CHAPTER 29 – THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7)

CHAPTER 29 – THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) CHAPTER 29 – THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) CHAPTER 29 – THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) CHAPTER 29 – THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) CHAPTER 29 – THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) CHAPTER 29 – THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7)

589 THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) JOHN W HANRAHAN, MARTINA GENTZSCH AND JOHN R RIORDAN CFTR MUTATION CAUSES CYSTIC FIBROSIS The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene mutated in patients with cystic fibrosis (CF), an autosomal recessive disease of relatively high frequency in the Caucasian population The large number of families with CF enabled linkage analysis, and ultimately identification of the gene by positional cloning Although ⌬F508, the first disease-associated mutation discovered, is present on at least one allele in approximately 90% of patients, more than a thousand different mutations in the CFTR gene have been detected worldwide (http://www.genet.sickkids.on.ca/cftr/) The absence or dysfunction of CFTR results in aberrant ion and liquid homeostasis at epithelial surfaces of the respiratory, intestinal and reproductive tracts as well as other secretory and reabsorptive epithelia CFTR is a channel that allows anions to diffuse through the membrane in either direction (absorptive or secretory) depending on the electrochemical gradients Since chloride is the predominant inorganic anion in vivo, the CFTR pore conducts mainly ClϪ ions under physiological conditions, although significant bicarbonate transport may also occur in some tissues (see below) ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 29 CHAPTER The principal example of ClϪ absorption occurs in the sweat gland, where failure to reabsorb NaCl results in the ‘salty sweat’ that is diagnostic of the disease More serious consequences, however, ensue in the gastrointestinal tract, where exocrine pancreatic function is lost owing to defective anion secretion by the small ducts, and reduced cAMP-stimulated chloride and H2O secretion by intestinal crypts leads to mucus accumulation and severe malabsorption Thus ClϪ ions move out of the cell through apical CFTR channels, which are the rate-limiting step during secretion (Figure 29.1) Indeed, the crucial role that the CFTR chloride channel plays in fluid secretion in the intestine may be at the basis of the so-called ‘heterozygote advantage’, which could account for the relatively high mutant gene frequency despite the fact that the mutations are generally not propagated by homozygotes Experiments with CFTR knockout mice provide some support for this theory (Cuthbert et al., 1995; Gabriel et al., 1994) According to this hypothesis heterozygotes may suffer less potentially fatal intestinal fluid loss and have better survival during toxic bacterial infections Of greater pathological significance to homozygous patients is the lack of adequate hydration of the airways of the lung, where macromolecular secretions also accumulate owing to inefficient muco-ciliary clearance Recurrent and persistent colonization by opportunistic microorganisms exacerbates inflammatory responses, leading to fibrosis and severely impaired airway function Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 590 ABC PROTEINS: FROM BACTERIA TO MAN Figure 29.1 Role of CFTR in transepithelial chloride secretion Cross-section of the epithelium, with basolateral side shown on the left and apical side on the right Monovalent ions Naϩ, Kϩ and 2ClϪ enter the cells from the blood side through the sodium, potassium, chloride cotransporter (NKCC) Naϩ is actively pumped out by Naϩ/Kϩ ATPase at the basolateral membrane, and Kϩ also leaves via a basolateral potassium channel ClϪ is raised above electrochemical equilibrium and flows passively to the lumenal side through the CFTR chloride channel Sodium ions follow paracellularly to maintain charge balance CFTR IS AN ION CHANNEL In addition to being at the basis of a common monogenic disease the other prominent feature of CFTR as an ABC protein is its function as an ion channel On the basis of present knowledge, this property makes it unique among ABC proteins and initially caused some consternation because it apparently did not fit perfectly into the ABC ‘transporter’ mold (Higgins, 1992) However, since these initial discussions, compelling evidence of its anion channel activity has accumulated (see the section on CFTR as an epithelial chloride channel, below) Although neither the three-dimensional (3-D) structure nor the mechanisms of permeation (ion flow through the open channel pore) and gating (spontaneous transitions of the channel pore between open and closed states) are known as yet, some of the primary structural features that contribute to its regulated channel activity and distinguish it from related members of the ABCC subfamily are apparent These features are very highly conserved in other mammals, amphibians, teleosts and cartilaginous fish, which are the most primitive organisms where CFTR homologues have been detected and where CFTR plays a central role in salt secretion (Figure 29.1) From this perspective, adaptation of the ABC structural architecture to the role of a nucleotide-regulated ion channel appears relatively recent when compared with other ABC proteins in much more ancient organisms (Saurin et al., 1999) This adaptation to achieve a highly specific function may have been quite unique as there is a single CFTR gene in all organisms where it has been examined, except in salmon, where it may have duplicated as part of a whole genome duplication (Chen et al., 2001) Moreover, there is a relative paucity of isoforms generated by alternate gene splicing or other mechanisms (Morales et al., 1996) This contrasts with the other ABCC family member involved in ion conduction, the sulfonylurea receptor component of KATP channels (see Chapter 27), for which there are at least two separate genes (Inagaki et al., 1996) It is important to place the findings from directly studying CFTR, once the gene had been cloned and expressed, into the context of the electrophysiological changes described much earlier in CF epithelia (Knowles et al., 1983; Quinton, 1983) Although there was increased sodium absorption in the airway epithelium of CF patients compared to normals (Knowles et al., 1983), Quinton found reduced chloride conductance in CF sweat ducts relative to the normal, and proposed that it was the primary electrophysiological alteration in CF (Quinton, 1983) This seminal finding was pursued, in the ensuing decade prior to the discovery of the CFTR gene, in assays of chloride channel activity in normal and CF epithelial cells, primarily employing the patch clamp technique (see below) A chloride channel activated by cyclic AMP was found to be absent from CF epithelia (Li et al., 1988; Schoumacher et al., 1987) This channel had properties different from those eventually identified as those of the CFTR chloride channel (Berger et al., 1991; Kartner et al., 1991) and has still not been identified at the molecular level However, this channel apparently requires a functional CFTR for its activation (Gabriel et al., 1993) In addition to CFTR’s primary, inherent chloride channel function, it has been reported to regulate the activity of THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) other channels and transporters This fascinating aspect of CFTR research has been reviewed elsewhere (Schwiebert et al., 1999) and will be dealt with only briefly in this chapter together with a consideration of other proteins interacting with CFTR Whether CFTR might also conduct other, as yet unidentified substrates will not be answered here as the experimental evidence is not sufficient to confirm or exclude this possibility Other important issues concerning the role of CFTR in human health and disease that will not be discussed include the contributions of mutations in the CFTR gene to clinical conditions other than cystic fibrosis (Noone and Knowles, 2001) These so-called ‘CFTR-opathies’ involve compromised functions of the major organs in which CFTR function is important, e.g pancreatitis (Sharer et al., 1998), disseminated bronchiectasis (Pignatti et al., 1996), and congenital bilateral absence of the vas deferens (Osborne et al., 1993) This chapter is not intended to provide a comprehensive review and evaluation of all knowledge gained about CFTR This has been done relatively recently (Supplement to Physiological Reviews 79 (1), 1999) Rather we consider it from the perspective of the other ABC proteins that are the subject of this book, emphasizing its common and distinguishing features To this we focus on what is known of its structure, its properties as a chloride channel, its phosphorylation and dephosphorylation, which control channel activity, its binding and hydrolysis of ATP and the consequences of these events for channel gating Finally, because the wild-type CFTR polypeptide itself matures inefficiently during its biosynthesis and because the most common disease-causing mutant form succumbs to degradation by the quality-control system of the endoplasmic reticulum, we discuss what is known of the features of CTFR that may contribute to this behavior CFTR STRUCTURE PRIMARY STRUCTURE The principal features of the CFTR protein sequence that distinguish it from other ABCC family members were recognized from the cDNA sequence (Riordan et al., 1989) The most striking difference from the known eukaryotic ABC transporters such as P-glycoprotein (Pgp) Figure 29.2 Cartoon of CFTR glycoprotein indicating its major distinguishing features: (1) the presence of the central highly charged R-domain, phosphorylation of which is necessary for nucleotide-regulated chloride channel activity, (2) the presence of charged amino acids within predicted membrane-spanning segments, several of which influence ion permeation is the presence of an extended hydrophilic region following nucleotide-binding domain (NBD1) This contains many charged residues and an extraordinarily large number of precise dibasic and monobasic consensus sequences for phosphorylation by protein kinase A (PKA) (Figure 29.2; Riordan et al., 1989) Phosphorylation and dephosphorylation of this region, named the R-domain, exerts major control over chloride channel activity as is discussed in detail later in the chapter The second apparent distinguishing feature at the time of CFTR discovery was the presence of a significantly higher number of amino acids with charged side-chains within putative transmembrane spanning (TMS) sequences, compared with other ABC proteins known at that time This feature is clearly not necessarily diagnostic of ion channels and there are now other ABC proteins known that have charged residues within the membrane spans (see Chapter 7) Nevertheless, several of these residues within TMS sequences of CFTR have been found to influence the properties of the ion pore, as described in the section on the CFTR channel pore, below It was of course the homology of the two NBD sequences with those of other known ABC proteins that placed CFTR in this family Each of the features of the NBDs that are described in length elsewhere in this book are present in CFTR, with most similarity to other members of the ABCC subfamily As was first pointed out for MRP1, the NBD1 of this protein, as well as 591 592 ABC PROTEINS: FROM BACTERIA TO MAN that of CFTR, has a deletion of approximately 13 residues between the Walker A and B motifs compared with NBD2 and the NBDs of many other ABC proteins (Deeley and Cole, 1997) Another notable feature of the CFTR NBD1 sequence is the substitution of the glutamic acid residue that follows the Walker B aspartic acid by a serine (Figure 29.3) This may be significant since this glutamate serves as a catalytic base in the hydrolysis of ATP by other NBDs, where it is present, and it is found in NBD2 of CFTR The LSGGQ signature motif is strictly conserved in NBD1 but is quite