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The ABC protein turned chloride channel whose failure causes cystic fibrosis

Abstract

CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

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Figure 1: Opening and closing of CFTR channels.
Figure 2: Possible structure and organization of domains in CFTR.
Figure 3: The conserved Walker A lysine is critical for ATP binding in each NBD.
Figure 4: ATP hydrolysis prompts channel closure.
Figure 5: Structures of the MalK homodimer.

References

  1. 1

    Rommens, J. M. et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245, 1059–1065 (1989).

    ADS  CAS  Google Scholar 

  2. 2

    Riordan, J. R. et al. Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066–1073 (1989).

    ADS  CAS  PubMed  Google Scholar 

  3. 3

    Quinton, P. M. Chloride impermeability in cystic fibrosis. Nature 301, 421–422 (1983).

    ADS  CAS  Google Scholar 

  4. 4

    Schoumacher, R. A. et al. Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells. Nature 330, 752–754 (1987)

    ADS  CAS  Google Scholar 

  5. 5

    Li, M. et al. Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium. Nature 331, 358–360 (1988).

    ADS  CAS  Google Scholar 

  6. 6

    Bear, C. E. et al. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell 68, 809–818 (1992).

    CAS  Google Scholar 

  7. 7

    Altschuler Y., Hodson C. & Milgram S. L. The apical compartment: trafficking pathways, regulators and scaffolding proteins. Curr. Opin. Cell Biol. 15, 423–429 (2003).

    CAS  Google Scholar 

  8. 8

    Li, C. & Naren, A. P. Macromolecular complexes of cystic fibrosis transmembrane conductance regulator and its interacting partners. Pharmacol. Ther. 108, 208–223 (2005).

    CAS  Google Scholar 

  9. 9

    Du, K., Sharma, M. & Lukacs, G. L. The DeltaF508 cystic fibrosis mutation impairs domain-domain interactions and arrests post-translational folding of CFTR. Nature Struct. Mol. Biol. 12, 17–25 (2005).

    CAS  Google Scholar 

  10. 10

    Wine, J. J. Acid in the airways. Focus on ‘Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis’. Am. J. Physiol. Cell Physiol. 290, C669–C671 (2006).

    CAS  Google Scholar 

  11. 11

    Davies, J. C. & Alton, E. W. Airway gene therapy. Adv. Genet. 54, 291–314 (2005).

    CAS  Google Scholar 

  12. 12

    Dean, M., Rzhetsky, A. & Allikmets, R. The human ATP-binding cassette (ABC) transporter superfamily. Genome Res. 11, 1156–1166 (2001).

    CAS  Google Scholar 

  13. 13

    Reyes, C. L. & Chang, G. Structure of the ABC transporter MsbA in complex with ADP.vanadate and lipopolysaccharide. Science 308, 1028–1031 (2005).

    ADS  CAS  Google Scholar 

  14. 14

    Locher, K. P., Lee, A. T. & Rees, D. C. The E. coli BtuCD structure: A framework for ABC transporter architecture and mechanism. Science 296, 1091–1098 (2002).

    ADS  CAS  Google Scholar 

  15. 15

    Rosenberg, M. F., Callaghan, R., Modok, S., Higgins, C. F. & Ford, R. C. Three-dimensional structure of P-glycoprotein: the transmembrane regions adopt an asymmetric configuration in the nucleotide-bound state. J. Biol. Chem. 280, 2857–2862 (2005).

    CAS  Google Scholar 

  16. 16

    Ramjeesingh, M., Kidd, J. F., Huan, L. J., Wang, Y. & Bear, C. E. Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane. Biochem. J. 374, 793–797 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Raghuram, V., Mak, D. D. & Foskett, J. K. Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction. Proc. Natl Acad. Sci. USA 98, 1300–1305 (2001).

    ADS  CAS  Google Scholar 

  18. 18

    Chen, J. H., Chang, X. B., Aleksandrov, A. A. & Riordan, J. R. CFTR is a misnomer: Biochemical and functional evidence. J. Membr. Biol. 188, 55–71 (2002).

    CAS  Google Scholar 

  19. 19

    Zhang, Z. R. et al. Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel: One polypeptide forms one pore. J. Biol. Chem. 280, 458–468 (2005).

    CAS  Google Scholar 

  20. 20

    Dawson, D. C., Liu, X., Zhang, Z. & McCarty, N. A. in Cystic Fibrosis Transmembrane Conductance Regulator (eds Kirk, K. L. & Dawson, D. C.) 1–34 (Kluwer/Plenum, New York, 2003).

