Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1996 Jun;70(6):2688–2695. doi: 10.1016/S0006-3495(96)79838-7

Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment.

M Cheung 1, M H Akabas 1
PMCID: PMC1225248  PMID: 8744306

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both alpha-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 A to allow the MTS reagents to reach these residues.

Full text

PDF

Images in this article

2689FIGURE 1
on p.2689
2691FIGURE 4
on p.2691
2691FIGURE 5
on p.2691

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akabas M. H., Karlin A. Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit. Biochemistry. 1995 Oct 3;34(39):12496–12500. doi: 10.1021/bi00039a002. [DOI] [PubMed] [Google Scholar]
  2. Akabas M. H., Kaufmann C., Archdeacon P., Karlin A. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit. Neuron. 1994 Oct;13(4):919–927. doi: 10.1016/0896-6273(94)90257-7. [DOI] [PubMed] [Google Scholar]
  3. Akabas M. H., Kaufmann C., Cook T. A., Archdeacon P. Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1994 May 27;269(21):14865–14868. [PubMed] [Google Scholar]
  4. Akabas M. H., Stauffer D. A., Xu M., Karlin A. Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science. 1992 Oct 9;258(5080):307–310. doi: 10.1126/science.1384130. [DOI] [PubMed] [Google Scholar]
  5. Altenbach C., Marti T., Khorana H. G., Hubbell W. L. Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants. Science. 1990 Jun 1;248(4959):1088–1092. doi: 10.1126/science.2160734. [DOI] [PubMed] [Google Scholar]
  6. Ames G. F., Mimura C. S., Holbrook S. R., Shyamala V. Traffic ATPases: a superfamily of transport proteins operating from Escherichia coli to humans. Adv Enzymol Relat Areas Mol Biol. 1992;65:1–47. doi: 10.1002/9780470123119.ch1. [DOI] [PubMed] [Google Scholar]
  7. Anderson M. P., Berger H. A., Rich D. P., Gregory R. J., Smith A. E., Welsh M. J. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell. 1991 Nov 15;67(4):775–784. doi: 10.1016/0092-8674(91)90072-7. [DOI] [PubMed] [Google Scholar]
  8. Anderson M. P., Gregory R. J., Thompson S., Souza D. W., Paul S., Mulligan R. C., Smith A. E., Welsh M. J. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science. 1991 Jul 12;253(5016):202–205. doi: 10.1126/science.1712984. [DOI] [PubMed] [Google Scholar]
  9. Audrézet M. P., Mercier B., Guillermit H., Quéré I., Verlingue C., Rault G., Férec C. Identification of 12 novel mutations in the CFTR gene. Hum Mol Genet. 1993 Jan;2(1):51–54. doi: 10.1093/hmg/2.1.51. [DOI] [PubMed] [Google Scholar]
  10. Baukrowitz T., Hwang T. C., Nairn A. C., Gadsby D. C. Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle. Neuron. 1994 Mar;12(3):473–482. doi: 10.1016/0896-6273(94)90206-2. [DOI] [PubMed] [Google Scholar]
  11. Bear C. E., Duguay F., Naismith A. L., Kartner N., Hanrahan J. W., Riordan J. R. Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. J Biol Chem. 1991 Oct 15;266(29):19142–19145. [PubMed] [Google Scholar]
  12. 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. 1993 Jan 25;268(3):2037–2047. [PubMed] [Google Scholar]
  13. 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. 1995 Jan 27;270(4):1711–1717. doi: 10.1074/jbc.270.4.1711. [DOI] [PubMed] [Google Scholar]
  14. Chang X. B., Hou Y. X., Jensen T. J., Riordan J. R. Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion. J Biol Chem. 1994 Jul 15;269(28):18572–18575. [PubMed] [Google Scholar]
  15. Chang X. B., Tabcharani J. A., Hou Y. X., Jensen T. J., Kartner N., Alon N., Hanrahan J. W., Riordan J. R. Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites. J Biol Chem. 1993 May 25;268(15):11304–11311. [PubMed] [Google Scholar]