degenerate in NBD2 The significance of other short sequence motifs within the NBDs is being increasingly appreciated from alignments with those NBDs of other ABC proteins for which 3-D structures have been determined Some of these motifs are structural elements that contribute to the stability of the domains and of the protein as a whole Four-residue hydrophobic ‘patches’ near the C-termini of NBDs of CFTR are essential for the maturation and stability of the protein (Gentzsch and Riordan, 2001) Other predictive suggestions have been made about conformation changes that may distinguish ATP- and ADP-bound forms of CFTR NBDs by alignment with ABC proteins where 3-D structures have been determined with either nucleotide bound (Berger et al., 2002) In addition to the R-domain and NBDs, which make up more than half of the protein mass, the N- and C-terminal tails and four cytoplasmic loops between predicted membrane spans also present on the cytoplasmic side of the plasma membrane Sequences within the N-terminal tail have been found to influence channel gating and interactions with other proteins (Naren et al., 1998) An amphipathic helix with acidic amino acids on one face between residues 46 and 60 influences channel gating (Naren et al., 1999), whereas another short sequence nearer the N-terminus apparently mediates interaction with an annexin (Nelson et al., 2001) The acidic helix also binds SNARE proteins and hence is suggestive of a role which provides a relationship between vesicular trafficking and CFTR channel gating (Naren et al., 1997) The C-terminal extension of CFTR beyond the end of NBD2, beginning at approximately residue 1424, is Figure 29.3 Patterns of similarity between NBD1 and NBD2 of CFTR and other ABC-ATPases Gapped-BLAST alignment of NBD1 (left) and NBD2 (right) with HisP, MalK, LolD (MJ0796) and LivG (MJ1267) using the program Cn3D 3.0 (National Center for Biotechnology) Similarities are shown on the crystal structure of LivG (MMDB 16953), catalytic domain to the left, helical domain to the right of each NBD Red represents identical residues, blue represents residues that are aligned and gray corresponds to sequences that not align The Mg2ϩ ion is shown as a gray sphere In the ATP-binding protein LivG, residue E179 following Walker B appears to function as a catalytic base activating the hydrolytic water for the attack on the ␥-phosphate of ATP (Karpowich et al., 2001) This glutamate is conserved in NBD2, HisP, MalK, LolD and LivG but is replaced by a serine in NBD1 of CFTR (red in the alignment with NBD2, but blue in the alignment with NBD1) The transport signature motif LSGGQ (residues 154–158 in LivG) is identical in all NBDs but not conserved in NBD2 of CFTR In dimers derived from the ABC ATPase Rad50, the LSGGQ sequence contacts the nucleotide bound to the other subunit If NBD1 and NBD2 of CFTR form a Rad50-like dimer, ATP bound to NBD1 would be contacted by an imprecise signature sequence of NBD2 THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) apparently not essential functionally but contains several motifs involved in the trafficking and localization of the molecule within the cell The best-characterized motif among these is the class I PDZ-domain binding sequence comprising the final three residues of the protein (Short et al., 1998) Interactions with several different PDZ-domain-containing proteins play roles in the apical localization in epithelial cells (Milewski et al., 2001), trafficking through the Golgi apparatus (Cheng et al., 2002) and channel regulation (Raghuram et al., 2001; Wang et al., 2000), possibly by localization in a regulatory complex in proximity to protein kinase A (Huang et al., 2001) The PDZ-binding domain is also involved in tethering together two CFTR polypeptides to modulate channel activity (Raghuram et al., 2001; Wang et al., 2000) Interestingly MRP2 (ABCC2), which also has a PDZ-binding C-terminus, also localizes to the apical membrane of polar cells whereas MRP1, which does not, resides in the basolateral membrane At the opposite N-terminal end of the C-terminal extension is tyrosine 1424, which is involved in endocytosis of CFTR through interaction with a subunit of the clathrin adaptor protein complex, AP-2 (Weixel and Bradbury, 2001) A dileucine motif at residues 1430 and 1431 may also be involved in internalization The C-terminal extension has additional motifs that are less well defined, including a site for phosphorylation by AMP activated protein kinase (Hallows et al., 2000) and an acidic cluster just upstream of the PDZ-binding terminus Mutagenesis of residues in the four cytoplasmic loops (CLs) between membrane-spanning helices generally has a greater effect on protein maturation than channel function (Seibert et al., 1996a, 1996b, 1997) although they are believed to mediate the influence of conformation changes in the NBDs affecting the activity of the ion pore Another set of short sequence motifs is composed of RXR tripeptides in the N-tail, NBD1 and the R-domain (Chang et al., 1999) These influence the fate of ⌬F508 CFTR, perhaps by directly contributing to, or signaling, misfolding and mislocalization The six extracytoplasmic loops (ELs) are much shorter than those on the cytoplasmic side, indicating that very little of the protein is exposed at the cell surface Disease-associated single residue substitutions in the ELs result primarily in decreases in the channel open state (Hämmerle et al., 2001) The longest of the ELs are the first in each half of the molecule, with that in the second half containing the two asparagine residues (894 and 900) that are the sites of N-glycosidic linkage of the two oligosaccharide chains on CFTR (Riordan et al., 1989) These carbohydrate chains are not essential to the function of the molecule SECONDARY STRUCTURE There has been very little experimental determination of secondary structure elements in CFTR, none on the whole purified protein The 12 membrane-spanning sequences are assumed to be ␣-helical but this is not confirmed in the intact molecule Reasonable predictions can be made about the segments of the NBDs that align well with those of other NBDs whose 3-D structures have been determined, and some of these have been discussed (Berger and Welsh, 2000; Thomas and Hunt, 2001) As one example, the four-residue hydrophobic ‘patch’ near the C-terminal end of NBDs of CFTR and other ABC proteins appear to be within the penultimate ␤-strand of the domains (Gentzsch et al., 2002) The ␣-helical structure of the acidic segment of the N-tail that modulates channel gating has been confirmed by nuclear magnetic resonance (NMR) spectroscopy of the corresponding synthetic peptide As a means of testing the hypothesis that phosphorylation by PKA of the R-domain may cause a conformational change, circular dichroism (CD) spectra of an isolated recombinant R-domain were recorded before and after phosphorylation (Dulhanty and Riordan, 1994) Calculation of the proportion of different secondary structure elements by deconvolution methods indicated that the overall domain is relatively unstructured but that phosphorylation causes a decrease in the relatively small ␣-helical content More recent considerations emphasized the lack of R-domain secondary structure (Ostedgaard et al., 2001) The content of ␣ and ␤ secondary structure in an NBD1–R-domain fusion (Ko et al., 1993) was considerably less than that of NBD1 alone (Neville et al., 1998) The relatively unordered structure of the R-domain is consistent with the fact that despite its essential functional role, the sequence of the R-domain is less conserved among different species than any other part of the molecule Only the consensus phosphorylation sites are conserved Whether the functionally relevant consequences of phosphorylation involve changes in secondary or tertiary structure is unknown 593 594 ABC PROTEINS: FROM BACTERIA TO MAN TERTIARY STRUCTURE As we have no 3-D structure information on CFTR, the only clues to its tertiary structure are provided by membrane topology, for which there is some experimental evidence (Chang et al., 1994) The 2-D segmentation discussed in the above section is probably correct; however, spatial relationships in the third dimension are all unknown The relationship between the 3-D structure and our current 2-D sketches are likely to be as dissimilar as in the case of the ClC chloride channel, for which a structure at atomic resolution was recently obtained (Dutzler et al., 2002) A 3-D image of the protein even at low resolution will be essential before great insight into the mechanism of action can be gained QUATERNARY STRUCTURE While the CFTR polypeptide alone can generate a PKA- and ATP-regulated chloride channel (Bear et al., 1992), it is not known with certainty how many of these polypeptides form the channel This uncertainty applies to most ABC proteins with the exception of SUR1, a monomer of which associates with each of the four subunits of the tetrameric Kir6.2 potassium channel to produce an octameric KATP (AguilarBryan et al., 1998; Chapter 22) A second ABCC protein, MRP1, appears as a dimer in 2-D crystalline arrays (Rosenberg et al., 2001), but there is no independent evidence that the conjugated organic anion exporter functions as a dimer In contrast, Pgp, which has been perhaps the most extensively studied ABC protein from the point of view of its oligomeric structure, appeared monomeric in the same type of 2-D crystal analysis by electron diffraction (Rosenberg et al., 1997) However, the Pgp particle diameters of 10–12 nm (Rosenberg et al., 1997), similar to estimates made earlier by freeze-fracture electron microscopy of membranes containing Pgp (Arsenault et al., 1988), are also quite similar to values obtained more recently (Eskandari et al., 1998) for CFTR in Xenopus oocyte membranes (ϳ9 nm) Despite these comparable dimensions of a monomeric Pgp and the CFTR particles, the authors of the latter study concluded that CFTR is dimeric This interpretation agreed with that from a study of concatemerized constructs containing one wild-type CFTR sequence and a variant with aberrant regulatory properties (Zerhusen et al., 1999) Detection of channels with intermediate regulatory behavior was believed to reflect the contribution of two CFTR polypeptides to a single channel Using a similar approach, in which variants with very different ionic conductances were coexpressed in equal amounts, no channels of intermediate conductance were formed (Chen et al., 2002) Although a portion of purified CFTR molecules chromatographed with a size corresponding to dimers, those in the size range of monomers yielded CFTR chloride channel activity (Ramjeesingh et al., 2001) Differentially epitopetagged CFTR species when coexpressed failed to be co-immunoprecipitated (Chen et al., 2002) or appreciably crosslinked to form dimers by chemical crosslinkers (Chen and Riordan, 2002) CFTR solubilized with different detergents from membranes of cells either heterologously or endogenously expressing the protein migrated as a monomer on velocity gradient centrifugation in sucrose gradients (Chen et al., 2002) Thus, at this stage definitive evidence of the stable quaternary structure of CFTR is not available However, the influence of bivalent PDZdomain proteins on CFTR channel activity has raised the possibility that two CFTR molecules might be tethered together at least transiently to yield channels of increased open probability compared to untethered individual molecules (Raghuram et al., 2001; Wang et al., 2000) It is not clear if this tethering promotes a more extensive interaction interface between two polypeptides and if so how this might relate to the formation or regulation of the ion pore This raises the issue of CFTR quaternary structure from the perspective of other ion channels, nearly all of which are oligomeric or assemblies of repeated units within a single polypeptide (Catterall, 2000) Cation channel pores are formed at interfaces between subunits It is unknown if the CFTR pore is formed employing this architectural strategy or if it is assembled by elements within individual subunits ClC chloride channels are dimeric but each