Control of Volume-Sensitive Chloride Channel Inactivation by the Coupled Action of Intracellular Chloride and Extracellular Protons

摘要

The volume-sensitive chloride current (IClVol) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of 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 pH_e was reduced from 7.3 to 5.5. Our data show that inactivation of I_ClVol 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.