VRAC: molecular identification as LRRC8 heteromers with differential functions

  • Thomas J. JentschEmail author
  • Darius Lutter
  • Rosa Planells-Cases
  • Florian Ullrich
  • Felizia K. Voss
Invited Review


A major player of vertebrate cell volume regulation is the volume-regulated anion channel (VRAC), which conducts halide ions and organic osmolytes to counteract osmotic imbalances. The molecular entity of this channel was unknown until very recently, although its biophysical characteristics and diverse physiological roles have been extensively studied over the last 30 years. On the road to the molecular identification of VRAC, experimental difficulties led to the proposal of a variety of false candidates. In 2014, in a final breakthrough, two groups independently identified LRRC8A as indispensable component of VRAC. LRRC8A is part of the leucine-rich repeat containing 8 family, which is comprised of five members (LRRC8A-E). Of those, LRRC8A is an obligatory subunit of VRAC but it needs at least one of the other family members to mediate the swelling-induced Cl current ICl,vol. This review discusses the remarkable journey which led to the molecular identification of VRAC, evidence for LRRC8 proteins forming the VRAC pore and their heteromeric assembly. Furthermore, first major insights on the role of LRRC8 proteins in cancer drug resistance and apoptosis and the role of LRRC8D in cisplatin and taurine transport will be summarized.


Chloride channel Swelling-activated VSOAC VSOR 


  1. 1.
    Abascal F, Zardoya R (2012) LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication. Bioessays 34:551–560. doi: 10.1002/bies.201100173 CrossRefPubMedGoogle Scholar
  2. 2.
    Akita T, Okada Y (2014) Characteristics and roles of the volume-sensitive outwardly rectifying (VSOR) anion channel in the central nervous system. Neuroscience 275C:211–231. doi: 10.1016/j.neuroscience.2014.06.015 CrossRefGoogle Scholar
  3. 3.
    Arreola J, Begenisich T, Nehrke K, Nguyen HV, Park K, Richardson L, Yang B, Schutte BC, Lamb FS, Melvin JE (2002) Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl channel gene. J Physiol 545:207–216. doi: 10.1113/jphysiol.2002.021980 PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Banderali U, Roy G (1992) Anion channels for amino acids in MDCK cells. Am J Physiol 263:C1200–C1207PubMedGoogle Scholar
  5. 5.
    Beneski DA, Catterall WA (1980) Covalent labeling of protein components of the sodium channel with a photoactivable derivative of scorpion toxin. Proc Natl Acad Sci U S A 77:639–643PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Berndtsson M, Hagg M, Panaretakis T, Havelka AM, Shoshan MC, Linder S (2007) Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA. Int J Cancer J Int Cancer 120:175–180. doi: 10.1002/ijc.22132 CrossRefGoogle Scholar
  7. 7.
    Buyse G, Voets T, Tytgat J, De Greef C, Droogmans G, Nilius B, Eggermont J (1997) Expression of human pICln and ClC-6 in Xenopus oocytes induces an identical endogenous chloride conductance. J Biol Chem 272:3615–3621. doi: 10.1074/jbc.272.6.3615 CrossRefPubMedGoogle Scholar
  8. 8.
    Canessa CM, Horisberger JD, Rossier BC (1993) Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361:467–470. doi: 10.1038/361467a0 CrossRefPubMedGoogle Scholar
  9. 9.
    Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, Pfeffer U, Ravazzolo R, Zegarra-Moran O, Galietta LJ (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322:590–594. doi: 10.1126/science.1163518 CrossRefPubMedGoogle Scholar
  10. 10.
    Catterall WA (2012) Voltage-gated sodium channels at 60: structure, function and pathophysiology. J Physiol 590:2577–2589. doi: 10.1113/jphysiol.2011.224204 PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Dolan J, Walshe K, Alsbury S, Hokamp K, O’Keeffe S, Okafuji T, Miller SF, Tear G, Mitchell KJ (2007) The extracellular leucine-rich repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns. BMC Genomics 8:320. doi: 10.1186/1471-2164-8-320 PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Emma F, McManus M, Strange K (1997) Intracellular electrolytes regulate the volume set point of the organic osmolyte/anion channel VSOAC. Am J Physiol 272:C1766–C1775PubMedGoogle Scholar
  13. 13.