monomer contains a functional pore that is removed from the dimer interface (Dutzler et al., 2002) One report has suggested that there may even be separate pores in each half of a single CFTR polypeptide (Yue et al., 2000) Resolution of the quaternary structure of CFTR, which is unique among both ABC proteins and ion channels, will be essential to achieve an understanding of its structure and function THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) CFTR IS AN EPITHELIAL CHLORIDE CHANNEL CHLORIDE CONDUCTANCE DEFECT IN CYSTIC FIBROSIS Electrophysiological experiments on isolated sweat ducts first revealed a chloride conductance defect in cystic fibrosis (Quinton, 1983) In normal ducts, transepithelial Naϩ reabsorption is active and involves diffusion through apical channels and extrusion by basolateral Naϩ/Kϩ ATPase pumps Transepithelial ClϪ reabsorption maintains electroneutrality and is passive, mediated by a parallel conductance The electrical effects of imposing transepithelial salt gradients across the duct suggested that ClϪ conductance is abnormally low in CF This was confirmed when normal ducts were found to behave like those from CF individuals when ClϪ on both sides of the epithelium was replaced with the less permeant anion sulfate (Quinton, 1983) When ClϪ is unable to follow Naϩ, the lumen-negative voltage hyperpolarizes by more than 30 mV and NaCl reabsorption is greatly reduced (Quinton, 1983; Quinton and Bijman, 1983) This low ClϪ conductance nicely explains why the sweat of CF patients is salty, a diagnostic feature of the disease In the airways ion transport is more complex; salt and fluid are absorbed under resting conditions and secreted when stimulated by epinephrine, acetylcholine, tachykinins and purine nucleotides The transepithelial potential across human nasal epithelium is Ϫ20 to Ϫ40 mV (lumen-negative) and, like the sweat duct, becomes hyperpolarized in CF patients The transepithelial potential in CF is more sensitive to block by amiloride, an antagonist of the epithelial Naϩ channel (Garty and Palmer, 1997) This would be consistent with both reduced ClϪ conductance and elevated Na absorption However, subsequent studies of cultured cells suggested a two- to fourfold increase in apical membrane Na conductance in CF (Boucher, 1994; Willumsen and Boucher, 1989) Although observed at the single channel level (Stutts et al., 1995), the mechanism by which CFTR normally downregulates the Naϩ channel is not understood and is not universal since CFTR is required for normal Naϩ conductance in the sweat duct (Reddy et al., 1999) CFTR IS A LOW-CONDUCTANCE, NON-RECTIFYING CHLORIDE CHANNEL IN EPITHELIAL CELLS The precise function of CFTR was not obvious when the gene was cloned However, a cAMPstimulated ClϪ channel with a nearly linear (i.e non-rectifying, Figure 29.4) current–voltage relationship and conductance of 7–10 pS appeared when CFTR was expressed in Sf9 insect cells (Kartner et al., 1991), fibroblasts (Berger et al., 1991), Chinese hamster ovary (CHO) cells (Tabcharani et al., 1991), and Xenopus oocytes Figure 29.4 Patch clamp recording of CFTR channel current A, Inside-out configuration showing pipette tip with excised patch that contains a single CFTR chloride channel B, Hypothetical trace illustrating unitary current amplitude and slow gating Openings are indicated by upward transitions, and result from nucleotide binding, probably at NBD2 Downward transitions are the closing events caused by dissociation of the nucleotide or its hydrolysis products C, Current–voltage relationship calculated for a single channel The reversal potential (Vr , zero-current potential) is mV in symmetrical, high-ClϪ solutions Vr shifts to a negative potential as predicted by the Nernst equation when bath ClϪ concentration is reduced 595 596 ABC PROTEINS: FROM BACTERIA TO MAN (Bear et al., 1991) These properties are identical to those of a channel reported in pancreatic ducts (Gray et al., 1988, 1989), rat thyroid epithelium (Champigny et al., 1990), and in T84 cells, the most widely used model cell line for epithelial ClϪ secretion (Tabcharani et al., 1990) The similarities in unitary conductance, regulation, gating and pharmacology led to the proposal that CFTR is itself the low-conductance ClϪ channel (Kartner et al., 1991) A different ClϪ channel with higher conductance that preferentially conducts anions into the cell (i.e exhibits ‘outward rectification’ of current flow) had been previously identified as defective in CF (Frizzell et al., 1986; Hwang et al., 1989; Li et al., 1988) Confusion was exacerbated when the macroscopic conductance first generated by heterologous CFTR expression was attributed to rectifying anion channels (Anderson et al., 1991c) However, mutagenesis and biochemical reconstitution soon established CFTR as a nonrectifying, low-conductance channel Mutations in the first and sixth predicted transmembrane segments of CFTR altered the selectivity of whole cell anion currents (Anderson et al., 1991b) When CFTR protein was purified to homogeneity from Sf9 insect cells and reconstituted into planar lipid bilayers, it generated channels having low conductance like those recorded previously on epithelial cells (Bear et al., 1992) What happened to the outwardly rectifying anion channel? Most attention has focused on other ClϪ channels that are consistently activated while still on the cell (as opposed to the less physiological conditions existing in isolated membrane patches) The outwardly rectifying anion channel is probably one of many membrane proteins that are influenced by the presence of CFTR, but there is no compelling evidence for a role in ClϪ secretion It has been difficult to establish the physiological roles of particular ClϪ channels because they cannot be pharmacologically dissected using available inhibitors For example, a cAMPstimulated, DIDS (4,4Ј-diisothiocyanatostilbene2,2Ј-disulfonate)-sensitive whole cell ClϪ current has been attributed to the outward rectifier, based on its sensitivity to external DIDS, which had little effect on CFTR in early studies of pancreatic ducts and T84 monolayers (Gray et al., 1990; Tabcharani et al., 1990) However, DIDS sensitivity depends on experimental conditions, and external DIDS does partially inhibit CFTR (Kartner et al., 1991) Since the action of DIDS on CFTR is enhanced by large voltage ramps such as those applied to whole cell patches, inhibition of CFTR probably explains the DIDS-sensitive currents during cAMP stimulation that have been ascribed to outwardly rectifying channels ROLE OF CFTR IN PATHOBIOLOGY OF CYSTIC FIBROSIS It is now widely accepted that CFTR is a nonrectifying ClϪ channel with low (i.e 7–10 pS) conductance, but its physiological role in the airways remains hotly debated In the airways, CFTR expression is highest in distal regions of the submucosal glands, where fluid secretion hydrates the mucus and helps to expel it onto the airway surface, and this may explain the early mucus impaction of glands seen in CF infants (Sturgess and Imrie, 1982) In one absorptive model, the ionic strength of airway surface liquid is increased in CF due to diminished salt absorption, which ultimately reduces the killing activity of antibacterial substances on the airway surface (Smith et al., 1996) In another, it has been proposed that pathology in the airways results from Naϩ and fluid hyperabsorption caused by dysregulated Naϩ absorption (Matsui et al., 1998) According to this scheme, CF symptoms are caused by the loss of CFTR protein and its effects on other proteins, particularly the epithelial Naϩ channel, rather than abnormally low chloride conductance The mechanisms that underlie these regulatory effects of CFTR are not understood, and whether they even occur when the channels are expressed in Xenopus oocytes remains highly controversial (Konig et al., 2001; Nagel et al., 2001) THE CFTR CHANNEL PORE STRUCTURE AND FUNCTION OF THE PORE Since a high-resolution structure of CFTR is not available, the identity of pore-lining amino acids has been inferred by comparing electrophysiological properties of wild-type and mutant channels A strong case can be made that the sixth transmembrane segment (TMS6) lines the pore, since mutations there affect anion selectivity (Anderson et al., 1991b; Linsdell et al., 1997a), conductance (Sheppard et al., 1993; THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) Tabcharani et al., 1993), multi-ion pore behavior (Tabcharani et al., 1993), and blocker sensitivities (Linsdell and Hanrahan, 1996; McDonough et al., 1994) Scanning cysteine accessibility mutagenesis (SCAM), in which TMS residues are systematically replaced by single cysteines and the effects of hydrophilic sulfhydryl reagents on permeation are assayed, suggested that TMS 1, and are mostly ␣-helical and pore-lining (Akabas, 2000) ␣-Helical structure was also demonstrated in TMS and when they were expressed as a helix-loop-helix construct and studied by circular dichroism, even when solubilized in SDS (Therien et al., 2001) A model in which the amino acids distal to TMS fold back into the membrane was proposed based on the accessibility of cysteines engineered in this region to externally applied hydrophilic methanesulfonate (MTS) reagents (Cheung and Akabas, 1997) Mutations that alter the charge at R352 in the proposed re-entrant loop dramatically alter anion:cation selectivity, consistent with the model (Guinamard and Akabas, 1999) However, further studies are needed as the interpretation of scanning cysteine accessibility results has been questioned, and the assumption that only pore-lining residues are accessible to such reagents has not been verified in other types of channel (Holmgren et al., 1996; Mansoura et al., 1998; Yang et al., 1996) Mutating G314 in TMS and S1118 in TMS 11 affects both anion permeation and channel gating, suggesting that they also contribute to the pore (McCarty, 2000; Zhang et al., 2000) A model of the CFTR pore has been proposed based on electrophysiological studies (McCarty, 2000), but there has not yet been direct biochemical confirmation for any porelining residue and the functional data obtained from some mutants are difficult to reconcile Deleting the amino terminus and TMSs 1–4 apparently has little effect on channel function when expressed in Xenopus oocytes (Carroll et al., 1995), yet there are several diseaseassociated mutations in this region that alter channel properties when expressed in mammalian cells or oocytes (Akabas et al., 1994; Anderson et al., 1991b; Mansoura et al., 1998) Mutations in TMS 12 affect sensitivity to the open channel blocker diphenylamine-2-carboxylic acid (DPC), suggesting that it lines the pore (McDonough et al., 1994), yet normal-looking channels are recorded when a mutant lacking the C-terminal half (i.e lacking TMS 7–12 and NBD2) is expressed (Sheppard et al., 1994) It was suggested that the N-terminal half of CFTR can form functional channels by dimerizing There is no experimental evidence for such homodimers, which would lack a hypothetical salt bridge between R347 in TMS and D924 in TMS 8, which was suggested to be essential for normal conductance (Cotten and Welsh, 1999) TMS 12 probably lines the pore, but its contribution must differ substantially from that of TMS since alanine substitutions at T1134, M1137, N1138, S1141 and T1142 (amino acids that correspond to important residues in TMS 6) have little, or no effect on permeation (Gupta et al., 2001) Flickery block of the CFTR channel by the pH buffer MOPS (3-morpholinopropanesulfonic acid) suggests that the CFTR pore may have two conformationally distinct open states with different blocker sensitivities (Ishihara and Welsh, 1997) This possibility awaits confirmation using other methods, but it is intriguing and could potentially be used to dissect steps in channel-gating models PERMEATION IN THE PORE CFTR is highly selective for monovalent anions over cations (PCl/Pcation Ͼ 8–14) (Bear and Reyes, 1992; Tabcharani et al., 1990) Permeability ratios (PX/PCl) generally follow the (inverse) lyotropic series, with large, weakly hydrated ions being most permeant Lyotropic anions such as iodide