    Google Scholar 

  21. 21

    Linsdell, P. Mechanism of chloride permeation in the cystic fibrosis transmembrane conductance regulator chloride channel. Exp. Physiol. 99, 123–129 (2006).

    Google Scholar 

  22. 22

    Tabcharani, J. A., Chang, X. B., Riordan, J. R. & Hanrahan, J. W. Phosphorylation-regulated Cl channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352, 628–631 (1991).

    ADS  CAS  Google Scholar 

  23. 23

    Hung, L. W. et al. Crystal structure of the ATP-binding subunit of an ABC transporter. Nature 396, 703–707 (1998).

    ADS  CAS  Google Scholar 

  24. 24

    Hopfner, K. P. et al. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell 101, 789–800 (2000).

    CAS  Google Scholar 

  25. 25

    Smith, P. C. et al. ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol. Cell 10, 139–149 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Chen, J., Lu, G., Lin, J., Davidson, A. L. & Quiocho, F. A. A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol. Cell 12, 651–661 (2003).

    CAS  Google Scholar 

  27. 27

    Zaitseva, J., Jenewein, S., Jumpertz, T., Holland, I. B. & Schmitt, L. H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. EMBO J. 24, 1901–1910 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Moody, J. E., Millen, L., Binns, D., Hunt, J. F. & Thomas, P. J. Cooperative, ATP-dependent association of the nucleotide-binding cassettes during the catalytic cycle of ATP-binding cassette transporters. J. Biol. Chem. 277, 21111–21114 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Lewis, H. A. et al. Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator. EMBO J. 23, 282–293 (2004) .

    CAS  Google Scholar 

  31. 31

    Lewis, H. A. et al. Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. J. Biol. Chem. 280, 1346–1353 (2005).

    CAS  Google Scholar 

  32. 32

    Thibodeau, P. H., Brautigam, C. A., Machius, M. & Thomas, P. J. Side chain and backbone contributions of Phe508 to CFTR folding. Nature Struct. Mol. Biol. 12, 10–16 (2005).

    CAS  Google Scholar 

  33. 33

    Rosenberg, M. F. et al. Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR). J. Biol. Chem. 279, 39051–39057 (2004).

    CAS  Google Scholar 

  34. 34

    Tabcharani, J. A. et al. Multi-ion pore behaviour in the CFTR chloride channel. Nature 366, 79–82 (1993).

    ADS  CAS  Google Scholar 

  35. 35

    Cotten, J. F. & Welsh, M. J. Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge. J. Biol. Chem. 274, 5429–5435 (1999).

    CAS  Google Scholar 

  36. 36

    Csanády, L., Chan, K. W., Nairn, A. C. & Gadsby, D. C. Functional roles of nonconserved structural segments in CFTR's NH2-terminal nucleotide binding domain. J. Gen. Physiol. 125, 43–55 (2005).

    PubMed  PubMed Central  Google Scholar 

  37. 37

    Cheng, S. H. et al. Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66, 1027–1036 (1991).

    CAS  Google Scholar 

  38. 38

    Picciotto, M., Cohn, J., Bertuzzi, G., Greengard, P. & Nairn, A.C. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 267, 12742–12752 (1992).

    CAS  Google Scholar 

  39. 39

    Csanády, L. et al. Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA. J. Gen. Physiol. 125, 171–186 (2005).

    PubMed  PubMed Central  Google Scholar 

  40. 40

    Neville, D. C. A. et al. Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry. Protein Sci. 6, 2436–2445 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Berger, H.A., Travis, S. M. & Welsh, M. J. Regulation of the cystic fibrosis transmembrane conductance regulator Cl channel by specific protein kinases and protein phosphatases. J. Biol. Chem. 268, 2037–2047 (1993).

    CAS  Google Scholar 

  42. 42

    Jia, Y., Mathews, C. J. & Hanrahan, J. W. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272, 4978–4984 (1997).

    CAS  Google Scholar 

  43. 43

    Chappe, V. et al. Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA. J. Physiol. 548, 39–52 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Rich, D. P. et al. Effect of deleting the R domain on CFTR-generated chloride channels. Science 253, 205–207 (1991).

    ADS  CAS  Google Scholar 

  45. 45

    Csanády, L. et al. Severed channels probe regulation of gating of CFTR by its cytoplasmic domains. J. Gen. Physiol. 116, 477–500 (2000).

