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

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 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.

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1)

“…Alternatively, when we recorded TMEM16A in the absence of intracellular Ca 2+ we used an external solution containing 140 mM SCN − and 0 Cl − . To prevent activation of endogenous volume-sensitive Cl − currents, the external solution was made hypertonic (Hernández-Carballo et al 2010) by adding D-mannitol. For PI(4,5)P2 recovery experiments the internal solution was supplemented with 1 mM MgCl 2 and freshly made 5 mM ATP.…”

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models

“…The pH of each solution was adjusted to 7.3 with TEA-OH or NaOH. ES was made hypertonic relative to the internal solutions to avoid activation of volume-sensitive Cl − channels present in HEK293 cells [23]. Osmolarity was adjusted by adding D-mannitol and measured using the vapour pressure point method (VAPRO, Wescor Inc., South Logan, UT, USA).…”

“…Gating of Cl − channels such as ClC-2, volume-sensitive channels, and CFTR are reported to be dependent on permeant anion [23, 25, 41, 51]. CaCCs are not the exception.…”

Identification of LRRC8 Heteromers as an Essential Component of the Volume-Regulated Anion Channel VRAC

“…Whereas I Cl(swell) shows prominent inactivation at positive potentials in HEK (23,24) (Fig. 1E, Fig.…”

“…2, B and D, and fig. S6D), it inactivates much less in blood cells like promyelocytic HL-60 cells, and in vascular smooth muscle and neurons (24)(25)(26). EST databases suggest that these cells express the 'decelerating' subunit LRRC8C, but lack LRRC8E that potently induces inactivation (Fig.…”