also bind tightly under some conditions and have given inconsistent results even within laboratories For example, reported PI/PCl values range from near unity (Gray et al., 1990; Kartner et al., 1991), to 0.8 (Linsdell et al., 2000), 0.4 (3; 10; 10; 22; 84; 92) and (Champigny et al., 1990) Inhibition of ClϪ current by IϪ has been observed in mixtures of both ions (Tabcharani et al., 1992) The properties of chimeras between human and Xenopus CFTR (which has inherently higher PI/PCl) suggest that determinants of PI/PCl may be situated in TMS 1–6 (Price et al., 1996) When analyzed from the extracellular side using mixed solutions, the permeability ratios for polyatomic anions follow the sequence Ϫ Ϫ NO Ϫ (1.73) Ͼ Cl (1.0) Ͼ HCO (0.25) ϾϾ gluconateϪ(0.03) (Gray et al., 1990) A similar Ϫ Ϫ Ϫ sequence NOϪ Ͼ Cl Ͼ HCO Ͼ formate Ͼ acetateϪ is obtained under bi-ionic conditions, although permeability to large kosmotropic anions was higher from the cytoplasmic than extracellular side (Linsdell et al., 1997a) Indeed, external pyruvate, propanoate, methane 597 598 ABC PROTEINS: FROM BACTERIA TO MAN sulfonate, ethane sulfonate and gluconate are not measurably permeant (i.e PX/PCl Ͻ 0.06) when macroscopic currents are measured using excised patches exposed to PKA and ATP Yet, currents carried by these anions are detectable from the cytoplasmic side (Linsdell and Hanrahan, 1998) The relationship between macroscopic permeability ratio and ion diameter suggests a minimum functional diameter of 5.3 Å from the outside (Linsdell et al., 1997a), and ϳ12 Å from the inside (Linsdell and Hanrahan, 1998; Linsdell et al., 1997a) HCOϪ is the major ion transported in the pancreatic duct and may also be important in airway and colonic fluid secretion (Quinton, 2001), accounting for about half the anion secretion by airway submucosal glands HCOϪ permeates through the CFTR channel pore, though less well than ClϪ (Gray et al., 1990; Hanrahan et al., 1993; Linsdell et al., 1997a; Poulsen et al., 1994) The physiological significance of this route for bicarbonate flux in pancreatic ducts has been questioned (Lee et al., 1999) Interestingly, external HCOϪ inhibits CFTR-mediated ClϪ current with an apparent Ki of mM (O’Reilly et al., 2000) Such inhibition may be important during pancreatic bicarbonate secretion since it would reduce dissipation Ϫ of the HCOϪ gradient when luminal HCO concentration is elevated to ϳ150 mM, which is sixfold higher than plasma and Ͼ10-fold higher than cytoplasm Several lines of evidence suggest that the CFTR pore holds more than one anion simultaneously The bi-ionic permeability ratio PI/PCl is concentration dependent (Tabcharani et al., 1992), a common feature of multi-ion pores (Eisenman and Horn, 1983; Hille, 1992) At least two different electrical distances have been calculated for voltage-dependent block by internal thiocyanate and gluconate respectively (0.2 and 0.4), and block by intracellular gluconate is relieved by raising the external ClϪ concentration, consistent with yet another external anion binding site When wild-type channels are bathed with ClϪ/SCNϪ mixtures, the conductance decreases from to pS as the SCNϪ mole fraction is elevated from to 7%, and then increases again as the SCNϪ mole fraction is elevated further to 97% (Tabcharani et al., 1993) This anomalous mole fraction effect (AMFE), and voltage-dependent block by cytoplasmic SCNϪ are abolished when R347 is mutated to aspartate, and become pH dependent in the R347H mutant The positive charge on R347 probably does not interact directly with anions since tight binding would slow permeation (e.g Dutzler et al., 2002) However, it may stabilize a binding site for anions R347 may also have an impact on NBD function, as the ATPase activity of R347D is significantly reduced compared to wild-type CFTR (Kogan et al., 2001) The permeability and conductance ratios measured for SCNϪ, IϪ, and BrϪ are very different (Linsdell et al., 1997a) Dawson and colleagues have emphasized the importance of anion binding in CFTR (e.g Dawson et al., 1999) Conductance ratios are more strongly affected by mutations in TMS 1, and than permeability ratios, and therefore may be more sensitive to pore structure (Mansoura et al., 1998) Mutating K335 near the extracellular end of TMS to a negatively charged glutamate reduces conductance by about 50% (Anderson et al., 1991b; Tabcharani et al., 1993) Covalent modification of cysteines substituted at R334 or K335 with the negatively charged methanethiosulfonate reagent MTSES caused a similar decrease, whereas modification by positively charged reagents had the opposite effect (Smith et al., 2001) Thus, the positive charge associated with R334 and K335 influences conductance, probably by elevating ClϪ concentration near the external mouth of the pore Permeability ratios depend on barrier heights and the relative ease with which anions can enter the pore from the bulk solution; therefore they are dominated by anion–water interactions (i.e hydration energy), whereas conductance ratios mainly reflect ion concentrations and ion binding within the pore Ion permeation is often interpreted by assuming that ions hop between sites, or wells, that are essentially fixed within the pore and are separated by energetically unfavorable regions, or barriers A four-barrier, three-site (4B3S) model can reproduce experimentally measured current– voltage relationships under a wide range of conditions, including variations in single channel conductance, reversal potentials, block by intracellular gluconate, and AMFEs in mixtures of SCNϪ and ClϪ (Linsdell et al., 1997b) Moreover, loss of the AMFE (as seen in the R347D mutant) can be simulated using this 4B3S model by adjusting the well that corresponds to an intracellular SCNϪ block and the adjacent barrier Admittedly such models are highly speculative and require many assumptions However, it is heartening that most inferences regarding selectivity, multi-ion pore behavior and other biophysical properties of Kϩ and ClϪ channels are strongly supported by the recent corresponding crystal structures (Doyle et al., 1998; Dutzler et al., 2002) 604 ABC PROTEINS: FROM BACTERIA TO MAN While photolabeling experiments have revealed a much stronger interaction of AMPPNP with NBD1 than NBD2, it is still not known which of the following three effects of AMPPNP are due to this high-affinity interaction: (1) reduction of the opening rate, (2) reduction of the closing rate, and (3) ability to open the channel in the absence of ATP In fact, all of these effects apparently require concentrations higher than those necessary to saturate NBD1 (Aleksandrov et al., 2002) Although we are still far from completely understanding nucleotide regulation of the channel, some aspects have been clarified It is very clear that the two NBDs are non-equivalent in many respects The principal nucleotide interaction at NBD1 involves stable binding of nucleoside triphosphate that is independent of vanadate This binding does not appear to be crucial for channel gating because it is prevented by the K464A mutation, which only slightly slows channel opening MgATP is bound, hydrolyzed and the products rapidly released at NBD2 of wild-type but not K1250A, which severely slows channel opening and closing, implying a direct role of this domain in gating Some role for ATP at NBD1 is anticipated but is not yet clear Unlike other studies (Zou et al., 2001) recent experiments indicate that binding at NBD1 and hydrolysis at NBD2 are not strictly interdependent, K464A abrogates the former without influencing the latter, and K1250A has just the opposite effect (Aleksandrov et al., 2002) This does not exclude the possibility of more elaborate allosteric interactions between the domains, which would require other kinds of experiments to detect In our current working hypothesis, CFTR is a ligand-gated channel in which reversibility of the gating process is made highly efficient by hydrolysis of the substrate Channel opening occurs due to a change in the configuration of the closed state as occurs on ligand binding by other ligand-gated channels It is not yet known if under physiological conditions, the conformational perturbation that is rate limiting for initiation of gating is due to the initial binding or formation of the transition state for hydrolysis In this model, hydrolysis and product dissociation are necessary for termination of gating and the latter step appears to be rate limiting Current evidence indicates that NBD2 is the more active player in this entire cycle The role of NBD1 remains to be more clearly elucidated but ATP binding there may influence the frequency of gating cycles CFTR MATURATION AND TRAFFICKING IN THE SECRETORY PATHWAY Attention was drawn to this topic because most CF patients have at least one copy of the ⌬F508 mutation, located in the helical domain of the NBD, which causes CFTR to fold inefficiently and be retained in the ER Thus, although there are many other mutations that compromise aspects of CFTR function as discussed above (gating, permeation, etc.), most CF is due to the failure of the protein to be transported to the apical membrane, where it is required for chloride conductance (Figure 29.6) Like other membrane and secreted glycoproteins (Figure 29.7), CFTR is core-glycosylated co-translationally Figure 29.6 Immunostaining of CFTR in sweat ducts in cryosections of skin biopsies from a non-CF individual and a homozygous ⌬F508 patient (upper panels) Fluorescence microscopy of wild-type and ⌬F508 green fluorescent protein fusions heterologously expressed in BHK-21 cells (lower panels) THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) in the ER, from which it is exported in COP IIcoated vesicles to vesicular-tubular clusters From there the protein is transferred to the Golgi apparatus, where oligosaccharide chain trimming and extension occurs to form complex structures (Kopito, 1999; Riordan, 1999) When first expressed heterologously in mammalian cells, ⌬F508 CFTR was observed to be synthesized as a full-length core-glycosylated molecule, which did not exit the ER or acquire complex oligosaccharide chains (Cheng et al., 1990) Initially it was not known if the lack of carbohydrate processing was the cause, or the result, of mislocalization However, there is Figure 29.7 Schematic representation of CFTR processing and trafficking CFTR molecules are synthesized on ER-associated ribosomes Core oligosaccharide chains are attached while CFTR is incorporated into the ER membrane At the ER the lumenal chaperone calnexin and the cytosolic chaperones Hsp90, Hsp70 and the Hsc70/Hdj-2 or Hsc70/CHIP complex interact transiently with unfolded or partially folded intermediates Mature CFTR molecules are exported from the ER and are transported to the Golgi apparatus by COP II vesicles An alternative pathway for mature CFTR molecules bypassing COP II vesicles and early Golgi compartments has been described recently (Yoo et al., 2002) The maturation of CFTR polypeptides is reflected by the attachment of complex oligosaccharide chains in the Golgi Secretory vesicles deliver CFTR from the trans-Golgi network (TGN) to the plasma membrane The PDZ-domain-containing protein CAL binds to the C-terminus of CFTR at the Golgi apparatus and may favor retention of CFTR Other PDZdomain proteins (EBP50, E3KARP, CAP70) bind to CFTR to anchor the protein to the plasma membrane or tether it in regulatory complexes Syntaxin 1A, a component of the membrane trafficking machinery, binds CFTR and inhibits channel activity Binding to the clathrin adaptor complex AP-2 mediates endocytosis in cathrin-coated vesicles Endocytosed CFTR can be recycled or degraded in the lysosome Molecules that fail to fold correctly at the ER are ubiquitinated and retrotranslocated through the Sec61 export pore Ubiquitination occurs with the help of ubiquitin conjugating enzymes (ubc) and ubiquitin ligases, such as CHIP In the cytosol, the ubiquitinated molecules are subjected to degradation by the proteasome This pathway eliminates not only misfolded mutant CFTR, but also about 70% of wild-type proteins that not attain a native conformation When degradation by the