    PubMed  PubMed Central  Google Scholar 

  46. 46

    Winter, M. C. & Welsh, M. J. Stimulation of CFTR activity by its phosphorylated R domain. Nature 389, 294–296 (1997).

    ADS  CAS  Google Scholar 

  47. 47

    Gadsby, D. C. & Nairn, A. C. Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol. Rev. 79, S77–S107 (1999).

    CAS  Google Scholar 

  48. 48

    Chang, X.-B. et al. Protein kinase A(PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites. J. Biol. Chem. 268, 11304–11311 (1993).

    CAS  Google Scholar 

  49. 49

    Ostedgaard, L. S., Baldursson, O., Vermeer, D. W., Welsh, M. J. & Robertson, A. D. A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution. Proc. Natl Acad. Sci. USA 97, 5657–5662 (2000).

    ADS  CAS  Google Scholar 

  50. 50

    Dulhanty, A. M. & Riordan, J. R. Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator. Biochemistry 33, 4072–4079 (1994).

    CAS  Google Scholar 

  51. 51

    Wilkinson, D. J. et al. CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. Am. J. Physiol. 273, L127–L133 (1997).

    CAS  Google Scholar 

  52. 52

    Rich, D. R. et al. Regulation of the cystic fibrosis transmembrane conductance regulator Cl channel by negative charge in the R domain. J. Biol. Chem. 268, 20259–20267 (1993).

    CAS  Google Scholar 

  53. 53

    Aleksandrov, A. A., Chang, X., Aleksandrov, L. & Riordan, J. R. The non-hydrolytic pathway of cystic fibrosis transmembrane conductance regulator ion channel gating. J. Physiol. 528, 259–265 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Dulhanty, A. M., Chang, X.-B. & Riordan, J. R. Mutation of potential phosphorylation sites in the recombinant R domain of the cystic fibrosis transmembrane conductance regulator has significant effects on domain conformation. Biochem. Biophys. Res. Commun. 206, 207–214 (1995).

    CAS  Google Scholar 

  55. 55

    Xie, J., Zhao, J., Davis, P. B. & Ma, J. Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings. Biophys. J. 78, 1293–1305 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Travis, S. M., Carson, M. R., Ries, D. R. & Welsh, M. J. Interaction of nucleotides with membrane-associated cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 268, 15336–15339 (1993).

    CAS  Google Scholar 

  57. 57

    Aleksandrov, L., Aleksandrov, A. A., Chang, X. B. & Riordan, J. R. The first nucleotide binding domain of cystic fibrosis transmembrane conductance regulator is a site of stable nucleotide interaction, whereas the second is a site of rapid turnover. J. Biol. Chem. 277, 15419–15425 (2002).

    CAS  Google Scholar 

  58. 58

    Basso, C., Vergani, P., Nairn, A. C. & Gadsby, D. C. Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating. J. Gen. Physiol. 122, 333–348 (2003) .

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Ostedgaard, L. S., Rich, D. P., DeBerg, L. G. & Welsh, M. J. Association of domains within the cystic fibrosis transmembrane conductance regulator. Biochemistry 36, 1287–1294 (1997) .

    CAS  Google Scholar 

  60. 60

    Naren, A. P. et al. CFTR chloride channel regulation by an interdomain interaction. Science 286, 544–548 (1999) .

    CAS  Google Scholar 

  61. 61

    Chappe, V., Irvine, T., Liao, J., Evagelidis, A. & Hanrahan, J. W. Phosphorylation of CFTR by PKA promotes binding of the regulatory domain. EMBO J. 24, 2730–2740 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Anderson, M. P. et al. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell 67, 775–784 (1991) .

    CAS  Google Scholar 

  63. 63

    Hwang, T.-C., Nagel, G. A., Nairn, A. C. & Gadsby, D. C. Regulation of the gating of CFTR Cl channels by phosphorylation and ATP hydrolysis. Proc. Natl Acad. Sci. USA 91, 4698–4702 (1994).

    ADS  CAS  Google Scholar 

  64. 64

    Vergani, P., Nairn, A. C. & Gadsby, D. C. On the mechanism of MgATP-dependent gating of CFTR Cl channels. J. Gen. Physiol. 121, 17–36 (2003) .

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    Powe, A. C. J., Al-Nakkash, L., Li M. & Hwang, T. C. Mutation of Walker-A lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide-binding domains. J. Physiol. 539, 333–346 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    Berger, A. L., Ikuma, M. & Welsh, M. J. Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain. Proc. Natl Acad. Sci. USA 102, 455–460 (2005).

