Control of volume-sensitive chloride channel inactivation by the coupled action of intracellular chloride and extracellular protons

  • Carmen Y. Hernández-Carballo
  • José A. De Santiago-Castillo
  • Teresa Rosales-Saavedra
  • Patricia Pérez-Cornejo
  • Jorge ArreolaEmail author
Ion Channels, Receptors and Transporters


The volume-sensitive chloride current (IClVol) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of chloride ions (Cl) and H+ ions on IClVol decay recorded in HEK-293 and HL-60 cells using the whole-cell patch clamp technique. Under control conditions ([Cl]e = [Cl]i = 140 mM and pHi = pHe = 7.3), IClVol in HEK cells shows a large decay at positive voltages but in HL-60 cells IClVol remained constant independently of time. In HEK-293 cells, simultaneously raising the [Cl]e and [Cl]i from 25 to 140 mM (with pHe = pHi = 7.3) increased the fraction of inactivated channels (FIC). This effect was reproduced by elevating [Cl]i while keeping the [Cl]e constant. Furthermore, a decrease in pHe from 7.3 to 5.5 accelerated current decay and increased FIC when [Cl] was 140 mM but not 25 mM. In HL-60 cells, a slight IClVol decay was seen when the pHe was reduced from 7.3 to 5.5. Our data show that inactivation of IClVol can be controlled by changing either the Cl or H+ concentration or both. Based on our results and previously published data, we have built a model that explains VRAC inactivation. In the model the H+ binding site is located outside the electrical field near the extracellular entry whilst the Cl binding site is intracellular. The model depicts inactivation as a pore constriction that happens by simultaneous binding of H+ and Cl ions to the channel followed by a voltage-dependent conformational change that ultimately causes inactivation.


Volume regulation Cl channels Inactivation pH dependence Chloride 



This work was supported by grants 79897, 59889, and 45895 (Consejo Nacional de Ciencia y Tecnologia, Mexico) and PO1-HL18208 (National Institutes of Health, USA). TRS and JADSC received a scholarship from Consejo Nacional de Ciencia y Tecnologia, Mexico.

Supplementary material

424_2010_842_MOESM1_ESM.ppt (153 kb)
Supplemental Fig. 1 Hypothetical V m-dependence of volume-sensitive chloride channels. a Simple barrier model representing the energy landscape along the VRAC pore. This energy profile plus the kinetic model shown in Fig. 7 were used to qualitatively explain the V m (a) and external Cl-dependence (b) of inactivation. The energy profiles depict the landscape along the pore that the permeant anions (with symmetrical [Cl]i = [Cl]e = 140 mM) experiment at −100, 0, and +100 mV. At each voltage, the pore occupancy changes and thus the probability that the pore is empty (P U) is greater at positive voltages. Moreover, P U changes as a function of the Cl gradient across the membrane. b P U becomes larger as the Cl gradient decreases. P U was calculated using the V m-dependent rate constants \( {\alpha_{\rm{V}}} = {\left[ {{\hbox{C}}{{\hbox{l}}^{-} }} \right]_{\rm{o}}} \times {k_1} + {\left[ {{\hbox{C}}{{\hbox{l}}^{-} }} \right]_{\rm{i}}} \times {k_{ - {2}}}\;{\hbox{and}}\;{\beta_{\rm{V}}} = {k_{ - {1} + }} \times {k_{ - {2}}} \) recorded in Table 2 (PPT 153 kb)