proteasome is inhibited or saturated, aggregation of export-incompetent CFTR molecules can occur The aggregated molecules are transported to structures named aggresomes near the centriole 605 606 ABC PROTEINS: FROM BACTERIA TO MAN now strong evidence in support of the latter possibility and the primary consequence of the absence of phe508 is defective folding of the polypeptide (Qu and Thomas, 1996) This behavior of the ⌬F508 molecule, and indeed many other variants with single amino acid substitutions or short insertions or deletions in cytoplasmic domains, has been confirmed in all mammalian cell expression systems tested (Kopito, 1999; Riordan, 1999) In amphibian (Drumm et al., 1991) and insect (Li et al., 1993) cell systems, however, both maintained at lower temperatures than mammalian cells, there is partial maturation and transport of the ⌬F508 protein to the cell surface Thus, the maturation is at least partially temperature sensitive for folding (Denning et al., 1992) Other means of promoting maturation are being intensively sought since development of a small molecule drug with this effect would provide a potential therapeutic strategy Some compounds at high concentrations, including osmolytes such as glycerol, can slightly improve maturation but are impractical for use in vivo (Brown et al., 1996) Preliminary clinical trials with phenyl butyrate, which is already used in the treatment of thalassemia (Zeitlin, 2000), have been undertaken with CF patients on the basis of reports that it improves maturation in cell culture systems (Rubenstein and Zeitlin, 2000) In several other instances, where misfolding of mutant proteins is at the basis of other genetic diseases, the binding of high-affinity ligands has been discovered to improve folding and at least partially rescue the mutant phenotype (Fan et al., 1999; Foster et al., 1999; Klabunde et al., 2000; Morello et al., 2000) Unfortunately, with one possible exception (Dormer et al., 2001), this approach has not yet been feasible because of a paucity of compounds known to specifically bind CFTR with high affinity (Schultz et al., 1999), although high-throughput screens for such reagents (Galietta et al., 2001) may still identify such drugs It is ironic that misfolding of other ABCC proteins such as Pgp caused by in vitro mutagenesis can be somewhat aleviated by drug binding (Loo and Clarke, 2000); unfortunately it is not the multidrug resistance phenotype that needs to be rescued Any such pharmacological approach to the rescue of ⌬F508 and other processing mutants in patients rests on the assumption that the abortive biogenesis observed in cultured cells also occurs in the epithelial cells of the affected tissues in patients The failure of the ⌬F508 protein to reach the apical membrane of sweat duct cells was clearly demonstrated (Kartner et al., 1992), but conflicting results have been reported in intestinal and airway cells (Bronsveld et al., 2001; Kalin et al., 1999) The low abundance of CFTR in the latter makes the resolution of this conflict very demanding However, the recent application of especially specific, high-affinity antibodies, which are capable of detecting mature and immature CFTR in biopsy and transplant specimens, detected both forms in non-CF tissues, but only the immature form in ⌬F508 homozygotes (Kreda et al., 2001) Mechanistically the very stringent localization quality control applied to CFTR is not well understood at the molecular or the cellular level The key issue to be understood is the recognition mechanism that distinguishes the mutant and wild-type nascent chains Unraveling the steps in this identification process is complicated by the fact that even wild-type CFTR matures inefficiently; as little at 25% of the nascent chain synthesized is converted to the mature form with the remainder degraded at the ER by the 26S proteasome (Gelman et al., 2002; Jensen et al., 1995; Lukacs et al., 1994; Ward et al., 1995) With mutants such as ⌬F508 this percent maturation is reduced to zero Ubiquitination occurs even before synthesis of the entire polypeptide is complete (Sato et al., 1998) Multiple molecular chaperones on both sides of the ER membrane interact with the nascent chain and there is evidence that they facilitate folding and also direct CFTR to the ubiquitin-proteasomal pathway (Loo et al., 1998; Meacham et al., 1999, 2001; Yang et al., 1993) The calnexin/UDP glucose-glycosyl transference conformation-sensing mechanism (Ritter and Helenius, 2000) does not appear to play an important role, since preventing the interaction of calnexin with CFTR neither prevents wildtype CFTR from maturing nor promotes maturation of ⌬F508 (Pind et al., 1994) Several cytoplasmic chaperones have an impact, especially Hsp70 and its co-chaperones (Meacham et al., 1999, 2001), and Hsp90 (Loo et al., 1998) That the principal recognition events occur on the cytoplasmic rather than the lumenal side of the ER membrane is supported by the finding that the majority of disease-associated mutations in cytoplasmic domains cause misprocessing while none of those analyzed in extracytoplasmic loops have this effect (Hämmerle et al., 2001; Seibert et al., 1996a, 1996b, 1997) The related fact that different missense mutations across the entire cytoplasmic THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) face of the protein prevent maturation indicates that achievement of a global native structure is required for ER export However, since it is unlikely that tertiary structure can be detected directly by the cellular machinery it may be that discrete short sequence motifs that are either exposed or buried are the actual recognition elements Positive export signals consisting of a short acidic consensus, DXE, play such a role in some secretory proteins (Nishimura et al., 1999) and could so in CFTR Negative retention/retrieval signals, arginine-framed tripeptides discovered in the channel and ABC protein components of KATP (Zerangue et al., 1999), apparently contribute to the ER retention of ⌬F508 (Chang et al., 1999) The receptors for these positive and negative traffic signals have not yet been identified The vesicular trafficking events that move CFTR through proximal steps in the secretory pathway are apparently conventional whereas late steps may be novel at more distal stages (Bannykh et al., 2000; Yoo et al., 2002) Thus, COP II vesicles are responsible for the ER export of CFTR as with other secretory proteins However, movement to the Golgi may TABLE 29.1 OTHER MISFOLDED MUTANT ABC PROTEINS IN HUMAN DISEASE Protein ABC family member Disease ABC1 ABCA1 ABCR MDR3 ABCA4 ABCB4 Familial high-density lipoprotein deficiency (FHD)a Stargardt diseaseb Progressive familial intrahepatic cholestasis type (PFIC) Intrahepatic cholestasis of pregnancy (ICP)c MRP2 SUR1 ABCC2 ABCC8 ALD ABCD1 a Brooks-Wilson et al., 1999 Lewis et al., 1999 c Dixon et al., 2000 d Keitel et al., 2000 e Cartier et al., 2001 f Smith et al., 1999 b Dubin–Johnson Syndromed Persistent hyperinsulinemic hypoglycemia of Infancy (PHHI)e X-linked adrenoleukodystrophyf take a less-well-characterized route that is not blocked by inhibitors of several of the small GTPases and the syntaxin SNARE protein, which are essential for the conventional pathway to the cis-Golgi (Yoo et al., 2002) Rather, the apparent involvement of the endosomal t-SNARE syntaxin 13 suggests that CFTR maturation may involve movement through the trans-Golgi-endosomal pathway (Yoo et al., 2002) It is not yet known if this routing contributes to the fragility of wild-type CFTR in the secretory pathway and the complete inability of many mutants to successfully traverse it Disease-associated mutations in other human ABC proteins also cause misfolding and failure to be transported out of the ER Examples that have been documented are listed in Table 29.1 However, many more will probably emerge as the mutations detected in patients are studied in heterologous expression systems The extensive research on this aspect of cystic fibrosis, while not yet yielding an effective treatment, provides a helpful framework for progress towards this end in other ABC protein diseases REFERENCES Aguilar-Bryan, L., Clement, J.P.t., Gonzalez, G., Kunjilwar, K., Babenko, A and Bryan, J (1998) Toward understanding the assembly and structure of KATP channels Physiol Rev 78(1), 227–245 Akabas, M.H (2000) Cystic fibrosis transmembrane conductance regulator Structure and function of an epithelial chloride channel J Biol Chem 275(6), 3729–3732 Akabas, M.H., Kaufmann, C., Cook, T.A and Archdeacon, P (1994) Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator J Biol Chem 269(21), 14865–14868 Aleksandrov, A.A and Riordan, J.R (1998) Regulation of CFTR ion channel gating by MgATP FEBS Lett 431(1), 97–101 Aleksandrov, A.A., Chang, X., Aleksandrov, L and Riordan, J.R (2000) The non-hydrolytic pathway of cystic fibrosis transmembrane conductance regulator ion channel gating J Physiol 528 (Pt 2), 259–265 Aleksandrov, L., Mengos, A., Chang, X., Aleksandrov, A and Riordan, J.R (2001) Differential interactions of nucleotides at the two nucleotide binding domains of 607 608 ABC PROTEINS: FROM BACTERIA TO MAN the cystic fibrosis transmembrane conductance regulator J Biol Chem 276(16), 12918–12923 Aleksandrov, L., Aleksandrov, A.A., Chang, X.-B and Riordan, J.R (2002) The first nucleotide binding domain of CFTR is a site of stable nucleotide interaction whereas the second is a site of rapid turnover J Biol Chem 3, 15419–15425 Anderson, M.P., Berger, H.A., Rich, D.P., Gregory, R.J., Smith, A.E and Welsh, M.J (1991a) Nucleoside triphosphates are required to open the CFTR chloride channel Cell 67, 775–784 Anderson, M.P., Gregory, R.J., Thompson, S., Souza, D.W., Paul, S., Mulligan, R.C., Smith, A.E and Welsh, M.J (1991b) Demonstration that CFTR is a chloride channel by alteration of its anion selectivity Science 253, 202–205 Anderson, M.P., Rich, D.P., Gregory, R.J., Smith, A.E and Welsh, M.J (1991c) Generation of cAMP-activated chloride currents by expression of CFTR Science 251, 679–682 Arsenault, A.L., Ling, V and Kartner, N (1988) Altered plasma membrane ultrastructure in multidrug-resistant cells Biochim Biophys Acta 938(2), 315–321 Bajnath, R.B., Groot, J.A., De Jonge, H.R., Kansen, M and Bijman, J (1993) Synergistic activation of non-rectifying smallconductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT29cl.19A Pflugers Arch 425(1–2), 100–108 Bannykh, S.I., Bannykh, G.I., Fish, K.N., Moyer, B.D., Riordan, J.R and Balch, W.E (2000) Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway Traffic 1(11), 852–870 Baukrowitz, T., Hwang, T.C., Nairn, A.C and Gadsby, D.C (1994) Coupling of CFTR ClϪ channel gating to an ATP hydrolysis cycle Neuron 12(3), 473–482 Bear, C.E and Reyes, E.F (1992) cAMPactivated chloride conductance in the colonic cell line, Caco-2 Am J Physiol 262(1 Pt 1), C251–C256 Bear, C.E., Duguay, F., Naismith, A.L., Kartner, N., Hanrahan, J.W and Riordan, J.R (1991) ClϪ channel activity in Xenopus oocytes expressing the cystic fibrosis gene J Biol Chem 266(29), 19142–19145 Bear, C.E., Li, C., Kartner, N., Bridges, R.J., Jensen, T.J., Ramjeesingh, M and Riordan, J.R (1992) Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR) Cell 68, 809–818 Becq, F., Jensen, T.J., Chang, X.B., Savoia, A., Rommens, J.M., Tsui, L.C., Buchwald, M., Riordan, J.R and Hanrahan, J.W (1994) Phosphatase inhibitors activate normal and defective CFTR chloride channels Proc Natl Acad Sci USA 91(19), 9160–9164 Berger, A.L and Welsh, M.J (2000) Differences between cystic fibrosis transmembrane conductance regulator and HisP in the interaction with the adenine ring of ATP J Biol Chem 275(38), 29407–29412 Berger, A.L., Ikuma, M., Hunt, J.F., Thomas, P.J and Welsh, M.J (2002) Mutations that change the position of the putative gammaphosphate linker in the nucleotide binding domains of CFTR alter channel gating J Biol Chem 277(3), 2125–2131 Berger, H.A., Anderson, M.P., Gregory, R.J., Thompson, S., Howard, P.W., Maurer, R.R., Mulligan, R., Smith, A.E and Welsh, M.J (1991) Identification and regulation of the CFTR-generated chloride channel J Clin Invest 88, 1422–1431 Borchardt, J., Kole, J