    Fischmeister R, Hartzell HC (2005) Volume sensitivity of the bestrophin family of chloride channels. J Physiol 562:477–491. doi: 10.1113/jphysiol.2004.075622 PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Frech GC, VanDongen AM, Schuster G, Brown AM, Joho RH (1989) A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning. Nature 340:642–645. doi: 10.1038/340642a0 CrossRefPubMedGoogle Scholar
  15. 15.
    Galietta LJ, Haggie PM, Verkman AS (2001) Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. FEBS Lett 499:220–224. doi: 10.1016/S0014-5793(01)02561-3 CrossRefPubMedGoogle Scholar
  16. 16.
    Gately DP, Howell SB (1993) Cellular accumulation of the anticancer agent cisplatin: a review. Br J Cancer 67:1171–1176. doi: 10.1038/bjc.1993.221 PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Gay NJ, Symmons MF, Gangloff M, Bryant CE (2014) Assembly and localization of Toll-like receptor signalling complexes. Nat Rev Immunol 14:546–558. doi: 10.1038/nri3713 CrossRefPubMedGoogle Scholar
  18. 18.
    Gill DR, Hyde SC, Higgins CF, Valverde MA, Mintenig GM, Sepúlveda FV (1992) Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein. Cell 71:23–32. doi: 10.1016/0092-8674(92)90263-C CrossRefPubMedGoogle Scholar
  19. 19.
    Gong W, Xu H, Shimizu T, Morishima S, Tanabe S, Tachibe T, Uchida S, Sasaki S, Okada Y (2004) ClC-3-independent, PKC-dependent activity of volume-sensitive Cl channel in mouse ventricular cardiomyocytes. Cell Physiol Biochem 14:213–224. doi: 10.1159/000080330 CrossRefPubMedGoogle Scholar
  20. 20.
    Grenningloh G, Rienitz A, Schmitt B, Methfessel C, Zensen M, Beyreuther K, Gundelfinger ED, Betz H (1987) The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature 328:215–220. doi: 10.1038/328215a0 CrossRefPubMedGoogle Scholar
  21. 21.
    Grinstein S, Clarke CA, Dupre A, Rothstein A (1982) Volume-induced increase of anion permeability in human lymphocytes. J Gen Physiol 80:801–823. doi: 10.1085/jgp.80.6.801 CrossRefPubMedGoogle Scholar
  22. 22.
    Gründer S, Thiemann A, Pusch M, Jentsch TJ (1992) Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume. Nature 360:759–762. doi: 10.1038/360759a0 CrossRefPubMedGoogle Scholar
  23. 23.
    Gschwentner M, Nagl UO, Woll E, Schmarda A, Ritter M, Paulmichl M (1995) Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels. Pflugers Arch 430:464–470. doi: 10.1007/BF00373882 CrossRefPubMedGoogle Scholar
  24. 24.
    Hazama A, Okada Y (1988) Ca2+ sensitivity of volume-regulatory K+ and Cl channels in cultured human epithelial cells. J Physiol 402:687–702. doi: 10.1113/jphysiol.1988.sp017229 PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Hernández-Carballo CY, De Santiago-Castillo JA, Rosales-Saavedra T, Pérez-Cornejo P, Arreola J (2010) Control of volume-sensitive chloride channel inactivation by the coupled action of intracellular chloride and extracellular protons. Pflugers Arch 460:633–644. doi: 10.1007/s00424-010-0842-0 PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Hoffmann EK, Simonsen LO, Lambert IH (1984) Volume-induced increase of K+ and Cl permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+. J Membr Biol 78:211–222. doi: 10.1007/BF01925969 CrossRefPubMedGoogle Scholar
  27. 27.
    Hoffmann EK, Lambert IH, Pedersen SF (2009) Physiology of cell volume regulation in vertebrates. Physiol Rev 89:193–277. doi: 10.1152/physrev.00037.2007 CrossRefPubMedGoogle Scholar
  28. 28.