  16. Cheng S. H., Rich D. P., Marshall J., Gregory R. J., Welsh M. J., Smith A. E. Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell. 1991 Sep 6;66(5):1027–1036. doi: 10.1016/0092-8674(91)90446-6. [DOI] [PubMed] [Google Scholar]
  17. Cowan S. W., Schirmer T., Rummel G., Steiert M., Ghosh R., Pauptit R. A., Jansonius J. N., Rosenbusch J. P. Crystal structures explain functional properties of two E. coli porins. Nature. 1992 Aug 27;358(6389):727–733. doi: 10.1038/358727a0. [DOI] [PubMed] [Google Scholar]
  18. Dean M., White M. B., Amos J., Gerrard B., Stewart C., Khaw K. T., Leppert M. Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell. 1990 Jun 1;61(5):863–870. doi: 10.1016/0092-8674(90)90196-l. [DOI] [PubMed] [Google Scholar]
  19. Férec C., Verlingue C., Guillermit H., Quéré I., Raguénès O., Feigelson J., Audrézet M. P., Moullier P., Mercier B. Genotype analysis of adult cystic fibrosis patients. Hum Mol Genet. 1993 Oct;2(10):1557–1560. doi: 10.1093/hmg/2.10.1557. [DOI] [PubMed] [Google Scholar]
  20. Gottesman M. M., Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem. 1993;62:385–427. doi: 10.1146/annurev.bi.62.070193.002125. [DOI] [PubMed] [Google Scholar]
  21. Gregory R. J., Cheng S. H., Rich D. P., Marshall J., Paul S., Hehir K., Ostedgaard L., Klinger K. W., Welsh M. J., Smith A. E. Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature. 1990 Sep 27;347(6291):382–386. doi: 10.1038/347382a0. [DOI] [PubMed] [Google Scholar]
  22. Gunderson K. L., Kopito R. R. Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis. Cell. 1995 Jul 28;82(2):231–239. doi: 10.1016/0092-8674(95)90310-0. [DOI] [PubMed] [Google Scholar]
  23. Hasegawa H., Skach W., Baker O., Calayag M. C., Lingappa V., Verkman A. S. A multifunctional aqueous channel formed by CFTR. Science. 1992 Nov 27;258(5087):1477–1479. doi: 10.1126/science.1279809. [DOI] [PubMed] [Google Scholar]
  24. Heemels M. T., Ploegh H. Generation, translocation, and presentation of MHC class I-restricted peptides. Annu Rev Biochem. 1995;64:463–491. doi: 10.1146/annurev.bi.64.070195.002335. [DOI] [PubMed] [Google Scholar]
  25. Hollmann M., Maron C., Heinemann S. N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1. Neuron. 1994 Dec;13(6):1331–1343. doi: 10.1016/0896-6273(94)90419-7. [DOI] [PubMed] [Google Scholar]
  26. Hwang T. C., Horie M., Gadsby D. C. Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart. J Gen Physiol. 1993 May;101(5):629–650. doi: 10.1085/jgp.101.5.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hwang T. C., Nagel G., Nairn A. C., Gadsby D. C. Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4698–4702. doi: 10.1073/pnas.91.11.4698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Javitch J. A., Fu D., Chen J., Karlin A. Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method. Neuron. 1995 Apr;14(4):825–831. doi: 10.1016/0896-6273(95)90226-0. [DOI] [PubMed] [Google Scholar]
  29. Jung K., Jung H., Wu J., Privé G. G., Kaback H. R. Use of site-directed fluorescence labeling to study proximity relationships in the lactose permease of Escherichia coli. Biochemistry. 1993 Nov 23;32(46):12273–12278. doi: 10.1021/bi00097a001. [DOI] [PubMed] [Google Scholar]
  30. Kürz L. L., Zühlke R. D., Zhang H. J., Joho R. H. Side-chain accessibilities in the pore of a K+ channel probed by sulfhydryl-specific reagents after cysteine-scanning mutagenesis. Biophys J. 1995 Mar;68(3):900–905. doi: 10.1016/S0006-3495(95)80266-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lü Q., Miller C. Silver as a probe of pore-forming residues in a potassium channel. Science. 1995 Apr 14;268(5208):304–307. doi: 10.1126/science.7716526. [DOI] [PubMed] [Google Scholar]
