conformation of the channel pore is different in phosphorylated versus non-phosphorylated channels (Fatehi and Linsdell 2008, Wang and Linsdell 2012b). While difficult to study directly, this idea could mean that phosphorylation itself causes some conformational rearrangement of the TMs independent of channel opening and closing. Potential structural/conformational differences between nonphosphorylated, inactive CFTR and phosphorylated, active (but closed) channels is an additional complication when considering different models of CFTR structure.

Comparing and reconciling structural and functional models of the pore As with other ion channels and proteins in general, true understanding of the molecular mechanism of CFTR function must come from studying the relationship between the atomic level structure of the protein and its molecular level ion channel function. While high resolution functional information on CFTR is available, high resolution structural information is not, and so deriving structure-function relationships – and in particular the effects of mutations on channel function and what this tells us about the molecular mechanism of Cl transport – requires a certain degree of guesswork or at least interpretation. At present, in the absence of direct structural information at the atomic level, this interpretation involves use of homology models based on the known structure of related ABC proteins (Figures 3 and 4), however, the relationship between modeled structure and real structure – in other words, the accuracy of the models – is not known. Nevertheless, these structural models can be most useful if they are able to interpret and predict functional information. This is, of course, a two-way street between structure and function. A good model can predict function and so suggest to experimentalists important future experiments. At the same time, functional information constrains and validates models, and informs, confirms, or refutes why observed structural features are important. A strong interrelationship between structural and functional information is particularly important as we begin to try to understand functionally important conformational changes in channel structure, and how dynamic changes in channel structure correlate with changes in channel function. This appears to be a particularly weak point currently in the link between CFTR model structures (Figure 3) and functional ideas about the CFTR channel pore (Figure 5). As an ion channel, we know that CFTR exists in distinct conformations with vastly different functional propeties – referred to as the open and closed states – and that it transitions almost instantaneously between these two functional conformations. As an ABC protein, the structure of the MSDs that make up the pore have been modeled as inward

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facing and outward facing (Figure 3). What is the relationship between (functionally) open and closed channels, and (structurally) inward facing and outward facing MSDs? Based on the relationship between NBD status and MSD orientation, it was originally assumed that the CFTR open channel pore would be outward facing, and the closed channel pore inward facing (Mornon et al. 2008, 2009, Serohijos et al. 2008, Gadsby 2009). Although this NBD:MSD relationship has recently become more blurred (Hohl et al. 2012, Shintre et al. 2013), the idea that the open CFTR channel presents outwardly facing MSDs has become accepted dogma (Miller 2010, Kirk and Wang 2011, Patrick and Thomas 2012, Chong et al. 2013, Hunt et al. 2013). However, functional evidence on the open channel pore is at odds with a strictly outwardly facing structure such as that shown in Figure 3(A). The narrow region of the pore, defined using a number of functional parameters as well as SCAM evidence, is located somewhat towards the outer aspect of the TMs (Figure 5), not at the cytoplasmic side of the membrane as in a strictly outward facing model (Figure 3A). The cytoplasmic ends of the TMs line an inner vestibule (Figure 5) that, rather than being a narrow region, appears to be wide open to the cytoplasm both in open channels and in closed channels (Bai et al. 2010, El Hiani and Linsdell 2010, Bai et al. 2011, Wang et al. 2011, 2014, Wang and Linsdell 2012c). Longstanding functional evidence suggests that the external entrance to the pore is narrower than the internal entrance (Linsdell and Hanrahan 1996, Hwang and Sheppard 1999, Linsdell 2006, Krasilnikov et al. 2011) (Figure 5A). Indeed, reduced access of large extracellular substances to numerous sites in the pore in open channels (Zhang et al. 2005, Beck et al. 2008, Wang and Linsdell 2012a, 2012b) has been used to suggest that the outer mouth of the pore actually constricts when the channel opens (Wang and Linsdell 2012a). In fact, a simple cartoon model of the open channel pore that recapitulates these wellaccepted key functional features (Figure 5A) – while not in itself a piece of structural information or evidence – looks very different from stereotypical (Figure 1A) or actual (Figures 2A and 3A) views of an outwardly facing transporter. Of course, the functional model of the pore does not look like a typical inwardly facing transporter either, not least because as an open ion channel it has to be open at both ends, not closed at one end as required of both outwardly facing and inwardly facing transporter models. Somewhat provocatively, our group went so far as to suggest that, based on functional investigation, the open channel pore appears to exist in an inwardly facing orientation (Wang and Linsdell 2012a). Indeed, it could be argued that the functional model of the open channel (Figure 5A) – cartoon though it is – superficially resembles the inward-facing MsbA-based CFTR model (Figure 3B) more than it does the original, outward-facing Sav1866-based model (Figure 3A). On the other hand, a low resolution electron crystallography structure of CFTR was described as showing a Sav1866-like outwardly facing orientation of MSDs, even though it was assumed to be in an inactive, closed state (Rosenberg et al. 2011). Why are structural (Figure 3) and functional (Figure 5) models of the CFTR pore so apparently at odds? There are many gaps in our understanding of both structural and functional aspects of CFTR. There is an almost total lack of

