Primary actions of opening of anion channels in either a KCC-dominant case (A) or an NKCC-dominant case (B) (see the text for details).

VSOR and Maxi-Cl currents in human monocytic U937 cells. (A) Macroscopic whole-cell VSOR currents recorded from U937 cells after osmotic swelling (left) and the corresponding current-to-voltage relationship (right). Experimental conditions, other than the cell line used, are the same as reported previously (Sabirov et al., 2001; Kurbannazarova et al., 2011). (B) Single VSOR currents recorded from cell-attached patches on preswollen cells. Holding membrane potentials are indicated on the left of each trace. The right panel shows the unitary current-voltage relationship. The calculated slope conductance is 84.5 ± 5.8 and 14.7 ± 2.4 pS for outward and inward currents, respectively. Experimental conditions, other than the cell line used, are the same as reported previously (Ternovsky et al., 2004; Kurbannazarova et al., 2011) with 100 mM TEACl-pipette and 100 mM KCl-bath solutions. (C) Activation of Maxi-Cl currents in U937 cells. (Left) Voltage- and time-dependent inactivation of the steady-state membrane current as observed in excised macropatches in response to voltage pulses (500 milliseconds) from a holding potential at 0 to ±50 mV in 10-mV increments. The pipette and bath solutions were normal Ringer’s solution. (Right) Single-channel I-V relationships for maxi-anion channel events recorded. Each symbol represents the mean ± S.E. (vertical bar). The solid line is a linear fit with a slope conductance of 445.3 ± 9.6 pS. Experimental conditions, other than the cell line used, are the same as reported previously (Islam et al., 2012).

Cl⁻ current amplitudes plotted against [ATP]_i, [Mg²⁺]_i, and voltages. Each symbol represents the mean current of four different Intestine 407 cells. Vertical bars represent the S.E. (A) [ATP]_i dependence of the peak Cl⁻ currents measured at +40 mV at a fixed [Mg²⁺]_i (1 mM). Data points were fitted by the following equation: I = I_max([ATP]_i/EC₅₀)/{1 + ([ATP]_i/EC₅₀)}. (B) [Mg²⁺]_i dependence of the peak Cl⁻ currents measured at +40 mV at a fixed [ATP]_i (0.1 or 0.01 mM). Data points were fitted by the following equation: I = I_max/{1 + ([Mg²⁺]_i/IC₅₀)}. (C) Current-voltage relationships. Normalized peak Cl⁻ currents (I_V/I_+40-mV) were measured at different [Mg²⁺]_i and fixed [ATP]_i. Mg²⁺ did not change the reversal potential, indicating that the Mg²⁺ effect is independent of voltage and that Mg²⁺ cannot permeate the channel. Experimental conditions are the same as reported previously (Oiki et al., 1994).

Dual mechanisms for VSOR activation. The bottom half represents the membrane unfolding-induced (type A) mechanism, which takes place upon cell swelling, and the top half represents the oxidation-induced (type B) mechanism, which takes place upon receptor stimulation (see the text for details and other abbreviations). BKR, bradykinin receptor; DAG, diacylglycerol; Gq, heterotrimeric G protein that activates phospholipase C; IP₃, inositol trisphosphate; mGluR, metabotropic glutamate receptor; P2YR, P2Y purinergic receptor; PKC, protein kinase C; PLC, phospholipase C; ROC, receptor-operated channel; SOC, store-operated channel.