  32. MacKinnon R. Pore loops: an emerging theme in ion channel structure. Neuron. 1995 May;14(5):889–892. doi: 10.1016/0896-6273(95)90327-5. [DOI] [PubMed] [Google Scholar]
  33. McDonough S., Davidson N., Lester H. A., McCarty N. A. Novel pore-lining residues in CFTR that govern permeation and open-channel block. Neuron. 1994 Sep;13(3):623–634. doi: 10.1016/0896-6273(94)90030-2. [DOI] [PubMed] [Google Scholar]
  34. Mindell J. A., Zhan H., Huynh P. D., Collier R. J., Finkelstein A. Reaction of diphtheria toxin channels with sulfhydryl-specific reagents: observation of chemical reactions at the single molecule level. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5272–5276. doi: 10.1073/pnas.91.12.5272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nunes V., Gasparini P., Novelli G., Gaona A., Bonizzato A., Sangiuolo F., Balassopoulou A., Giménez F. J., Dognini M., Ravnik-Glavac M. Analysis of 14 cystic fibrosis mutations in five south European populations. Hum Genet. 1991 Oct;87(6):737–738. doi: 10.1007/BF00201737. [DOI] [PubMed] [Google Scholar]
  36. Pascual J. M., Shieh C. C., Kirsch G. E., Brown A. M. K+ pore structure revealed by reporter cysteines at inner and outer surfaces. Neuron. 1995 May;14(5):1055–1063. doi: 10.1016/0896-6273(95)90344-5. [DOI] [PubMed] [Google Scholar]
  37. Reisin I. L., Prat A. G., Abraham E. H., Amara J. F., Gregory R. J., Ausiello D. A., Cantiello H. F. The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel. J Biol Chem. 1994 Aug 12;269(32):20584–20591. [PubMed] [Google Scholar]
  38. Riordan J. R., Rommens J. M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J. L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  39. Roberts D. D., Lewis S. D., Ballou D. P., Olson S. T., Shafer J. A. Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry. 1986 Sep 23;25(19):5595–5601. doi: 10.1021/bi00367a038. [DOI] [PubMed] [Google Scholar]
  40. Saba L., Leoni G. B., Meloni A., Faà V., Cao A., Rosatelli M. C. Two novel mutations in the transmembrane domains of the CFTR gene in subjects of Sardinian descent. Hum Mol Genet. 1993 Oct;2(10):1739–1740. doi: 10.1093/hmg/2.10.1739. [DOI] [PubMed] [Google Scholar]
  41. Schirmer T., Keller T. A., Wang Y. F., Rosenbusch J. P. Structural basis for sugar translocation through maltoporin channels at 3.1 A resolution. Science. 1995 Jan 27;267(5197):512–514. doi: 10.1126/science.7824948. [DOI] [PubMed] [Google Scholar]
  42. Schultz B. D., Venglarik C. J., Bridges R. J., Frizzell R. A. Regulation of CFTR Cl- channel gating by ADP and ATP analogues. J Gen Physiol. 1995 Mar;105(3):329–361. doi: 10.1085/jgp.105.3.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schwiebert E. M., Egan M. E., Hwang T. H., Fulmer S. B., Allen S. S., Cutting G. R., Guggino W. B. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell. 1995 Jun 30;81(7):1063–1073. doi: 10.1016/s0092-8674(05)80011-x. [DOI] [PubMed] [Google Scholar]
  44. Seibert F. S., Tabcharani J. A., Chang X. B., Dulhanty A. M., Mathews C., Hanrahan J. W., Riordan J. R. cAMP-dependent protein kinase-mediated phosphorylation of cystic fibrosis transmembrane conductance regulator residue Ser-753 and its role in channel activation. J Biol Chem. 1995 Feb 3;270(5):2158–2162. doi: 10.1074/jbc.270.5.2158. [DOI] [PubMed] [Google Scholar]