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information concerning the relationship between NBD function and conformational changes in the MSDs. Even among active transporter ABCs, the idea that dimerized, nucleotidebound NBDs are associated with outwardly facing MSDs, and separated, nucleotide-free NBDs are associated with inwardly facing MSDs is now coming into question (Hohl et al. 2012, Shintre et al. 2013). If CFTR is a ‘broken pump’, then the relationship between NBD status and MSD orientation may be even less concrete. In CFTR, even during repeated rounds of channel opening and closing, the NBDs are thought not to completely separate and dissociate, and ATP is bound to the NBDs both in open and in closed channels (Muallem and Vergani 2009, Jih and Hwang 2012). At the same time, we do not understand the conformational changes inside the MSDs that are important for the transition between open and closed states. The location of the presumed gate that opens and closes the channel is not known. Because CFTR is an ion channel with a single functional gate controlled by ATP action at the NBDs, it is possible that relatively small, localized conformational change within the MSDs is sufficient to open and close the channel. It could be, therefore, that the ‘broken pump’ CFTR does not have to undergo a dramatic change between inward facing to outward facing MSDs during each open-closed transition. Alternatively, it could be that the MSDs do alternate between quite different conformations that resemble the inward facing and outward facing conformations of other, active transporter ABC proteins, but that the functionally important change in pore structure that opens and closes the channel is highly localized, with most of the larger-scale movements being almost inconsequential in terms of overall pore function. Overall, there is a lack of understanding concerning the degree of structural and functional homology or divergence between CFTR and its active transporter ABC relatives, most of which transport organic substrates that are very much larger than Cl ions. Recent work on other families of transport proteins, in particular ClC proteins that function as either Cl channels or as Cl/H+ exchangers, has emphasized the functional similarities between ion channels and active transporters (Miller 2006, Chen and Hwang 2008, Gadsby 2009). In structural terms, ClC proteins that function as channels or secondary active transporters appear indistinguishable (Dutzler et al. 2002, 2003, Miller 2006, Jayaram et al. 2008), emphasizing that minor structural changes can allow a protein to cross the function divide between channel and active transporter. However, the substrate selectivity of these proteins is very similar (Cl permeation versus Cl/H+ exchange), and the rate of transport by some ClC Cl/H+ exchangers is conspicuously high for an active transporter (42000 s1) (Walden et al. 2007, Jayaram et al. 2008, Picollo et al. 2009). On the other hand, CFTR’s closest relatives in the ABCC subfamily are active transporters of large organic compounds in which transport is coupled to ATP hydrolysis and likely occurs at a rate of 51 s1 (Deeley et al. 2006). Therefore, our lack of understanding concerning the functional relationship between CFTR and structurally related ABC proteins should urge caution in terms of making direct parallels in terms of either structure or function. Functional models of the CFTR pore, such as that shown in Figure 5(A), relate to the channel open state; much less is

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known about the closed state, which is invisible to most forms of electrophysiological investigation. Nevertheless, functional work is now beginning to provide some information on changes in channel pore conformation that occur during channel opening and closing. At the same time, homologybased models can be used to describe conformational changes in the protein using approaches such as molecular dynamics simulation (Norimatsu et al. 2012). Functional data has suggested that different kinds of conformational rearrangements may occur during channel opening and closing (see Conformational changes of the pore during channel gating). Future modeling and molecular dynamics efforts will need to incorporate such functional information, and not rely too exclusively on what has been learned from the structure of other ABC proteins, in order to accurately reproduce these important conformational changes.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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conductance regulator chloride channel pore. J Membr Biol 216(2–3): 129–142. Zhou J-J, Fatehi M, Linsdell P. 2008. Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore. Pflugers Arch 457(2):351–360. Zhou J-J, Li M-S, Qi J, Linsdell P. 2010. Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore. J Gen Physiol 135(3):229–245.

Functional architecture of the CFTR chloride channel.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) f...
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