and Cohn, J (1996) Phosphorylation of CFTR Ser-737 by protein kinase A Pediatr Pulm (Suppl 13), 212 (Abstract) Boucher, R.C (1994) Human airway ion transport Part two Am J Respir Crit Care Med 150(2), 581–593 Bronsveld, I., Mekus, F., Bijman, J., Ballmann, M., de Jonge, H.R., Laabs, U., et al (2001) Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings J Clin Invest 108(11), 1705–1715 Brooks-Wilson, A., Marcil, M., Clee, S.M., Zhang, L.H., Roomp, K., van Dam, M., et al (1999) Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency Nat Genet 22(4), 336–345 Brown, C.R., Hong-Brown, L.Q., Biwersi, J., Verkman, A.S and Welch, W.J (1996) Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein Cell Stress Chaperon 1(2), 117–125 Button, B., Reuss, L and Altenberg, G.A (2001) PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site: insertion of this site THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) confers PKC sensitivity to human CFTR J Gen Physiol 117(5), 457–468 Carroll, T.P., Morales, M.M., Fulmer, S.B., Allen, S.S., Flotte, T.R., Cutting, G.R and Guggino, W.B (1995) Alternate translation initiation codons can create functional forms of cystic fibrosis transmembrane conductance regulator J Biol Chem 270(20), 11941–11946 Carson, M.R and Welsh, M.J (1993) 5Ј-adenylylimidodiphosphate does not activate CFTR chloride channels in cell-free patches of membrane Am J Physiol 265(1 Pt 1), L27–L32 Carson, M.R., Travis, S.M and Welsh, M.J (1995) The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity J Biol Chem 270(4), 1711–1717 Cartier, E.A., Conti, L.R., Vandenberg, C.A and Shyng, S.L (2001) Defective trafficking and function of KATP channels caused by a sulfonylurea receptor mutation associated with persistent hyperinsulinemic hypoglycemia of infancy Proc Natl Acad Sci USA 98(5), 2882–2887 Catterall, W.A (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels Neuron 26(1), 13–25 Champigny, G., Verrier, B., Gerard, C., Mauchamp, J and Lazdunski, M (1990) Small conductance chloride channels in the apical membrane of thyroid cells FEBS Lett 259(2), 263–268 Chang, X.-B., Tabcharani, J.A., Hou, Y.-X., Jensen, T.J., Kartner, N., Alon, N., Hanrahan, J.W and Riordan, J.R (1993) Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all ten PKA consensus phosphorylation sites J Biol Chem 268(15), 11304–11311 Chang, X.-B., Hou, Y.-X., Jensen, T and Riordan, J.R (1994) Mapping of the cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion J Biol Chem 269(28), 18572–18575 Chang, X.-B., Cui, L., Hou, Y., Jensen, T.J., Aleksandrov, A.A., Mengos, A and Riordan J.R (1999) Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis Mol Cell 4(1), 137– 142 Chappe, V., Hinkson, D.A.R., Chang, X.-B., Riordan, J.R and Hanrahan, J.W (2002) Phosphorylation of protein kinase C consensus sites in NBD1 and the R domain control CFTR channel activation by PKA J Physiol (in press) Chen, J.-H and Riordan, J.R (2002) Chemical cross-linking reveals different CFTR associations when the protein is heterologously and endogenously expressed Arch Biochem Biophys (submitted) Chen, J.-H., Chang, X.-B., Aleksandrov, A.A and Riordan, J.R (2002) CFTR is a monomer: biochemical and functional evidence J Membrane Biol (on line DOI: 10.1007/ 500232-001-0174-2 (07/10/02) Chen, J.M., Cutler, C., Jacques, C., Boeuf, G., Denamur, E., Lecointre, G., Mercier, B., Cramb, G and Ferec, C (2001) A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models Mol Biol Evol 18(9), 1771–1788 Cheng, J., Moyer, B.D., Milewski, M., Loffing, J., Ikeda, M., Mickle, J.E., Cutting, G.R., Li, M., Stanton, B.A and Guggino, W.B (2002) A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression J Biol Chem 277(5), 3520–3529 Cheng, S.H., Gregory, R.J., Marshall, J., Paul, S., Souza, D.W., White, G.A., O’Riordan, C.R and Smith, A.E (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis Cell 63, 827–834 Cheng, S.H., Rich, D.P., Marshall, J., Gregory, R.J., Welsh, M.J and Smith, A.E (1991) Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel Cell 66, 1027–1036 Cheung, M and Akabas, M.H (1997) Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel J Gen Physiol 109(3), 289–299 Cotten, J.F and Welsh, M.J (1999) Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR Evidence for disruption of a salt bridge J Biol Chem 274(9), 5429–5435 Cuthbert, A.W., Halstead, J., Ratcliff, R., Colledge, W.H and Evans, M.J (1995) The genetic advantage hypothesis in cystic fibrosis heterozygotes: a murine study J Physiol 482(Pt 2), 449–454 609 610 ABC PROTEINS: FROM BACTERIA TO MAN Dahan, D., Zhu, T., Evaglelidis, A and Hanrahan, J.W (1999) CFTR-protein phosphatase 2C (PP2C) association is independent of the carboxyl terminus PDZ binding motif FASEB J 13, A71(Abstract) Dawson, D.C., Smith, S.S and Mansoura, M.K (1999) CFTR: mechanism of anion conduction Physiol Rev 79(1 Suppl.): S47–S75 Dechecchi, M.C., Rolfini, R., Tamanini, A., Gamberi, C., Berton, G and Cabrini, G (1992) Effect of modulation of protein kinase C on the cAMP-dependent chloride conductance in T84 cells FEBS Lett 311(1), 25–28 Deeley, R.G and Cole S.P (1997) Function, evolution and structure of multidrug resistance protein (MRP) Semin Cancer Biol 8(3), 193–204 Denning, G.M., Anderson, M.P., Amara, J.F., Marshall, J., Smith, A.E and Welsh, M.J (1992) Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive Nature 358(6389), 761–764 Dixon, P.H., Weerasekera, N., Linton, K.J., Donaldson, O., Chambers, J., Egginton, E., et al (2000) Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking Hum Mol Genet 9(8), 1209–1217 Dormer, R.L., Derand, R., McNeilly, C.M., Mettey, Y., Bulteau-Pignoux, L., Metaye, T., et al (2001) Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells J Cell Sci 114(Pt 22), 4073–4081 Doyle, D.A., Morais Cabral, J., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T and MacKinnon, R (1998) The structure of the potassium channel: molecular basis of Kϩ conduction and selectivity Science 280(5360), 69–77 Drumm, M.L., Wilkinson, D.J., Smit, L.S., Worrell, R.T., Strong, T.V., Frizzell, R.A., Dawson, D.C and Collins, F.S (1991) Chloride conductance expressed by ÐF508 and other mutant CFTRs in Xenopus oocytes Science 254, 1797–1799 Duffieux, F., Annereau, J.P., Boucher, J., Miclet, E., Pamlard, O., Schneider, M., Stoven, V and Lallemand, J.Y (2000) Nucleotide-binding domain of cystic fibrosis transmembrane conductance regulator production of a suitable protein for structural studies Eur J Biochem 267(17), 5306–5312 Dulhanty, A.M and Riordan J.R (1994) Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator Biochemistry 33(13), 4072–4079 Dutzler, R., Campbell, E.B., Cadene, M., Chait, B.T and MacKinnon, R (2002) X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity Nature 415(6869), 287–294 Eisenman, G and Horn, R (1983) Ionic selectivity revisited: the role of kinetic and equilibrium processes in ion permeation through channels J Membr Biol 76(3), 197–225 Eskandari, S., Wright, E.M., Kreman, M., Starace, D.M and Zampighi, G.A (1998) Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy Proc Natl Acad Sci USA 95(19), 11235–11240 Fan, J.Q., Ishii, S., Asano, N and Suzuki, Y (1999) Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor Nat Med 5(1), 112–115 Foster, B.A., Coffey, H.A., Morin, M.J and Rastinejad, F (1999) Pharmacological rescue of mutant p53 conformation and function Science 286(5449), 2507–2510 Frizzell, R.A., Rechkemmer, G and Shoemaker, R.L (1986) Altered regulation of airway epithelial cell chloride channels in cystic fibrosis Science 233, 558–560 Gabriel, S.E., Clarke, L.L., Boucher, R.C and Stutts, M.J (1993) CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship Nature 363, 263–268 Gabriel, S.E., Brigman, K.N., Koller, B.H., Boucher, R.C and Stutts, M.J (1994) Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model Science 266(5182), 107–109 Gadsby, D.C and Nairn A.C (1999a) Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis Physiol Rev 79(1 Suppl.): S77–S107 Gadsby, D.C and Nairn A.C (1999b) Regulation of CFTR ClϪ ion channels by phosphorylation and dephosphorylation Adv Sec Mess Phosph 33, 79–106 Galietta, L.J., Springsteel, M.F., Eda, M., Niedzinski, E.J., By, K., Haddadin, M.J., THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) Kurth, M.J., Nantz, M.H and Verkman, A.S (2001) Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds J Biol Chem 276(23), 19723–19728 Gao, M., Cui, H.R., Loe, D.W., Grant, C.E., Almquist, K.C., Cole, S.P and Deeley, R.G (2000) Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein J Biol Chem 275(17), 13098–13108 Garty, H and Palmer, L.G (1997) Epithelial sodium channels: function, structure, and regulation Physiol Rev 77(2), 359–396 Gelman, M.S., Kannegaard, E.S and Kopito, R.R (2002) A principal role for the proteasome in ER-associated degradation of misfolded intracellular CFTR J Biol Chem 277, 11709–11714 Gentzsch, M and Riordan J.R (2001) Localization of sequences within the C-terminal domain of the cystic fibrosis transmembrane conductance regulator which impact maturation and stability J Biol Chem 276(2), 1291–1298 Gentzsch, M., Aleksandrov, A., Aleksandrov, L and Riordan, J.R (2002) Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications Biochem J 05/20/02 DOI: 10.1042/BJ20020511 Gray, M.A., Greenwell, J.R and Argent, B.E (1988) Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells J Mem Biol 105(2), 131–142 Gray, M.A., Harris, A., Coleman, L., Greenwell, J.R and Argent, B.E (1989) Two types of chloride channel on duct cells cultured from human fetal pancreas Am J Physiol 257, C240–C251 Gray, M.A., Pollard, C.E., Harris, A., Coleman, L., Greenwell, J.R and Argent, B.E (1990) Anion selectivity and block of the small-conductance chloride channel on pancreatic duct cells Am J Physiol 259(5 Pt 1), C752–C761 Guinamard, R and Akabas, M.H (1999) Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel Biochemistry 38(17), 5528–5537 Gunderson, K.L and Kopito, R.R (1994) Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating J Biol Chem 269(30), 19349–19353 Gupta, J., Evagelidis, A., Hanrahan, J.W and Linsdell, P (2001) Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region Biochemistry 40(22), 6620–6627 Hallows, K.R., Raghuram, V., Kemp, B.E., Witters, L.A and Foskett, J.K (2000) Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase J Clin Invest 105(12), 1711–1721 Hämmerle, M.M., Aleksandrov, A.A and Riordan, J.R (2001) Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator not impede biosynthetic processing but impair chloride channel stability J Biol Chem 276(18), 14848–14854 Hanrahan, J.W., Tabcharani, J.A and Grygorczyk, R (1993) Patch clamp studies of apical membrane chloride channels In: Cystic Fibrosis – Current Topics (ed J.A Dodge, D.J.H Brock and J.H Widdicombe), pp 93–137 London: John Wiley and Sons Hartman, J., Huong, Z., Rado, T.A., Peng, S., Jilling, T., Muccio, D.D and Sorscher, E.J (1992) Recombinant synthesis purification and nucleotide binding characteristics of the first nucleotide binding domains of the cystic fibrosis gene product J Biol Chem 267, 6455–6458 Higgins, C.F (1992) ABC transporters: from microorganisms to man Annu Rev Cell Biol 8, 67–113 Hille, B (1992) Ionic Channels of Excitable Membranes Sunderland, MA: Sinauer Associates Holmgren, M., Liu, Y., Xu, Y and Yellen, G (1996) On the use of thiol-modifying agents to determine channel topology Neuropharmacology 35(7), 797–804 Hopfner, K.P., Karcher, A., Shin, D.S., Craig, L., Arthur, L.M., Carney, J.P and Tainer, J.A (2000) Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABCATPase superfamily Cell 101(7), 789–800 Hou, Y.-X., Cui, L., Riordan, J.R and Chang, X.-B (2000) Allosteric interactions 611 612 ABC PROTEINS: FROM BACTERIA TO MAN between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1 J Biol Chem 275, 20280–20287 Howell, L.D., Borchardt, R and Cohn, J.A (2000) ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation Biochem Biophys Res Commun 271(2), 518–525 Huang, P., Lazarowski, E.R., Tarran, R., Milgram, S.L., Boucher, R.C and Stutts, M.J (2001) Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells Proc Natl Acad Sci USA 98(24), 14120–14125 Hwang, T., Lu, L., Zeitlin, P.L., Gruenert, D.C., Huganir, R and Guggino, W.B (1989) ClϪ channels in CF: Lack of activation by protein kinase C and cAMP-dependent protein kinase Science 244, 1351–1353 Hwang, T.-C., Baukrowtiz, G., Nagel, G., Horie, A.C and Gadsby, D.C (1994) Regulation of gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis Proc Natl Acad Sci USA 91, 4698–4702 Ikuma, M and Welsh, M.J (2000) Regulation of CFTR Cl– channel gating by ATP binding and hydrolysis Proc Natl Acad Sci USA 97(15), 8675–8680 Inagaki, N., Gonoi, T., Clement, J.P., Wang, C.Z., Aguilar-Bryan, L., Bryan, J and Seino, S (1996) A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive Kϩ channels Neuron 16(5), 1011–1017 Ishihara, H and Welsh, M.J (1997) Block by MOPS reveals a conformation change in the CFTR pore produced by ATP hydrolysis Am J Physiol 273(4 Pt 1), C1278–C1289 Jensen, T.J., Loo, M.A., Pind, S., Williams, D.B., Goldberg, A.L and Riordan, J.R (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing Cell 83, 129–135 Jia, Y., Mathews, C.J and Hanrahan, J.W (1997) Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A J Biol Chem 272(8), 4978–4984 Kalin, N., Claass, A., Sommer, M., Puchelle, E and Tummler, B (1999) DeltaF508 CFTR protein expression in tissues from patients with cystic fibrosis [see comments] J Clin Invest 103(10), 1379–1389 Karpowich, N., Martsinkevich, O., Millen, L., Yuan, Y.R., Dai, P.L., MacVey, K., Thomas, P.J and Hunt, J.F (2001) Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter Structure(Camb) 9(7), 571–586 Kartner, N., Hanrahan, J.W., Jensen, T.J., Naismith, A.L., Sun, S., Ackerley, C.A., et al (1991) Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance Cell 64(4), 681–691 Kartner, N., Augustinas, O., Jensen, T.J., Naismith, A.L and Riordan, J.R (1992) Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland Nat Genet 1(5), 321–327 Keitel, V., Kartenbeck, J., Nies, A.T., Spring, H., Brom, M and Keppler, D (2000) Impaired protein maturation of the conjugate export pump multidrug resistance protein as a consequence of a deletion mutation in Dubin–Johnson syndrome Hepatology 32(6), 1317–1328 Klabunde, T., Petrassi, H.M., Oza, V.B., Raman, P., Kelly, J.W and Sacchettini, J.C (2000) Rational design of potent human transthyretin amyloid disease inhibitors Nat Struct Biol 7(4), 312–321 Knowles, M.R., Stutts, M.J., Spock, A., Fischer, N., Gatzy, J.T and Boucher, R.C (1983) Abnormal ion permeation through cystic fibrosis respiratory epithelium Science 221, 1067–1070 Ko, Y.H and Pedersen P.L (1995) The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase J Biol Chem 270(38), 22093–22096 Ko, Y.H., Thomas, P.J., Delannoy, M.R and Pedersen, P.L (1993) The cystic fibrosis transmembrane conductance regulator Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein J Biol Chem 268(32), 24330–24338 Kogan, I., Ramjeesingh, M., Huan, L.J., Wang, Y and Bear, C.E (2001) Perturbation of the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits its ATPase activity J Biol Chem 276(15), 11575–11581 THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) Konig, J., Schreiber, R., Voelcker, T., Mall, M and Kunzelmann, K (2001) The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular ClϪ concentration EMBO Rep 2(11), 1047–1051 Kopito, R.R (1999) Biosynthesis and degradation of CFTR Physiol Rev 79(1 Suppl.): S167–S173 Kreda, J.M., Mall, M., Seydewitz, H., Kuehr, J., Mengos, A., Jensen, T.J., Riordan, J.R and Boucher, R (2001) CFTR expression in epithelia from cystic fibrosis patients Mol Biol Cell (Suppl 12)(Abstract 2137) Lee, M.G., Choi, J.Y., Luo, X., Strickland, E., Thomas, P.J and Muallem, S (1999) Cystic fibrosis transmembrane conductance regulator regulates luminal ClϪ/HCOϪ exchange in mouse submandibular and pancreatic ducts J Biol Chem 274(21), 14670–14677 Lewis, R.A., Shroyer, N.F., Singh, N., Allikmets, R., Hutchinson, A., Li, Y., Lupski, J.R., Leppert, M and Dean, M (1999) Genotype/Phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease Am J Hum Genet 64(2), 422–434 Li, C., Ramjeesingh, M., Reyes, E., Jensen, T., Chang, X., Rommens, J.M and Bear, C.E (1993) The cystic fibrosis mutation (delta F508) does not influence the chloride channel activity of CFTR Nat Genet 3(4), 311–316 Li, C., Ramjeesingh, M., Wang, W., Garami, E., Hewryk, M., Lee, D., Rommens, J.M., Galley, K and Bear, C.E (1996) ATPase activity of the cystic fibrosis transmembrane conductance regulator J Biol Chem 271(45), 28463–28468 Li, M., McCann, J.D., Liedtke, C.M., Nairn, A.C., Greengard, P and Welsh, M.J (1988) Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium Nature 331, 358–360 Linsdell, P and Hanrahan, J.W (1996) Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator ClϪ channels expressed in a mammalian cell line and its regulation by a critical pore residue J Physiol 496(Pt 3), 687–693 Linsdell, P and Hanrahan, J.W (1998) Adenosine triphosphate-dependent asymmetry of anion permeation in the cystic fibrosis transmembrane conductance regulator chloride channel J Gen Physiol 111(4), 601–614 Linsdell, P., Tabcharani, J.A., Rommens, J.M., Hou, Y.X., Chang, X.B., Tsui, L.C., Riordan, J.R and Hanrahan, J.W (1997a) Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions J Gen Physiol 110(4), 355–364 Linsdell, P., Tabcharani, J.A and Hanrahan, J.W (1997b) Multi-ion mechanism for ion permeation and block in the cystic fibrosis transmembrane conductance regulator chloride channel J Gen Physiol 110(4), 365–377 Linsdell, P., Evagelidis, A and Hanrahan, J.W (2000) Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore Biophys J 78(6), 2973–2982 Loo, M.A., Jensen, T.J., Cui, L., Hou, Y.-X., Chang, X.-B and Riordan, J.R (1998) Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome EMBO J 17, 6879–6887 Loo, T.W and Clarke, D.M (2000) Blockage of drug resistance in vitro by disulfiram, a drug used to treat alcoholism J Natl Cancer Inst 92(11), 898–902 Lukacs, G.L., Mohamed, A., Kartner, N., Chang, X.-B., Riordan, J.R and Grinstein, S (1994) Confirmational maturation of CFTR but not its mutant counterpart (ÐF508) occurs in the endoplasmic reticulum and requires ATP EMBO J 13, 6076–6086 Luo, J., Pato, M.D., Riordan, J.R and Hanrahan, J.W (1998) Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases Am J Physiol 274(5 Pt 1), C1397–C1410 Mansoura, M.K., Smith, S.S., Choi, A.D., Richards, N.W., Strong, T.V., Drumm, M.L., Collins, F.S and Dawson, D.C (1998) Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore Biophys J 74(3), 1320–1332 Mathews, C.J., Tabcharani, J.A and Hanrahan, J.W (1998) The CFTR chloride channel: nucleotide interactions and temperature-dependent gating J Membr Biol 163(1), 55–66 Matsui, H., Grubb, B.R., Tarran, R., Randell, S.H., Gatzy, J.T., Davis, C.W and Boucher, R.C (1998) Evidence for periciliary liquid layer depletion, not abnormal ion 613 614 ABC PROTEINS: FROM BACTERIA TO MAN composition, in the pathogenesis of cystic fibrosis airways disease Cell 95(7), 1005–1015 McCarty, N.A (2000) Permeation through the CFTR chloride channel J Exp Biol 203 (Pt 13), 1947–1962 McDonough, S., Davidson, N., Lester, H.A and McCarty, N.A (1994) Novel pore-lining residues in CFTR that govern permeation and open-channel block Neuron 13(3), 623–634 Meacham, G.C., Lu, Z., King, S., Sorscher, E., Tousson, A and Cyr, D.M (1999) The Hdj-2/Hsc 70 chaperone pair facilitates early steps in CFTR biogenesis EMBO J 18, 1492–1505 Meacham, G.C., Patterson, C., Zhang, W., Younger, J.M and Cyr, D.M (2001) The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation Nat Cell Biol 3(1), 100–105 Middleton, L.M and Harvey, R.D (1998) PKC regulation of cardiac CFTR ClϪ channel function in guinea pig ventricular myocytes Am J Physiol 275(1 Pt 1), C293–C302 Milewski, M.I., Mickle, J.E., Forrest, J.K., Stafford, D.M., Moyer, B.D., Cheng, J., Guggino, W.B., Stanton, B.A and Cutting, G.R (2001) A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane J Cell Sci 114(Pt 4), 719–726 Morales, M.M., Carroll, T.P., Morita, T., Schwiebert, E.M., Devuyst, O., Wilson, P.D., et al (1996) Both the wild type and a functional isoform of CFTR are expressed in kidney Am J Physiol 270(6 Pt 2), F1038–F1048 Morello, J.P., Salahpour, A., Laperriere, A., Bernier, V., Arthus, M.F., Lonergan, M., et al (2000) Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants J Clin Invest 105(7), 887–895 Nagata, K., Nishitani, M., Matsuo, M., Kioka, N., Amachi, T and Ueda, K (2000) Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1 J Biol Chem 275(23), 17626–17630 Nagel, G (1999) Differential function of the two nucleotide binding domains on cystic fibrosis transmembrane conductance regulator Biochim Biophys Acta 1461(2), 263–274 Nagel, G., Szellas, T., Riordan, J.R., Friedrich, T and Hartung, K (2001) Non-specific activation of the epithelial sodium channel by the CFTR chloride channel EMBO Rep 2(3), 249–254 Nairn, A.C., Qin, J., Chait, B.T and Gadsby, D.C (1996) Identification of sites in the R-domain of CFTR phosphorylated by cAMP-dependent protein kinase and dephosphorylated by protein phosphatases 2A and 2C Pediatr Pulm (Suppl 13), 211 (Abstract) Naren, A.P., Nelson, D.J., Xie, W., Jovov, B., Pevsner, J., Bennett, M.K., Benos, D.J., Quick, M.W and Kirk, K.L (1997) Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms Nature 390(6657), 302–305 Naren, A.P., Quick, M.W., Collawn, J.F., Nelson, D.J and Kirk, K.L (1998) Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein–protein interactions Proc Natl Acad Sci USA 95(18), 10972–10977 Naren, A.P., Cormet-Boyaka, E., Fu, J., Villain, M., Blalock, J.E., Quick, M.W and Kirk, K.L (1999) CFTR chloride channel regulation by an interdomain interaction Science 286(5439), 544–548 Nelson, D.J., et al (2001) CFTR associates with the P11 subunit of the annexin II heterotetramer Pediatr Pulm (Suppl 18), 178 (abstract) Neville, D.C., Rozanas, C.R., Price, E.M., Gruis, D.B., Verkman, A.S and Townsend, R.R (1997) Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrixassisted laser desorption mass spectrometry Protein Sci 6(11), 2436–2445 Neville, D.C., Rozanas, C.R., Tulk, B.M., Townsend, R.R and Verkman, A.S (1998) Expression and characterization of the NBD1-R domain region of CFTR: evidence for subunit–subunit interactions Biochemistry 37(8), 2401–2409 Nishimura, N., Bannykh, S., Slabough, S., Matteson, J., Altschuler, Y., Hahn, K and Balch, W.E (1999) A di-acidic (DXE) code directs concentration of cargo during export from the endoplasmic reticulum J Biol Chem 274(22), 15937–15946 Noone, P.G and Knowles, M.R (2001) ‘CFTRopathies’: disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations Respir Res 2(6), 328–332 O’Reilly, C.M., Winpenny, J.P., Argent, B.E and Gray, M.A (2000) Cystic fibrosis THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) transmembrane conductance regulator currents in guinea pig pancreatic duct cells: inhibition by bicarbonate ions Gastroenterology 118(6), 1187–1196 Osborne, L.R., Lynch, M., Middleton, P.G., Alton, E.W., Geddes, D.M., Pryor, J.P., Hodson, M.E and Santis, G.K (1993) Nasal epithelial ion transport and genetic analysis of infertile men with congenital bilateral absence of the vas deferens Hum Mol Genet 2(10), 1605–1609 Ostedgaard, L.S., Baldursson, O and Welsh, M.J (2001) Regulation of the cystic fibrosis transmembrane conductance regulator ClϪ channel by its R domain J Biol Chem 276(11), 7689–7692 Picciotto, M.R., Cohn, J.A., Bertuzzi, G., Greengard, P and Nairn, A.C (1992) Phosphorylation of the cystic fibrosis transmembrane conductance regulator J Biol Chem 267, 12742–12752 Pignatti, P.F., Bombieri, C., Benetazzo, M., Casartelli, A., Trabetti, E., Gile, L.S., Martinati, L.C., Boner, A.L and Luisetti, M (1996) CFTR gene variant IVS8–5T in disseminated bronchiectasis Am J Hum Genet 58(4), 889–892 Pind, S., Riordan, J.R and Williams, D.B (1994) Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator J Biol Chem 269(12), 12784–12788 Poulsen, J.H., Fischer, H., Illek, B and Machen, T.E (1994) Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator Proc Natl Acad Sci USA 91(12), 5340–5344 Price, M.P., Ishihara, H., Sheppard, D.N and