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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 Arreola
Ion Channels, Receptors and Transporters

Abstract

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.

Keywords

Volume regulation Cl channels Inactivation pH dependence Chloride 

Notes

Acknowledgements

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)

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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
  1. 1.Instituto de FísicaUniversidad Autónoma de San Luis PotosíSan Luis PotosíMéxico
  2. 2.Facultad de MedicinaUniversidad Autónoma de San Luis PotosíSan Luis PotosíMéxico

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