  1. 1.
    Almac J, Tian Y, Aldehni F et al (2009) TMEM16 proteins produce volume-regulated chloride currents that are reduced in mice lacking TMEM16A. J Biol Chem 284:28571–28578CrossRefGoogle Scholar
  2. 2.
    Arreola J, Begenisich T, Nehrke K et al (2002) Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl- channel gene. J Physiol 545:207–216CrossRefPubMedGoogle Scholar
  3. 3.
    Arreola J, Hallows KR, Knauf PA (1995) Volume-activated chloride channels in HL-60 cells: potent inhibition by an oxonol dye. Am J Physiol 269:C1063–C1072PubMedGoogle Scholar
  4. 4.
    Arreola J, Melvin JE, Begenisich T (1995) Volume-activated chloride channels in rat parotid acinar cells. J Physiol 484:677–687PubMedGoogle Scholar
  5. 5.
    Arreola J, Park K, Melvin JE et al (1996) Three distinct chloride channels control anion movements in rat parotid acinar cells. J Physiol 490:351–362PubMedGoogle Scholar
  6. 6.
    Coca-Prados M, Sánchez-Torres J, Peterson-Yantorno K et al (1996) Association of ClC-3 channel with Cl- transport by human nonpigmented ciliary epithelial cells. J Membr Biol 150:197–208CrossRefPubMedGoogle Scholar
  7. 7.
    Duan D, Winter C, Cowley S et al (1997) Molecular identification of a volume-regulated chloride channel. Nature 390:417–421CrossRefPubMedGoogle Scholar
  8. 8.
    Duan D, Zhong J, Hermoso M et al (2001) Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody. J Physiol 531:437–444CrossRefPubMedGoogle Scholar
  9. 9.
    Fürst J, Gschwentner M, Ritter M et al (2002) Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells. Pflugers Arch 444:1–25CrossRefPubMedGoogle Scholar
  10. 10.
    Hanrahan JW, Tabcharini JA (1990) Inhibition of an outwardly rectifying anion channel by HEPES and related buffers. J Membr Biol 116:65–77CrossRefPubMedGoogle Scholar
  11. 11.
    Hille B (2001) Ion channels of excitable membranes. Sinauer Associates, Inc., USAGoogle Scholar
  12. 12.
    Lewis RS, Ross PE, Cahalan MD (1993) Chloride channels activated by osmotic stress in T lymphocytes. J Gen Physiol 101:801–26CrossRefPubMedGoogle Scholar
  13. 13.
    Meyer K, Korbmacher C (1996) Cell swelling activates ATP-dependent voltage-gated chloride channels in M-1 mouse cortical collecting duct cells. J Gen Physiol 108:177–193CrossRefPubMedGoogle Scholar
  14. 14.
    Miller C (2006) ClC chloride channels viewed through a transporter lens. Nature 440:484–489CrossRefPubMedGoogle Scholar
  15. 15.
    Nilius B, Eggermont J, Voets T et al (1997) Properties of volume-regulated anion channels in mammalian cells. Prog Biophys Mol Biol 68:69–119CrossRefPubMedGoogle Scholar
  16. 16.
    Nilius B, Prenen J, Droogmans G (1998) Modulation of volume-regulated anion channels by extra- and intracellular pH. Pflugers Arch 436:742–748CrossRefPubMedGoogle Scholar
  17. 17.
    Niemeyer MI, Cid LP, Yusef YR et al (2009) Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protons. J Physiol 587:1387–1400CrossRefPubMedGoogle Scholar
  18. 18.
    Okada Y (2006) Cell volume-sensitive chloride channels: phenotypic properties and molecular identity. Contrib Nephrol 152:9–24CrossRefPubMedGoogle Scholar
  19. 19.
    Okada Y, Sato K, Numata T (2009) Pathophysiology and puzzles of the volume-sensitive outwardly rectifying anion channel. J Physiol 587:2141–2149PubMedGoogle Scholar
  20. 20.
    Perez-Cornejo P, Arreola J, Law FY et al (2004) Volume-sensitive chloride channels do not mediate activation-induced chloride efflux in human neutrophils. J Immunol 172:6988–6993PubMedGoogle Scholar
  21. 21.
    Poletto Chaves LA, Varanda WA (2008) Volume-activated chloride channels in mice Leydig cells. Pflugers Arch 457:493–504CrossRefPubMedGoogle Scholar
  22. 22.
    Rossow CF, Duan D, Hatton WJ et al (2006) Functional role of amino terminus in ClC-3 chloride channel regulation by phosphorylation and cell volume. Acta Physiol (Oxf) 187:5–19CrossRefGoogle Scholar
  23. 23.
    Sabirov RZ, Prenen J, Droogmans G et al (2000) Extra- and Intracellular Proton-Binding Sites of Volume-Regulated Anion Channels. J Membr Biol 177:13–22CrossRefPubMedGoogle Scholar
  24. 24.
    Stobrawa SM, Breiderhoff T, Takamori S et al (2001) Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 29:185–196CrossRefPubMedGoogle Scholar
  25. 25.
    Stoddard JS, Steinbach JH, Simchowitz L (1993) Whole cell Cl- currents in human neutrophils induced by cell swelling. Am J Physiol 265:C156–C165PubMedGoogle Scholar
  26. 26.
    Voets T, Droogmans G, Nilius B (1997) Modulation of Voltage-dependent Properties of a Swelling-activated Cl Current. J Gen Physiol 110:313–325CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Carmen Y. Hernández-Carballo
    • 1
  • José A. De Santiago-Castillo
    • 2
  • Teresa Rosales-Saavedra
    • 2
  • Patricia Pérez-Cornejo
    • 2
  • Jorge Arreola
    • 1
    Email author
  1. 1.Instituto de FísicaUniversidad Autónoma de San Luis PotosíSan Luis PotosíMéxico
  2. 2.Facultad de MedicinaUniversidad Autónoma de San Luis PotosíSan Luis PotosíMéxico

Personalised recommendations