  45. Sheppard D. N., Rich D. P., Ostedgaard L. S., Gregory R. J., Smith A. E., Welsh M. J. Mutations in CFTR associated with mild-disease-form Cl- channels with altered pore properties. Nature. 1993 Mar 11;362(6416):160–164. doi: 10.1038/362160a0. [DOI] [PubMed] [Google Scholar]
  46. Slatin S. L., Qiu X. Q., Jakes K. S., Finkelstein A. Identification of a translocated protein segment in a voltage-dependent channel. Nature. 1994 Sep 8;371(6493):158–161. doi: 10.1038/371158a0. [DOI] [PubMed] [Google Scholar]
  47. Smit L. S., Wilkinson D. J., Mansoura M. K., Collins F. S., Dawson D. C. Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963–9967. doi: 10.1073/pnas.90.21.9963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stauffer D. A., Karlin A. Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. Biochemistry. 1994 Jun 7;33(22):6840–6849. doi: 10.1021/bi00188a013. [DOI] [PubMed] [Google Scholar]
  49. Steinhoff H. J., Mollaaghababa R., Altenbach C., Hideg K., Krebs M., Khorana H. G., Hubbell W. L. Time-resolved detection of structural changes during the photocycle of spin-labeled bacteriorhodopsin. Science. 1994 Oct 7;266(5182):105–107. doi: 10.1126/science.7939627. [DOI] [PubMed] [Google Scholar]
  50. Sun Z. P., Akabas M. H., Goulding E. H., Karlin A., Siegelbaum S. A. Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating. Neuron. 1996 Jan;16(1):141–149. doi: 10.1016/s0896-6273(00)80031-8. [DOI] [PubMed] [Google Scholar]
  51. 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. 1991 Aug 15;352(6336):628–631. doi: 10.1038/352628a0. [DOI] [PubMed] [Google Scholar]
  52. Tabcharani J. A., Rommens J. M., Hou Y. X., Chang X. B., Tsui L. C., Riordan J. R., Hanrahan J. W. Multi-ion pore behaviour in the CFTR chloride channel. Nature. 1993 Nov 4;366(6450):79–82. doi: 10.1038/366079a0. [DOI] [PubMed] [Google Scholar]
  53. Todd A. P., Cong J., Levinthal F., Levinthal C., Hubbell W. L. Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation. Proteins. 1989;6(3):294–305. doi: 10.1002/prot.340060312. [DOI] [PubMed] [Google Scholar]
  54. 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. 1993 Jul 25;268(21):15336–15339. [PubMed] [Google Scholar]
  55. Tsui L. C. Mutations and sequence variations detected in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a report from the Cystic Fibrosis Genetic Analysis Consortium. Hum Mutat. 1992;1(3):197–203. doi: 10.1002/humu.1380010304. [DOI] [PubMed] [Google Scholar]
  56. Weiss M. S., Abele U., Weckesser J., Welte W., Schiltz E., Schulz G. E. Molecular architecture and electrostatic properties of a bacterial porin. Science. 1991 Dec 13;254(5038):1627–1630. doi: 10.1126/science.1721242. [DOI] [PubMed] [Google Scholar]
  57. Winter M. C., Sheppard D. N., Carson M. R., Welsh M. J. Effect of ATP concentration on CFTR Cl- channels: a kinetic analysis of channel regulation. Biophys J. 1994 May;66(5):1398–1403. doi: 10.1016/S0006-3495(94)80930-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Xu M., Akabas M. H. Amino acids lining the channel of the gamma-aminobutyric acid type A receptor identified by cysteine substitution. J Biol Chem. 1993 Oct 15;268(29):21505–21508. [PubMed] [Google Scholar]
  59. Xu M., Akabas M. H. Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA(A) receptor alpha1 subunit. J Gen Physiol. 1996 Feb;107(2):195–205. doi: 10.1085/jgp.107.2.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Xu M., Covey D. F., Akabas M. H. Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants. Biophys J. 1995 Nov;69(5):1858–1867. doi: 10.1016/S0006-3495(95)80056-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES