European Biophysics Journal

, 39:129 | Cite as

Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane

  • Sophia C. Goodchild
  • Michael W. Howell
  • Nicole M. Cordina
  • Dene R. Littler
  • Samuel N. Breit
  • Paul M. G. Curmi
  • Louise Jennifer BrownEmail author
Original Paper


Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.


CLIC1 Fluorescence quenching Membrane protein Ion channel 



Chloride intracellular channel




Sucrose-loaded vesicles


Putative transmembrane region encompassing Cys24 through Val46 in CLIC1



This work was supported by an Australian Research Council (ARC) grant and an ARC APD fellowship to LJB.


  1. Berry KL, Hobert O (2006) Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo. J Mol Biol 359:1316–1333. doi: 10.1016/j.jmb.2006.04.046 CrossRefPubMedGoogle Scholar
  2. Berry KL, Bulow HE, Hall DH et al (2003) A C. elegans CLIC-like protein required for intracellular tube formation and maintenance. Science 302:2134–2137. doi: 10.1126/science.1087667 CrossRefPubMedGoogle Scholar
  3. Berryman MA, Goldenring JR (2003) CLIC4 is enriched at cell–cell junctions and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells. Cell Motil Cytoskeleton 56:159–172. doi: 10.1002/cm.10141 CrossRefPubMedGoogle Scholar
  4. Cromer BA, Gorman MA, Hansen G et al (2007) Structure of the Janus protein human CLIC2. J Mol Biol 374:719–731. doi: 10.1016/j.jmb.2007.09.041 CrossRefPubMedGoogle Scholar
  5. Duncan RR, Westwood PK, Boyd A et al (1997) Rat brain p64H1, expression of a new member of the p64 chloride channel protein family in endoplasmic reticulum. J Biol Chem 272:23880–23886. doi: 10.1074/jbc.272.38.23880 CrossRefPubMedGoogle Scholar
  6. Elter A, Hartel A, Sieben C et al (2007) A plant homolog of animal CLICs generates an ion conductance in heterologous systems. J Biol Chem 282:8786–8792. doi: 10.1074/jbc.M607241200 CrossRefPubMedGoogle Scholar
  7. Fanucchi S, Adamson RJ, Dirr HW (2008) Formation of an unfolding intermediate state of soluble chloride intracellular channel protein CLIC1 at acidic pH. Biochemistry 47:11674–11681. doi: 10.1021/bi801147r CrossRefPubMedGoogle Scholar
  8. Fernandez-Salas E, Sagar M, Cheng C et al (1999) p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein. J Biol Chem 274:36488–36497. doi: 10.1074/jbc.274.51.36488 CrossRefPubMedGoogle Scholar
  9. Harrop SJ, DeMaere MZ, Fairlie WD et al (2001) Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution. J Biol Chem 276:44993–45000. doi: 10.1074/jbc.M107804200 CrossRefPubMedGoogle Scholar
  10. Kaplan RS, Pedersen PL (1989) Sensitive protein assay in presence of high levels of lipid. Methods Enzymol 172:393–399. doi: 10.1016/S0076-6879(89)72025-5 CrossRefPubMedGoogle Scholar
  11. Laudi S, Steudel W, Jonscher K et al (2007) Comparison of lung proteome profiles in two rodent models of pulmonary arterial hypertension. Proteomics 7:2469–2478. doi: 10.1002/pmic.200600848 CrossRefPubMedGoogle Scholar
  12. Littler DR, Harrop SJ, Fairlie WD et al (2004) The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition. J Biol Chem 279:9298–9305. doi: 10.1074/jbc.M308444200 CrossRefPubMedGoogle Scholar
  13. Littler DR, Assaad NN, Harrop SJ et al (2005) Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4. FEBS J 272:4996–5007. doi: 10.1111/j.1742-4658.2005.04909.x CrossRefPubMedGoogle Scholar
  14. Littler DR, Harrop SJ, Brown LJ et al (2008) Comparison of vertebrate and invertebrate CLIC proteins: the crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC. Proteins 71:364–378. doi: 10.1002/prot.21704 CrossRefPubMedGoogle Scholar
  15. Mosior M, Epand RM (1993) Mechanism of activation of protein kinase C: roles of diolein and phosphatidylserine. Biochemistry 32:66–75. doi: 10.1021/bi00052a010 CrossRefPubMedGoogle Scholar
  16. Murzin AG (2008) Biochemistry. Metamorphic proteins. Science 320:1725–1726. doi: 10.1126/science.1158868 CrossRefPubMedGoogle Scholar
  17. Nishizawa T, Nagao T, Iwatsubo T et al (2000) Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells. J Biol Chem 275:11164–11173. doi: 10.1074/jbc.275.15.11164 CrossRefPubMedGoogle Scholar
  18. Novarino G, Fabrizi C, Tonini R et al (2004) Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity. J Neurosci 24:5322–5330. doi: 10.1523/JNEUROSCI.1170-04.2004 CrossRefPubMedGoogle Scholar
  19. Rebecchi M, Peterson A, McLaughlin S (1992) Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4, 5-bisphosphate. Biochemistry 31:12742–12747. doi: 10.1021/bi00166a005 CrossRefPubMedGoogle Scholar
  20. Rønnov-Jessen L, Villadsen R, Edwards JC et al (2002) Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1-mediated conversion of fibroblasts to myofibroblasts. Am J Pathol 161:471–480PubMedGoogle Scholar
  21. Schlesinger PH, Blair HC, Teitelbaum SL et al (1997) Characterization of the osteoclast ruffled border chloride channel and its role in bone resorption. J Biol Chem 272:18636–18643. doi: 10.1074/jbc.272.30.18636 CrossRefPubMedGoogle Scholar
  22. Shiio Y, Suh KS, Lee H et al (2006) Quantitative proteomic analysis of myc-induced apoptosis: a direct role for Myc induction of the mitochondrial chloride ion channel, mtCLIC/CLIC4. J Biol Chem 281:2750–2756. doi: 10.1074/jbc.M509349200 CrossRefPubMedGoogle Scholar
  23. Singh H, Ashley RH (2006) Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore. Biophys J 90:1628–1638. doi: 10.1529/biophysj.105.072678 CrossRefPubMedGoogle Scholar
  24. Singh H, Ashley RH (2007) CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels. Mol Membr Biol 24:41–52. doi: 10.1080/09687860600927907 CrossRefPubMedGoogle Scholar
  25. Suh KS, Yuspa SH (2005) Intracellular chloride channels: critical mediators of cell viability and potential targets for cancer therapy. Curr Pharm Des 11:2753–2764. doi: 10.2174/1381612054546806 CrossRefPubMedGoogle Scholar
  26. Suh KS, Mutoh M, Mutoh T et al (2007) CLIC4 mediates and is required for Ca2 +-induced keratinocyte differentiation. J Cell Sci 120:2631–2640. doi: 10.1242/jcs.002741 CrossRefPubMedGoogle Scholar
  27. Tonini R, Ferroni A, Valenzuela SM et al (2000) Functional characterization of the NCC27 nuclear protein in stable transfected CHO-K1 cells. FASEB J 14:1171–1178PubMedGoogle Scholar
  28. Tulk BM, Schlesinger PH, Kapadia SA et al (2000) CLIC-1 functions as a chloride channel when expressed and purified from bacteria. J Biol Chem 275:26986–26993PubMedGoogle Scholar
  29. Tulk BM, Kapadia S, Edwards JC (2002) CLIC1 inserts from the aqueous phase into phospholipid membranes, where it functions as an anion channel. Am J Physiol Cell Physiol 282:C1103–C1112PubMedGoogle Scholar
  30. Valenzuela SM, Martin DK, Por SB et al (1997) Molecular cloning and expression of a chloride ion channel of cell nuclei. J Biol Chem 272:12575–12582. doi: 10.1074/jbc.272.19.12575 CrossRefPubMedGoogle Scholar
  31. Valenzuela SM, Mazzanti M, Tonini R et al (2000) The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle. J Physiol 529:541–552. doi: 10.1111/j.1469-7793.2000.00541.x CrossRefPubMedGoogle Scholar
  32. Warton K, Tonini R, Fairlie WD et al (2002) Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1. J Biol Chem 277:26003–26011. doi: 10.1074/jbc.M203666200 CrossRefPubMedGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • Sophia C. Goodchild
    • 1
  • Michael W. Howell
    • 1
  • Nicole M. Cordina
    • 1
  • Dene R. Littler
    • 2
    • 4
  • Samuel N. Breit
    • 3
  • Paul M. G. Curmi
    • 2
    • 3
  • Louise Jennifer Brown
    • 1
    Email author
  1. 1.Department of Chemistry and Biomolecular SciencesMacquarie UniversitySydneyAustralia
  2. 2.School of PhysicsUniversity of New South WalesSydneyAustralia
  3. 3.Centre for ImmunologySt Vincent’s Hospital, University of New South WalesSydneyAustralia
  4. 4.Division of Molecular CarcinogenesisThe Netherlands Cancer InstituteAmsterdamThe Netherlands

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