    ADS  CAS  Google Scholar 

  67. 67

    Bompadre, S. G. CFTR gating II: effects of nucleotide binding on the stability of open states. J. Gen. Physiol. 125, 377–394 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Szabó, K., Szakács, G., Hegedus, T. & Sarkadi, B. Nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 274, 12209–12212 (1999) .

    Google Scholar 

  69. 69

    Davidson, A. L. & Chen, J. ATP-binding cassette transporters in bacteria. Annu. Rev. Biochem. 73, 241–268 (2004).

    CAS  Google Scholar 

  70. 70

    Higgins, C. F. & Linton, K. J. The ATP switch model for ABC transporters. Nature Struct. Mol. Biol. 11, 918–926 (2004).

    CAS  Google Scholar 

  71. 71

    Ramjeesingh, M. et al. Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator). Biochemistry 38, 1463–1468 (1999) .

    CAS  Google Scholar 

  72. 72

    Gunderson, K. L. & Kopito, R. R. Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis. Cell 82, 231–239 (1995).

    CAS  Google Scholar 

  73. 73

    Carson, M. R., Travis, S. M. & Welsh, M. J. The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity. J. Biol. Chem. 270, 1711–1717 (1995).

    CAS  Google Scholar 

  74. 74

    Dousmanis, A. G., Nairn, A. C. & Gadsby, D. C. Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl() channel. J. Gen. Physiol. 119, 545–559 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. 75

    Mathews, C. J., Tabcharani, J. A. & Hanrahan, J. W. The CFTR chloride channel: nucleotide interactions and temperature-dependent gating. J. Membr. Biol. 163, 55–66 (1998).

    CAS  Google Scholar 

  76. 76

    Gunderson, K. L. & Kopito, R. R. Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating. J. Biol. Chem. 269, 19349–19353 (1994).

    CAS  Google Scholar 

  77. 77

    Baukrowitz, T., Hwang, T.-C., Nairn, A. C. & Gadsby, D. C. Coupling of CFTR Cl channel gating to an ATP hydrolysis cycle. Neuron 12, 473–482 (1994).

    CAS  Google Scholar 

  78. 78

    Urbatsch, I. L., Sankaran, B., Weber, J. & Senior, A. E. P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. J. Biol. Chem. 270, 19383–19390 (1995).

    CAS  Google Scholar 

  79. 79

    Aleksandrov, A. A. & Riordan, J. R. Regulation of CFTR ion channel gating by MgATP. FEBS Lett. 431, 97–101 (1998).

    CAS  Google Scholar 

  80. 80

    Vergani, P., Lockless, S. W., Nairn, A. C. & Gadsby, D. C. CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. Nature 433, 876–880 (2005).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  81. 81

    Tombline, G., Bartholomew, L. A., Urbatsch, I. L. & Senior, A. E. Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly. J. Biol. Chem. 279, 31212–31220 (2004).

    CAS  Google Scholar 

  82. 82

    Mense, M., Nairn, A. C. & Gadsby, D. C. CFTR chloride channel activation in Xenopus oocytes by forskolin/IBMX promotes formation of a Rad50-like NBD1/NBD2 dimer. Biophys. J. 90, 310a (2006).

    Google Scholar 

  83. 83

    Lockless, S. W. & Ranganathan, R. Evolutionarily conserved pathways of energetic connectivity in protein families. Science 286, 295–299 (1999).

    CAS  Google Scholar 

  84. 84

    Kogan I. et al. CFTR directly mediates nucleotide-regulated glutathione flux. EMBO J. 22, 1981–1989 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. 85

    Randak, C. O. & Welsh, M. J. Adenylate kinase activity in ABC transporters. J. Biol. Chem. 280, 34385–34388 (2005).

    CAS  Google Scholar 

  86. 86

    Gross, C. H. et al. Nucleotide binding domains of cystic fibrosis transmembrane conductance regulator, an ABC-transporter, catalyze adenylate kinase activity but not ATP hydrolysis. J. Biol. Chem. (in the press).

  87. 87

    Muanprasat, C. et al. Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy. J. Gen. Physiol. 124, 125–137 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This review and our research on CFTR were supported by grants from the NIH and Fogarty International Center (to D.C.G). We dedicate this review to our late colleague Benjamin Angel, MA, MB BS (3rd December 1978 to 2nd October 2005), whose courage and grace in the face of CF continue to inspire us.

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Gadsby, D., Vergani, P. & Csanády, L. The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 440, 477–483 (2006). https://doi.org/10.1038/nature04712

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