Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin

Abstract

The possible role of Cl- currents in regulatory volume decrease processes has been explored in HeLa cells using the whole-cell recording mode of the patch-clamp technique. Cells showed very small currents in voltage-clamp experiments performed with Cl(-)-rich, permeant-cation-free (N-methyl-D-glucamine replacement) intracellular and bathing solutions. Exposure of the cells to hypotonic solutions visibly swelled the cells and activated, reversibly, an outward rectifying Cl- current, which decayed at the most depolarised voltages used. Replacement of extracellular Cl- by a series of halide anions, SCN- and gluconate was consistent with an anion selectivity sequence: SCN- > I- > Br- > Cl- > F- > gluconate. The volume-regulated Cl- current was effectively inhibited by 100 microM 5-nitro-2-(3-phenyl-propylamino)-benzoic acid and by 100 microM 4,4'-diisothiocyanotostilbene-2,2-disulphonic acid, substances known to block Cl- channels in a variety of cells. Chloride current activation by hypotonicity was dependent on the presence of ATP in the intracellular solution and this requirement could be replaced by the non-hydrolysable analogue ATP[gamma S] and Mg(2+)-free ATP. The data suggest that the channels responsible for the current described are involved in the regulatory volume decrease in HeLa cells. The characteristics of this Cl- current are similar to those of the current associated with expression of multidrug resistance P-glycoprotein. Furthermore, the currents in HeLa cells were inhibited rapidly and reversibly by verapamil and 1,9-dideoxyforskolin, which are known to inhibit P-glycoprotein function.

A role of reactive oxygen species in apoptotic activation of volume-sensitive Cl-channel

“…Among the volume-regulatory Cl Ϫ channels, the most ubiquitous type is the VSOR Cl Ϫ channel (14), also called the volume-sensitive organic osmolyte and anion channel (VSOAC) (15) or the volume-regulated anion channel (VRAC) (16), which exhibits a number of phenotypical properties distinct from those of other types of anion channel (14)(15)(16). Human cervix epithelial HeLa cells also express this channel activity (17). Osmotic swelling induced by a hypotonic challenge (92% osmolality) brought about the activation of whole-cell Cl Ϫ currents with properties phenotypical of VSOR Cl Ϫ channel currents (14-16), including outward rectification, voltage-dependent inactivation gating at large positive potentials, inhibition by osmotic shrinkage, sensitivity to classic Cl Ϫ channel blockers such as 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4,4Ј-diisothiocyanatestilbene-2,2Ј-disulfonic acid (DIDS), and dependence on cytosolic ATP (Fig.…”

Extracellular acidification elicits a chloride current that shares characteristics with ICl(swell)

“…The relative anion selectivity for the swelling-activated Cl Ϫ channel is SCN Ϫ Ͼ I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ Ͼ F Ϫ Ͼ gluconate; this sequence is referred to as "Eisenman sequence I" and indicates a weak interaction between the channel pore and the permeation anion. A number of different substances, including the stilbene derivative 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid (DIDS) (9,35), the anti-inflammatory drug niflumic acid (20), the antiestrogen drug tamoxifen (43), diphenylamine-2-carboxylic acid (DPC) (22), and 1,9-dideoxyforskolin (DDFSK) (9), inhibit I Cl(swell) . Furthermore I Cl(swell) is insensitive to changes in calcium concentrations (33).…”

ClC-3 Is a Fundamental Molecular Component of Volume-sensitive Outwardly Rectifying Cl− Channels and Volume Regulation in HeLa Cells and Xenopus laevis Oocytes

“…Blockade of VSOACs were found to suppress RVD in a variety of cell types (5). Macroscopic outwardly rectifying VSOAC currents are characterized by activation after cell volume increase (6), a SCN Ͼ I Ͼ Br Ͼ Cl Ͼ F Ͼ gluconate permeability sequence (6), time-dependent inactivation at positive potentials (7), inhibition by tamoxifen, 1,9-dideoxyforskolin, and stilbene derivatives (8), dependence on intracellular ATP (6), and a single channel conductance in the range of 20 -40 pS (9). These biophysical and pharmacological characteristics are considered to represent classic characteristics of VSOACs.…”