Cystic Fibrosis Transmembrane Conductance Regulator

cystic fibrosis transmembrane conductance regulator cystic fibrosis 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid multiple drug resistance protein nucleotide-binding domain cAMP-dependent protein kinase protein kinase C endoplasmic reticulum The cystic fibrosis transmembrane conductance regulator (CFTR)1 forms a Cl− channel that is an essential component of epithelial Cl− transport systems in many organs, including the intestines, pancreas, lungs, sweat glands, and kidneys. In the Cl− secretory intestinal epithelium, Cl−enters the cells through a Na+-K+-2Cl− cotransporter in the basolateral membrane and exits through CFTR in the apical membrane; water follows osmotically (1.Field M. Semrad C.E. Annu. Rev. Physiol. 1993; 55: 631-655Crossref PubMed Scopus (80) Google Scholar). Absorptive epithelia use similar transporters and channels, but their polarized distribution between the apical and basolateral membranes is usually reversed. A major determinant of the transepithelial Cl− transport rate is the level of activation of CFTR (2.Gabriel S.E. Brigman K.N. Koller B.H. Boucher R.C. Stutts M.J. Science. 1994; 266: 107-109Crossref PubMed Scopus (379) Google Scholar, 3.Cuthbert A.W. Hickman M.E. MacVinish L.J. Evans M.J. Colledge W.H. Ratcliff R. Seale P.W. Humphrey P.P. Br. J. Pharmacol. 1994; 112: 31-36Crossref PubMed Scopus (92) Google Scholar), which depends on the extent to which it is phosphorylated. This is determined by the relative activities of kinases and phosphatases, the activities of which are often hormonally regulated (1.Field M. Semrad C.E. Annu. Rev. Physiol. 1993; 55: 631-655Crossref PubMed Scopus (80) Google Scholar). Defects in the gene encoding CFTR that reduce either its Cl− transport capacity or its level of cell surface expression cause cystic fibrosis (CF) (4.Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1266) Google Scholar, 5.Pilewski J.M. Frizzell R.A. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (387) Google Scholar, 6.Wine J.J. J. Clin. Invest. 1999; 103: 309-312Crossref PubMed Scopus (219) Google Scholar) as well as a form of male sterility due to congenital bilateral absence of the vas deferens (7.De Braekeleer M. Ferec C. Mol. Hum. Reprod. 1996; 2: 669-677Crossref PubMed Scopus (91) Google Scholar). CF is the most common lethal genetic disease in Caucasians, with about 30,000 CF patients in the United States. In contrast, in intestinal epithelial cells overstimulation of CFTR because of the activation of protein kinases by bacterial enterotoxins causes secretory diarrhea (1.Field M. Semrad C.E. Annu. Rev. Physiol. 1993; 55: 631-655Crossref PubMed Scopus (80) Google Scholar,8.Hansen M.B. Skadhauge E. Physiol. Res. 1995; 44: 61-78PubMed Google Scholar). Secretory diarrhea is the second largest cause of infant mortality in the developing world, causing 3 million deaths per year of children under the age of 5. Thus, although CFTR was named because of its association with CF, as a cause of disease, its relationship to secretory diarrhea is a more widespread public health problem. The cloning of CFTR in 1989 (9.Riordan J.R. Rommens J.M. Kerem B.S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.T. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 254: 1066-1073Crossref Scopus (6210) Google Scholar) has facilitated studies of its structure, function, regulation, biogenesis, and degradation, which will be reviewed in this article. Issues reviewed elsewhere and not discussed here include the mechanisms by which mutations in CFTR cause CF (5.Pilewski J.M. Frizzell R.A. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (387) Google Scholar, 6.Wine J.J. J. Clin. Invest. 1999; 103: 309-312Crossref PubMed Scopus (219) Google Scholar) and the possible role of CFTR in regulating the pH within intracellular organelles (10.al-Awqati Q. Curr. Opin. Cell Biol. 1995; 7: 504-508Crossref PubMed Scopus (97) Google Scholar). CFTR is a member of the ATP-bindingcassette (ABC) membrane transporter gene superfamily that includes both eukaryotic and bacterial proteins, such as the multiple drug resistance protein (MDR), the sulfonylurea receptor, the transporter for antigen presentation, and the bacterial periplasmic permeases (9.Riordan J.R. Rommens J.M. Kerem B.S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.T. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 254: 1066-1073Crossref Scopus (6210) Google Scholar, 11.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3434) Google Scholar). Being an ion channel makes CFTR unique in this gene superfamily in which most other members are ATP-driven membrane transporters. CFTR contains 1480 amino acids and consists of two homologous halves (Fig. 1). Each half contains six membrane-spanning segments and a nucleotide-binding domain (NBD). The two halves of CFTR are linked by a cytoplasmic regulatory domain (R-domain) that contains a number of consensus phosphorylation sites (9.Riordan J.R. Rommens J.M. Kerem B.S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.T. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 254: 1066-1073Crossref Scopus (6210) Google Scholar). In the proposed transmembrane topology (Fig. 1), which is supported by experimental evidence (9.Riordan J.R. Rommens J.M. Kerem B.S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.T. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 254: 1066-1073Crossref Scopus (6210) Google Scholar, 12.Riordan J.R. Annu. Rev. Physiol. 1993; 55: 609-630Crossref PubMed Scopus (309) Google Scholar, 13.Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar), 77% of the protein is in the cytoplasm, 19% in membrane-spanning segments, and 4% in extracellular loops, which (except for the M1–M2 and M7–M8 loops) are very short. The M7–M8 loop contains two N-linked glycosylation sites that are used in vivo (12.Riordan J.R. Annu. Rev. Physiol. 1993; 55: 609-630Crossref PubMed Scopus (309) Google Scholar, 13.Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar). Covalent linkage of the two halves is not required for assembly and function. When expressed as separate proteins in the same cells the halves assembled into functional channels and could be co-immunoprecipitated. This assembly required interactions within the membrane-spanning domains (13.Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar, 14.Ostedgaard L.S. Rich D.P. DeBerg L.G. Welsh M.J. Biochemistry. 1997; 36: 1287-1294Crossref PubMed Scopus (61) Google Scholar). CFTR was inferred to be a monomer because following solubilization, with either ionic or nonionic detergents, biochemically distinct forms of CFTR, expressed in the same cells, could not be co-immunoprecipitated (15.Marshall J. Fang S. Ostedgaard L.S. O'Riordan C.R. Ferrara D. Amara J.F. Hoppe H., IV Scheule R.K. Welsh M.J. Smith A.E. Cheng S.H. J. Biol. Chem. 1994; 269: 2987-2995Abstract Full Text PDF PubMed Google Scholar). In contrast, two studies concluded that CFTR is a homodimer in situ based on the size of CFTR particles in freeze-fracture electron micrographs (16.Eskandari S. Wright E.M. Kreman M. Starace D.M. Zampighi G.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11235-11240Crossref PubMed Scopus (163) Google Scholar) and on the functional effects of coexpressing mutants with distinct functional properties (17.Zerhusen B. Zhao J. Xie J. Davis P.B. Ma J. J. Biol. Chem. 1999; 274: 7627-7630Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In the homologous MDR transporter, however, dimers were not seen in cryoelectron microscopic images (18.Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). The channel lining is formed, at least in part, by residues from among the 12 membrane-spanning segments. The major determinants of the channel's functional properties are likely to be among the channel-lining residues. Water-accessible residues inferred to line the channel were identified in the M1, M3, and M6 segments using the substituted cysteine accessibility method (19.Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 20.Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 21.Akabas M.H. Biochemistry. 1998; 37: 12233-12240Crossref PubMed Scopus (37) Google Scholar). These segments' secondary structure was inferred to be largely α-helical based on the patterns formed by the channel-lining residues (19.Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 20.Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 21.Akabas M.H. Biochemistry. 1998; 37: 12233-12240Crossref PubMed Scopus (37) Google Scholar). Further support for an α-helical secondary structure of the membrane-spanning segments was provided by studies of synthetic peptides, the sequences of which corresponded to those of the M1–M6 segments. These peptides were largely α-helical in liposomes and in detergent micelles (22.Peng S. Liu L.P. Emili A.Q. Deber C.M. FEBS Lett. 1998; 431: 29-33Crossref PubMed Scopus (19) Google Scholar, 23.Wigley W.C. Vijayakumar S. Jones J.D. Slaughter C. Thomas P.J. Biochemistry. 1998; 37: 844-853Crossref PubMed Scopus (38) Google Scholar). The minimum channel diameter was inferred to be ∼5.3 Å based on the size of the largest permeant anion (24.Linsdell P. Tabcharani J.A. Rommens J.M. Hou Y.X. Chang X.B. Tsui L.C. Riordan J.R. Hanrahan J.W. J. Gen. Physiol. 1997; 110: 355-364Crossref PubMed Scopus (181) Google Scholar). Further studies using patches with larger numbers of channels showed that anions as large as lactobionate (10–13 Å in diameter) were slightly permeable. Thus, at least transiently, the diameter of the channel must be 10–13 Å (25.Linsdell P. Hanrahan J.W. J. Gen. Physiol. 1998; 111: 601-614Crossref PubMed Scopus (126) Google Scholar). In the presence of cytoplasmic ATP the large anions were only permeable from the cytoplasmic side (25.Linsdell P. Hanrahan J.W. J. Gen. Physiol. 1998; 111: 601-614Crossref PubMed Scopus (126) Google Scholar). The molecular basis for asymmetric permeation by large anions is unknown. There is no asymmetry in the conduction of small anions; the channel has a linear current-voltage relationship in Cl− solutions (13.Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar). Although some studies suggested that ATP was permeable through CFTR (reviewed in Ref. 26.Schwiebert E.M. Benos D.J. Egan M.E. Stutts M.J. Guggino W.B. Physiol. Rev. 1999; 79: S145-S166Crossref PubMed Scopus (383) Google Scholar), it has been convincingly demonstrated that there is no measurable electrogenic ATP flux through CFTR (27.Grygorczyk R. Tabcharani J.A. Hanrahan J.W. J. Membr. Biol. 1996; 151: 139-148Crossref PubMed Scopus (78) Google Scholar, 28.Li C. Ramjeesingh M. Bear C.E. J. Biol. Chem. 1996; 271: 11623-11626Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 29.Reddy M.M. Quinton P.M. Haws C. Wine J.J. Grygorczyk R. Tabcharani J.A. Hanrahan J.W. Gunderson K.L. Kopito R.R. Science. 1996; 271: 1876-1879Crossref PubMed Scopus (163) Google Scholar). The ability to discriminate between Cl− and cations is essential for the role of CFTR in epithelial Cl− transport. The extent of anion selectivity, as measured by the Cl− to Na+permeability ratio (30.Dawson D.C. Smith S.S. Mansoura M.K. Physiol. Rev. 1999; 79: S47-S75Crossref PubMed Scopus (121) Google Scholar), ranges between 10 and 300 (31.Anderson M.P. Gregory R.J. Thompson S. Souza D.W. Paul S. Mulligan R.C. Smith A.E. Welsh M.J. Science. 1991; 253: 202-205Crossref PubMed Scopus (954) Google Scholar, 32.Tabcharani J.A. Linsdell P. Hanrahan J.W. J. Gen. Physiol. 1997; 110: 341-354Crossref PubMed Scopus (103) Google Scholar). We showed that the charge selectivity filter that discriminates between anions and cations is located at the cytoplasmic end of the channel and that both anions and cations can enter the extracellular end of the channel (33.Cheung M. Akabas M.H. J. Gen. Physiol. 1997; 109: 289-300Crossref PubMed Scopus (86) Google Scholar). Arg-352, at the cytoplasmic end of M6, is a major determinant of charge selectivity. Removal of the positive charge at this position reduced the Cl− to Na+ permeability ratio approximately 10-fold (34.Guinamard R. Akabas M.H. Biochemistry. 