Effects of H₂O₂ stimulation on the subcellular localization of ACTN4 and molecular interaction between ACTN4 and ABCF2. (A and B) Accumulation of ACTN4, but not ABCF2, in the RIPA-insoluble fraction with 0.5 mM H₂O₂ stimulation. Five micrograms of RIPA-soluble and RIPA-insoluble fractions of the 100,000g pellet prepared from the cells transiently overexpressing ACTN4/FLAG and ABCF2/HA were subjected to SDS-PAGE and immunoblotting with anti-FLAG M2 mAb and anti-HA and anti–caveolin-1 antibodies. (A) Immunoblots. ACTN4/FLAG, ABCF2/HA, and caveolin-1 were detected. Molecular weight markers (in kilodaltons) are indicated on the left. (B) Densitometric analysis of the immunoblot data shown in (A). The ratio of the ACTN4/FLAG contents (normalized with caveolin-1 content) in the presence of 0.5 mM H₂O₂ to that in the absence of 0.5 mM H₂O₂ is plotted. *P < 0.05 (significant difference between the data from cells with or without H₂O₂ stimulation). (C and D) H₂O₂ stimulation enhances the interaction between ACTN4 and ABCF2 in the RIPA-insoluble fraction. (C) RIPA-soluble and RIPA-insoluble proteins from the 100,000g pellets were immunoprecipitated using anti-FLAG M2 mAb and were immunoblotted with anti-FLAG mAb and anti-HA antibody (right). Transfected ACTN4/FLAG and ABCF2/HA were shown in the input (left). (D) Densitometric analysis of the immunoblot data shown in (C). The ratio of the coprecipitated ABCF2/HA content (normalized with the precipitated ACTN4/FLAG content) with H₂O₂ stimulation (black columns) to that of control (white columns) is plotted. *P < 0.05 (significant difference between the data from cells with or without H₂O₂ stimulation). Experimental conditions are the same as reported previously (Ando-Akatsuka et al., 2012). HA, hemagglutinin; IB, immunoblotted; IP, immunoprecipitated; mAb, monoclonal antibody; RIPA, radioimmunoprecipitation assay buffer.

Effects of NOX expression on VSOR currents in HEK293T cells. (A) Representative VSOR whole-cell currents elicited by step pulses from −100 to +100 mV in 20-mV increments during application of hypotonic (83% osmolality) solution in vector-transfected cells (upper panel) and NOX1-transfected cells (lower panel). (B) Mean VSOR currents recorded at +100 and −100 mV in vector-, NOX1-, NOX2-, NOX4-, and NOX5-transfected cells (n = 5–14). Experimental conditions are the same as reported previously (Okada et al., 2009a; Ando-Akatsuka et al., 2002).

Strategy adopted in our study for molecular identification of the core component of Maxi-Cl. By performing bleb membrane proteomics followed by gene silencing and disruption, mutagenesis, pharmacology, heterologous expression, and recombinant protein reconstitution, SLCO2A1 protein was identified as a core component of the ATP-conductive Maxi-Cl channel (see the text and Sabirov et al., 2017 for details). LC, liquid chromatography; MS/MS, tandem mass spectrometry.

Dual modes of SLCO2A1 functions as a transporter, PGT (left), and an ATP-permeable maxi-anion channel, Maxi-Cl (right).

Chemical structures of some VSOR blockers. (A) Etacrynic acid derivatives. The blue dashed line depicts the 2,3-dichlorophenoxy fragment, which is identical for all of these molecules. The red dashed line delineates a fragment (short-chain oxy-carbonic acid combined with chlorinated cyclopentyl-oxo-indanyl) common for VSOR blockers (see the text for details) but not for etacrynic acid. (B) VSOR blockers that have two aromatic rings connected with a chain of one to four atoms (carbon, nitrogen, or mixed). The blue dashed lines mark the first aromatic ring (see the text for details). IAA-94, indanyloxyacetic acid 94.

Voltage-dependent inhibition by DIDS of volume-sensitive outwardly rectifying Cl⁻ currents in human cervical carcinoma HeLa cells. (A) Representative current traces before and after application of DIDS. Step pulses were applied from −100 to +100 mV in 20-mV increments, with a prepulse of −100 mV and a postpulse of −60 mV. (B) Concentration dependence of DIDS on the currents recorded at +100 mV (filled circles) and −100 mV (open circles) (n = 3–6). The IC₅₀ values at +100 and −100 mV were 22.7 and 88.5 µM, respectively. Experimental conditions are the same as reported previously (Shimizu et al., 2004).

Pharmacological profile of Maxi-Cl currents in patches excised from C127 cells. All of the columns represent the mean currents recorded at +25 mV. (A) Inhibitory effects of extracellular application of Gd³⁺ (50 μM) and intracellular application of arachidonic acid (20 μM). (B) Insensitivity to a VSOR blocker, DCPIB (10 μM), added from the intracellular or extracellular side. (C) Insensitivity to pannexin hemichannel antagonists, probenecid (1 mM) and ¹⁰Panx1 (8 μg/ml), added to the extracellular (pipette) solution. (D) Insensitivity to gap junction antagonists, CBX (100 μM), 1-octanol (2 mM), and GAP27 (4 μg/ml), added to the extracellular (pipette) solution. Experimental conditions, other than the cell line used, are the same as reported previously (Islam et al., 2012; Sabirov et al., 2017). AA, arachidonic acid; Probe, probenecid.