Welsh, M.J (1996) Function of Xenopus cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and use of human–Xenopus chimeras to investigate the pore properties of CFTR J Biol Chem 271(41), 25184–25191 Qu, B.H and Thomas, P.J (1996) Alteration of the cystic fibrosis transmembrane conductance regulator folding pathway J Biol Chem 271(13), 7261–7264 Quinton, P.M (1983) Chloride impermeability in cystic fibrosis Nature 301, 421–422 Quinton, P.M (2001) The neglected ion: HCO3 Nat Med 7(3), 292–293 Quinton, P.M and Bijman, J (1983) Higher bioelectric potentials due to decreased chloride absorption in the sweat glands of patients with cystic fibrosis N Engl J Med 308, 1185–1189 Quinton, P.M and Reddy, M.M (1992) Control of CFTR chloride conductance by ATP levels through non- hydrolytic binding Nature 360(6399), 79–81 Raghuram, V., Mak, D.D and Foskett, J.K (2001) Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction Proc Natl Acad Sci USA 98(3), 1300–1305 Ramjeesingh, M., Li, C., Garami, E., Huan, L.J., Galley, K., Wang, Y and Bear, C.E (1999) Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator) Biochemistry 38(5), 1463–1468 Ramjeesingh, M., Li, C., Kogan, I., Wang, Y., Huan, L.J and Bear, C.E (2001) A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator Biochemistry 40(35), 10700–10706 Randak, C., Roscher, A.A., Hadorn, H.B., Assfalg-Machleidt, I., Auerswald, E.A and Machleidt, W (1995) Expression and functional properties of the second predicted nucleotide binding fold of CFTR fused to GST FEBS Lett 363, 189–194 Randak, C., Neth, P., Auerswald, E.A., AssfalgMachleidt, I., Roscher, A.A., Hadorn, H.B and Machleidt, W (1996) A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein FEBS Lett 398(1), 97–100 Randak, C., Neth, P., Auerswald, E.A., Eckerskorn, C., Assfalg-Machleidt, I and Machleidt, W (1997) A recombinant polypeptide model of the second nucleotidebinding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase FEBS Lett 410(2–3), 180–186 Reddy, M.M., Light, M.J and Quinton, P.M (1999) Activation of the epithelial Naϩ channel (ENaC) requires CFTR ClϪ channel function Nature 402(6759), 301–304 Riordan, J.R (1999) Cystic fibrosis as a disease of misprocessing of the cystic fibrosis transmembrane conductance regulator glycoprotein Am J Hum Genet 64(6), 1499–1504 615 616 ABC PROTEINS: FROM BACTERIA TO MAN Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., et al (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA Science 245(4922), 1066–1073 Ritter, C and Helenius, A (2000) Recognition of local glycoprotein misfolding by the ER folding sensor UDP-glucose:glycoprotein glucosyltransferase Nat Struct Biol 7(4), 278–280 Rosenberg, M.F., Callaghan, R., Ford, R.C and Higgins, C.F (1997) Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis J Biol Chem 272(16), 10685–10694 Rosenberg, M.F., Mao, Q., Holzenburg, A., Ford, R.C., Deeley, R.G and Cole, S.P (2001) The structure of the multidrug resistance protein (MRP1/ABCC1) crystallization and single-particle analysis J Biol Chem 276(19), 16076–16082 Rubenstein, R.C and Zeitlin, P.L (2000) Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR Am J Physiol Cell Physiol 278(2), C259–C267 Sato, S., Ward, C.L and Kopito, R.R (1998) Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro J Biol Chem 273(13), 7189–7192 Saurin, W., Hofnung, M and Dassa, E (1999) Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters J Mol Evol 48(1), 22–41 Schoumacher, R.A., Shoemaker, R.L., Halm, D.R., Tallant, E.A., Wallace, R.W and Frizzell, R.A (1987) Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells Nature 330, 752–754 Schultz, B.D., Bridges, R.J and Frizzell, R.A (1996) Lack of conventional ATPase properties in CFTR chloride channel gating J Membr Biol 151(1), 63–75 Schultz, B.D., Singh, A.K., Devor, D.C and Bridges, R.J (1999) Pharmacology of CFTR chloride channel activity Physiol Rev 79(1 Suppl.): S109–S144 Schwiebert, E.M., Benos, D.J., Egan, M.E., Stutts, M.J and Guggino, W.B (1999) CFTR is a conductance regulator as well as a chloride channel Physiol Rev 79, 145 Seibert, F.S., Tabcharani, J.A., Chang, X.-B., Dulhanty, A.M., Mathews, C., Hanrahan, J.W and Riordan, J.R (1995) cAMP-dependent protein kinase-mediated phosphorylation of cystic fibrosis transmembrane conductance regulator residue ser-753 and its role in channel activation J Biol Chem 270(5), 2158–2162 Seibert, F.S., Linsdell, P., Loo, T.W., Hanrahan, J.W., Clarke, D.M and Riordan, J.R (1996a) Disease-associated mutations in the fourth cytoplasmic loop of CFTR compromise biosynthetic processing and chloride channel activity J Biol Chem 271, 15139–15143 Seibert, F.S., Linsdell, P., Loo, T.W., Hanrahan, J.W., Riordan, J.R and Clarke, D.M (1996b) Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity J Biol Chem 271(44), 27493–27499 Seibert, F.S., Jia, Y., Mathews, C.J., Hanrahan, J.W., Riordan, J.R., Loo, T.W and Clarke, D.M (1997) Disease-associated mutations in cytoplasmic loops and of cystic fibrosis transmembrane conductance regulator impede processing or opening of the channel Biochemistry 36(39), 11966–11974 Senior, A.E and Gadsby, D.C (1997) ATP hydrolysis cycles and mechanism in P-glycoprotein and CFTR Semin Cancer Biol 8(3), 143–150 Sharer, N., Schwarz, M., Malone, G., Howarth, A., Painter, J., Super, M and Braganza, J (1998) Mutations of the cystic fibrosis gene in patients with chronic pancreatitis N Engl J Med 339(10), 645–652 Sheppard, D.N and Welsh, M.J (1999) Structure and function of the CFTR chloride channel Physiol Rev 79(1 Suppl.): S23–S45 Sheppard, D.N., Rich, D.P., Ostedgaard, L.S., Gregory, R.J., Smith, A.E and Welsh, M.J (1993) Mutations in CFTR associated with mild-disease-form ClϪ channels with altered pore properties Nature 362(6416), 160–164 Sheppard, D.N., Ostedgaard, L.S., Rich, D.P and Welsh, M.J (1994) The amino-terminal portion of CFTR forms a regulated ClϪ channel Cell 76(6), 1091–1098 Short, D.B., Trotter, K.W., Reczek, D., Kreda, S.M., Bretscher, A., Boucher, R.C., Stutts, M.J and Milgram, S.L (1998) An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton J Biol Chem 273(31), 19797–19801 Smith, J.J., Travis, S.M., Greenberg, E.P and Welsh, M.J (1996) Cystic fibrosis airway epithelia fail to kill bacteria because of THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) abnormal airway surface fluid [published erratum appears in Cell 1996 Oct 18; 87(2), following 355] Cell 85(2), 229–236 Smith, K.D., Kemp, S., Braiterman, L.T., Lu, J.F., Wei, H.M., Geraghty, M., Stetten, G., Bergin, J.S., Pevsner, J and Watkins, P.A (1999) X-linked adrenoleukodystrophy: genes, mutations, and phenotypes Neurochem Res 24(4), 521–535 Smith, S.S., Liu, X., Zhang, Z.R., Sun, F., Kriewall, T.E., McCarty, N.A and Dawson, D.C (2001) CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction J Gen Physiol 118(4), 407–431 Sturgess, J and Imrie, J (1982) Quantitative evaluation of the development of tracheal submucosal glands in infants with cystic fibrosis and control infants Am J Pathol 106(3), 303–311 Stutts, M.J., Canessa, C.M., Olsen, J.C., Hamrick, M., Cohn, J.A., Rossier, B.C and Boucher, R.C (1995) CFTR as a cAMPdependent regulator of sodium channels Science 269, 847–850 Szabo, K., Szakacs, G., Hegeds, T and Sarkadi, B (1999) Nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator Different patterns in the two nucleotide binding domains J Biol Chem 274(18), 12209–12212 Tabcharani, J.A., Low, D., Elie, D and Hanrahan, J.W (1990) Low conductance chloride channel activated by cAMP in the epithelial cell line T84 FEBS Lett 270, 157–164 Tabcharani, J.A., Chang, X.-B., Riordan, J.R and Hanrahan, J.W (1991) Phosphorylationregulated ClϪ channel in CHO cells stably expressing the cystic fibrosis gene Nature 352, 628–631 Tabcharani, J.A., Chang, X.-B., Riordan, J.R and Hanrahan, J.W (1992) The cystic fibrosis transmembrane conductance regulator chloride channel: Iodide block and permeation Biophys J 62, 1–4 Tabcharani, J.A., Rommens, J.M., Hou, Y.X., Chang, X.B., Tsui, L.C., Riordan, J.R and Hanrahan, J.W (1993) Multi-ion pore behaviour in the CFTR chloride channel Nature 366(6450), 79–82 Therien, A.G., Grant, F.E and Deber, C.M (2001) Interhelical hydrogen bonds in the CFTR membrane domain Nat Struct Biol 8(7), 597–601 Thomas, P.J and Hunt, J.F (2001) A snapshot of Nature’s favorite pump Nat Struct Biol 8(11), 920–923 Travis, S.M., Carson, M.R., Ries, D.R and Welsh, M.J (1993) Interaction of nucleotides with membrane-associated cystic fibrosis transmembrane conductance regulator J Biol Chem 268(21), 15336–15339 Travis, S.M., Berger, H.A and Welsh, M.J (1997) Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator Proc Natl Acad Sci USA 94(20), 11055–11060 Urbatsch, I.L., Sankaran, B., Weber, J and Senior, A.E (1995) P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site J Biol Chem 270(33), 19383–19390 Wang, S., Yue, H., Derin, R.B., Guggino, W.B and Li, M (2000) Accessory protein facilitated CFTR–CFTR interaction, a molecular mechanism to potentiate the chloride channel activity Cell 103(1), 169–179 Ward, C.L., Omura, S and Kopito, R.R (1995) Degradation of CFTR by the ubiquitinproteasome pathway Cell 83, 121–127 Weixel, K.M and Bradbury, N.A (2001) Mu binding directs the cystic fibrosis transmembrane conductance regulator to the clathrinmediated endocytic pathway J Biol Chem 276(49), 46251–46259 Welsh, M.J., Robertson, A.D and Ostegaard L.S (1998) Structural biology The ABC of a versatile engine Nature 396(6712), 623–624 Wilkinson, D.J., Strong, T.V., Mansoura, M.K., Wood, D.L., Smith, S.S., Collins, F.S and Dawson, D.C (1997) CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain Am J Physiol 273(1 Pt 1), L127–L133 Willumsen, N.J and Boucher, R.C (1989) Shunt resistance and ion permeabilities in normal and cystic fibrosis airway epithelia Am J Physiol 256, C1054–C1063 Yamazaki, J., Britton, F., Collier, M.L., Horowitz, B and Hume, J.R (1999) Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C Biophys J 76(4), 1972–1987 Yang, N., George, A.L., Jr and Horn, R (1996) Molecular basis of charge movement in voltage-gated sodium channels Neuron 16(1), 113–122 617 618 ABC PROTEINS: FROM BACTERIA TO MAN Yang, Y., Janich, S., Cohn, J.A and Wilson, J.M (1993) The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment Proc Natl Acad Sci USA 90, 9480–9484 Yoo, J.-S., Moyer, B.D., Bannykh, S.I., Yoo, H.-M., Riordan, J.R and Balch, W.E (2002) Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) through the early secretory pathway J Biol Chem 277, 1140–1149 Yue, H., Devidas, S and Guggino, W.B (2000) The two halves of CFTR form a dual-pore ion channel J Biol Chem 275(14), 10030–10034 Zeitlin, P.L (2000) Pharmacologic restoration of delta F508 CFTR-mediated chloride current Kidney Int 57(3), 832–837 Zerangue, N., Schwappach, B., Jan, Y.N and Jan, L.Y (1999) A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane KATP channels Neuron 22, 537–548 Zerhusen, B., Zhao, J., Xie, J., Davis, P.B and Ma, J (1999) A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules J Biol Chem 274(12), 7627–7630 Zhang, Z.R., McDonough, S.I and McCarty, N.A (2000) Interaction between permeation and gating in a putative pore domain mutant in the cystic fibrosis transmembrane conductance regulator Biophys J 79(1), 298–313 Zhu, T., Dahan, D., Evagelidis, A., Zheng, S., Luo, J and Hanrahan, J.W (1999) Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C J Biol Chem 274(41), 29102–29107 Zou, X and Hwang, T.C (2001) ATP hydrolysis-coupled gating of CFTR chloride channels: structure and function Biochemistry 40(19), 5579–5586 ... Winpenny, J.P., Argent, B.E and Gray, M.A (2000) Cystic fibrosis THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) transmembrane conductance regulator currents in guinea pig pancreatic... inhibition of hydrolysis there, which in one version of the model is required for THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) the dissociation of the product of hydrolysis... its structure and function THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (ABCC7) CFTR IS AN EPITHELIAL CHLORIDE CHANNEL CHLORIDE CONDUCTANCE DEFECT IN CYSTIC FIBROSIS Electrophysiological