1999; 38: 5528-5537Crossref PubMed Scopus (36) Google Scholar). We hypothesized that the positive charge of Arg-352 creates an electrostatic barrier to cation permeation. Near Arg-352 the channel must be narrow enough so that the electrostatic barrier extends across the channel lumen (34.Guinamard R. Akabas M.H. Biochemistry. 1999; 38: 5528-5537Crossref PubMed Scopus (36) Google Scholar). The channel displays modest selectivity between halides. The halide permeability sequence is I− (1.8) > Br− (1.2) > Cl− (1.0) > F− (0.2), indicative of weak interactions between permeating anions and channel binding sites (30.Dawson D.C. Smith S.S. Mansoura M.K. Physiol. Rev. 1999; 79: S47-S75Crossref PubMed Scopus (121) Google Scholar, 32.Tabcharani J.A. Linsdell P. Hanrahan J.W. J. Gen. Physiol. 1997; 110: 341-354Crossref PubMed Scopus (103) Google Scholar). Multiple anions can simultaneously occupy the channel, thereby resulting in anomalous mole fraction effects in the single channel conductance in mixtures of Cl− and SCN−. These effects were eliminated in the M6 mutant R347D, and Arg-347, therefore, was hypothesized to be at or near an anion binding site (35.Tabcharani J.A. Rommens J.M. Hou Y.X. Chang X.B. Tsui L.C. Riordan J.R. Hanrahan J.W. Nature. 1993; 366: 79-82Crossref PubMed Scopus (210) Google Scholar). Arg-347, however, has been shown to form a salt bridge with Asp-924 (36.Cotten J.F. Welsh M.J. J. Biol. Chem. 1999; 274: 5429-5435Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Therefore, it is possible that rather than Arg-347 being a binding site itself, structural perturbation caused by the loss of the salt bridge disrupted an anion binding site elsewhere (30.Dawson D.C. Smith S.S. Mansoura M.K. Physiol. Rev. 1999; 79: S47-S75Crossref PubMed Scopus (121) Google Scholar). CFTR has few inhibitors and none are of high affinity or specificity (13.Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar). The affinity of the channel blocker diphenylamine 2-carboxylate was reduced by mutating two water-accessible residues in M6. Mutations of the aligned M12 residues increased diphenylamine 2-carboxylate affinity suggesting that the channel is lined by residues from both halves of CFTR (37.McDonough S. Davidson N. Lester H.A. McCarty N.A. Neuron. 1994; 13: 623-634Abstract Full Text PDF PubMed Scopus (161) Google Scholar). In contrast to their actions on other Cl− channels and transporters, disulfonic stilbenes such as DIDS inhibit only when applied to the cytoplasmic side (38.Linsdell P. Hanrahan J.W. J. Physiol. (Lond.). 1996; 496: 687-693Crossref Scopus (119) Google Scholar). Cytoplasmic application of other large anions also caused flickery block of CFTR. This is further evidence that the channel is asymmetric and contains an anion binding site in the cytoplasmic vestibule (39.Linsdell P. Hanrahan J.W. Am. J. Physiol. 1996; 271: C628-C634Crossref PubMed Google Scholar). The residues forming the cytoplasmic vestibule remain to be identified, but the cytoplasmic loops probably do not contribute to the vestibule because none of the CF-related loop mutations affected ion conduction (40.Seibert F.S. Jia Y. Mathews C.J. Hanrahan J.W. Riordan J.R. Loo T.W. Clarke D.M. Biochemistry. 1997; 36: 11966-11974Crossref PubMed Scopus (67) Google Scholar). The picture that is emerging is of a channel with a large extracellular vestibule that extends into the plain of the membrane and is accessible to anions and cations from the extracellular side (Fig.2). The channel narrows toward the cytoplasmic end where the charge selectivity filter is located. Finally, there is a short cytoplasmic vestibule that contains an anion binding site. The channel lining is formed, in part, by residues from the M1, M3, M6, and M12 segments. Little is known about whether the other eight membrane-spanning segments contribute to the channel lining. The location of the gate that blocks ion conduction through the channel in the closed state is unknown. The gate, however, is controlled by conformational changes in the cytoplasmic domains. Two separate processes control the gating of CFTR: 1) phosphorylation and 2) binding and hydrolysis of ATP. Phosphorylation is necessary for activation, but it is not sufficient. After phosphorylation, gating between the closed and open states is controlled by ATP hydrolysis (12.Riordan J.R. Annu. Rev. Physiol. 