    Hoshi T, Zagotta WN, Aldrich RW (1990) Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250:533–538. doi: 10.1126/science.2122519 CrossRefPubMedGoogle Scholar
  29. 29.
    Hyzinski-García MC, Rudkouskaya A, Mongin AA (2014) LRRC8A protein is indispensable for swelling-activated and ATP-induced release of excitatory amino acids in rat astrocytes. J Physiol 592:4855–4862. doi: 10.1113/jphysiol.2014.278887 PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Ise T, Shimizu T, Lee EL, Inoue H, Kohno K, Okada Y (2005) Roles of volume-sensitive Cl channel in cisplatin-induced apoptosis in human epidermoid cancer cells. J Membr Biol 205:139–145. doi: 10.1007/s00232-005-0779-y CrossRefPubMedGoogle Scholar
  31. 31.
    Jackson PS, Strange K (1993) Volume-sensitive anion channels mediate swelling-activated inositol and taurine efflux. Am J Physiol 265:C1489–C1500PubMedGoogle Scholar
  32. 32.
    Jackson PS, Morrison R, Strange K (1994) The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding. Am J Physiol 267:C1203–C1209PubMedGoogle Scholar
  33. 33.
    Juul CA, Grubb S, Poulsen KA, Kyed T, Hashem N, Lambert IH, Larsen EH, Hoffmann EK (2014) Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca2+. Pflugers Arch. doi: 10.1007/s00424-013-1428-4 PubMedCentralPubMedGoogle Scholar
  34. 34.
    Kondratskyi A, Yassine M, Slomianny C, Kondratska K, Gordienko D, Dewailly E, Lehen’kyi V, Skryma R, Prevarskaya N (2014) Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death. Cell Death Dis 5, e1193. doi: 10.1038/cddis.2014.156 PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Kumar L, Chou J, Yee CS, Borzutzky A, Vollmann EH, von Andrian UH, Park SY, Hollander G, Manis JP, Poliani PL, Geha RS (2014) Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function. J Exp Med 211:929–942. doi: 10.1084/jem.20131379 PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    Lambert IH, Kristensen DM, Holm JB, Mortensen OH (2015) Physiological role of taurine – from organism to organelle. Acta Physiol (Oxf) 213:191–212. doi: 10.1111/apha.12365 CrossRefGoogle Scholar
  37. 37.
    Lang F, Hoffmann EK (2012) Role of ion transport in control of apoptotic cell death. Compr Physiol 2:2037–2061. doi: 10.1002/cphy.c110046 PubMedGoogle Scholar
  38. 38.
    Leaney JL, Marsh SJ, Brown DA (1997) A swelling-activated chloride current in rat sympathetic neurones. J Physiol 501(3):555–564. doi: 10.1111/j.1469-7793.1997.555bm.x PubMedCentralCrossRefPubMedGoogle Scholar
  39. 39.
    Lee EL, Shimizu T, Ise T, Numata T, Kohno K, Okada Y (2007) Impaired activity of volume-sensitive Cl channel is involved in cisplatin resistance of cancer cells. J Cell Physiol 211:513–521. doi: 10.1002/jcp.20961 CrossRefPubMedGoogle Scholar
  40. 40.
    Lee CC, Freinkman E, Sabatini DM, Ploegh HL (2014) The protein synthesis inhibitor blasticidin s enters mammalian cells via leucine-rich repeat-containing protein 8D. J Biol Chem. doi: 10.1074/jbc.M114.571257 Google Scholar
  41. 41.
    Mongin AA (2007) Disruption of ionic and cell volume homeostasis in cerebral ischemia: the perfect storm. Pathophysiol: Off J Int Soc Pathophysiol/ISP 14:183–193. doi: 10.1016/j.pathophys.2007.09.009 CrossRefGoogle Scholar
  42. 42.
    Morin XK, Bond TD, Loo TW, Clarke DM, Bear CE (1995) Failure of P-glycoprotein (MDR1) expressed in Xenopus oocytes to produce swelling-activated chloride channel activity. J Physiol 486(Pt 3):707–714. doi: 10.1113/jphysiol.1995.sp020846 PubMedCentralCrossRefPubMedGoogle Scholar
  43. 43.
    Nilius B, Sehrer J, Viana F, De Greef C, Raeymaekers L, Eggermont J, Droogmans G (1994) Volume-activated Cl currents in different mammalian non-excitable cell types. Pflugers Arch 428:364–371. doi: 10.1007/BF00724520 CrossRefPubMedGoogle Scholar
  44. 44.
    Nilius B, Eggermont J, Voets T, Buyse G, Manolopoulos V, Droogmans G (1997) Properties of volume-regulated anion channels in mammalian cells. Prog Biophys Mol Biol 68:69–119. doi: 10.1016/S0079-6107(97)00021-7 CrossRefPubMedGoogle Scholar
  45. 45.
    Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, Morishima S (2001) Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 532:3–16. doi: 10.1111/j.1469-7793.2001.0003g.x PubMedCentralCrossRefPubMedGoogle Scholar
  46. 46.
    Okada Y, Shimizu T, Maeno E, Tanabe S, Wang X, Takahashi N (2006) Volume-sensitive chloride channels involved in apoptotic volume decrease and cell death. J Membr Biol 209:21–29. doi: 10.1007/s00232-005-0836-6 CrossRefPubMedGoogle Scholar
  47. 47.
    Okada Y, Sato K, Numata T (2009) Pathophysiology and puzzles of the volume-sensitive outwardly rectifying anion channel. J Physiol 587:2141–2149. doi: 10.1113/jphysiol.2008.165076 PubMedCentralPubMedGoogle Scholar
  48. 48.
    Paulmichl M, Li Y, Wickman K, Ackerman M, Peralta E, Clapham D (1992) New mammalian chloride channel identified by expression cloning. Nature 356:238–241. doi: 10.1038/356238a0 CrossRefPubMedGoogle Scholar
  49. 49.
    Pedersen SF, Klausen TK, Nilius B (2015) The identification of a volume-regulated anion channel: an amazing Odyssey. Acta Physiol (Oxf) 213:868–881. doi: 10.1111/apha.12450 CrossRefGoogle Scholar
  50. 50.
    Planells-Cases R, Lutter D, Guyader C, Gerhards NM, Ullrich F, Elger DA, Kucukosmanoglu A, Xu G, Voss FK, Reincke SM, Stauber T, Blomen VA, Vis DJ, Wessels LF, Brummelkamp TR, Borst P, Rottenberg S, Jentsch TJ (2015) Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. EMBO J. doi: 10.15252/embj.201592409 PubMedCentralPubMedGoogle Scholar
  51. 51.
    Poulsen KA, Andersen EC, Hansen CF, Klausen TK, Hougaard C, Lambert IH, Hoffmann EK (2010) Deregulation of apoptotic volume decrease and ionic movements in multidrug-resistant tumor cells: role of chloride channels. Am J Physiol Cell Physiol 298:C14–C25. doi: 10.1152/ajpcell.00654.2008 CrossRefPubMedGoogle Scholar
  52. 52.
    Pu WT, Krapivinsky GB, Krapivinsky L, Clapham DE (1999) pICln inhibits snRNP biogenesis by binding core spliceosomal proteins. Mol Cell Biol 19:4113–4120. doi: 10.1128/MCB.19.6.4113 PubMedCentralCrossRefPubMedGoogle Scholar
  53. 53.
    Qiu Z, Dubin AE, Mathur J, Tu B, Reddy K, Miraglia LJ, Reinhardt J, Orth AP, Patapoutian A (2014) SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel. Cell 157:447–458. doi: 10.1016/j.cell.2014.03.024 PubMedCentralCrossRefPubMedGoogle Scholar
  54. 54.
    Rettig J, Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JO, Pongs O (1994) Inactivation properties of voltage-gated K+ channels altered by presence of β-subunit. Nature 369:289–294. doi: 10.1038/369289a0 CrossRefPubMedGoogle Scholar
  55. 55.
    Sawada A, Takihara Y, Kim JY, Matsuda-Hashii Y, Tokimasa S, Fujisaki H, Kubota K, Endo H, Onodera T, Ohta H, Ozono K, Hara J (2003) A congenital mutation of the novel gene LRRC8 causes agammaglobulinemia in humans. J Clin Invest 112:1707–1713. doi: 10.1172/JCI18937 PubMedCentralCrossRefPubMedGoogle Scholar
  56. 56.
    Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134:1019–1029. doi: 10.1016/j.cell.2008.09.003 PubMedCentralCrossRefPubMedGoogle Scholar
  57. 57.
    Shennan DB (2008) Swelling-induced taurine transport: relationship with chloride channels, anion-exchangers and other swelling-activated transport pathways. Cell Physiol Biochem 21:15–28. doi: 10.1159/000113743 CrossRefPubMedGoogle Scholar
  58. 58.
    Shimizu T, Numata T, Okada Y (2004) A role of reactive oxygen species in apoptotic activation of volume-sensitive Cl channel. Proc Natl Acad Sci U S A 101:6770–6773. doi: 10.1073/pnas.0401604101 PubMedCentralCrossRefPubMedGoogle Scholar
  59. 59.
    Shimizu T, Lee EL, Ise T, Okada Y (2008) Volume-sensitive Cl- channel as a regulator of acquired cisplatin resistance. Anticancer Res 28:75--83Google Scholar
  60. 60.
    Stobrawa SM, Breiderhoff T, Takamori S, Engel D, Schweizer M, Zdebik AA, Bösl MR, Ruether K, Jahn H, Draguhn A, Jahn R, Jentsch TJ (2001) Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 29:185–196. doi: 10.1016/S0896-6273(01)00189-1 CrossRefPubMedGoogle Scholar
  61. 61.
    Sturman JA, Hepner GW, Hofmann AF, Thomas PJ (1975) Metabolism of [35S]taurine in man. J Nutr 105:1206–1214PubMedGoogle Scholar
  62. 62.
    Tominaga M, Tominaga T, Miwa A, Okada Y (1995) Volume-sensitive chloride channel activity does not depend on endogenous P-glycoprotein. J Biol Chem 270:27887–27893. doi: 10.1074/jbc.270.46.27887 CrossRefPubMedGoogle Scholar
  63. 63.
    Valverde MA, Díaz M, Sepúlveda FV, Gill DR, Hyde SC, Higgins CF (1992) Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein. Nature 355:830–833. doi: 10.1038/355830a0 CrossRefPubMedGoogle Scholar
  64. 64.
    Viana F, Van Acker K, De Greef C, Eggermont J, Raeymaekers L, Droogmans G, Nilius B (1995) Drug-transport and volume-activated chloride channel functions in human erythroleukemia cells: relation to expression level of P-glycoprotein. J Membr Biol 145:87–98. doi: 10.1007/BF00233309 CrossRefPubMedGoogle Scholar
  65. 65.
    Voets T, Droogmans G, Nilius B (1997) Modulation of voltage-dependent properties of a swelling-activated Cl current. J Gen Physiol 110:313–325. doi: 10.1085/jgp.110.3.313 PubMedCentralCrossRefPubMedGoogle Scholar
  66. 66.
    Voets T, Droogmans G, Raskin G, Eggermont J, Nilius B (1999) Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels. Proc Natl Acad Sci U S A 96:5298–5303PubMedCentralCrossRefPubMedGoogle Scholar
  67. 67.
    Voss FK, Ullrich F, Münch J, Lazarow K, Lutter D, Mah N, Andrade-Navarro MA, von Kries JP, Stauber T, Jentsch TJ (2014) Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science 344:634–638. doi: 10.1126/science.1252826 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Thomas J. Jentsch
    • 1
    Email author
  • Darius Lutter
    • 1
  • Rosa Planells-Cases
    • 1
  • Florian Ullrich
    • 1
  • Felizia K. Voss
    • 1
  1. 1.Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC)BerlinGermany

Personalised recommendations