1993; 55: 609-630Crossref PubMed Scopus (309) Google Scholar, 13.Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar, 41.Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (375) Google Scholar). The contains multiple consensus phosphorylation sites for cAMP-dependent protein kinase protein kinase C and protein kinase (9.Riordan J.R. Rommens J.M. Kerem B.S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.T. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 254: 1066-1073Crossref Scopus (6210) Google Scholar, 12.Riordan J.R. Annu. Rev. Physiol. 1993; 55: 609-630Crossref PubMed Scopus (309) Google D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar, 41.Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (375) Google Scholar, A. U. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar). The more the phosphorylation, the the channel open D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (375) Google Scholar, M. D.C. J. Gen. Physiol. 1993; PubMed Scopus Google Scholar). There to be in the regulatory phosphorylation sites are in but of sites or 10 sites reduced but not activation Tabcharani J.A. Hou N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). CFTR is of the 10 sites that phosphorylation at sites is for channel such site has been identified (12.Riordan J.R. Annu. Rev. Physiol. 1993; 55: 609-630Crossref PubMed Scopus (309) Google Scholar, 13.Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar, 41.Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (375) Google Scholar). phosphorylation be a for activation by Y. Mathews C.J. Hanrahan J.W. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, although a kinase CFTR this through a with CFTR and not a kinase (reviewed in Ref. 41.Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (375) Google Scholar). After phosphorylation, CFTR is by protein D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (375) Google Scholar). is largely by protein and H.A. Welsh M.J. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: PubMed Google Scholar). is linked to CFTR in the membrane as it could be co-immunoprecipitated with CFTR as well as to CFTR by and could not D. A. S. J. Hanrahan J.W. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation, and of the channel is controlled by ATP binding and hydrolysis (12.Riordan J.R. Annu. Rev. Physiol. 1993; 55: 609-630Crossref PubMed Scopus (309) Google Scholar, 13.Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar, 41.Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (375) Google Scholar). ATP do not support channel M.P. H.A. Rich D.P. Gregory R.J. Smith A.E. Welsh M.J. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). CFTR ATP with a of and ATP at a rate of about C. Ramjeesingh M. E. M. D. Rommens J.M. Bear C.E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), similar to the rate of channel ATP hydrolysis in the which A and in other (9.Riordan J.R. Rommens J.M. Kerem B.S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.T. Iannuzzi M.C. 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Rev. 1999; 79: PubMed Scopus Google Hou Y. Chang X.B. Riordan J.R. J. 1998; PubMed Scopus Google Scholar). The molecular basis for the of CFTR in the protein synthetic to be CFTR has an role in epithelial both as a Cl− channel and as a regulator of other channels and transporters. of the channel to a picture of its structure and CFTR with other channels and transporter will into the of epithelial transport and the that from in CFTR or from its overstimulation by bacterial that further studies will a basis for the or of high affinity CFTR inhibitors that be for studies of the role of CFTR in epithelial Cl− transport and as a for secretory a major cause of and infant mortality in the developing