Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

Abstract

The broadly expressed volume-sensitive outwardly rectifying anion channel (VSOR, also called VRAC) plays essential roles in cell survival and death. Recent findings have suggested that LRRC8A is a core component of VSOR in human cells. In the present study, VSOR currents were found to be largely reduced by siRNA against LRRC8A in mouse C127 cells as well. In contrast, LRRC8A knockdown never affected activities of 4 other types of anion channel activated by acid, Ca²⁺, patch excision or cAMP. While cisplatin-resistant KCP-4 cells poorly expressed endogenous VSOR activity, molecular expression levels of LRRC8A, LRRC8D and LRRC8E were indistinguishable between VSOR-deficient KCP-4 cells and the parental VSOR-rich KB cells. Furthermore, overexpression of LRRC8A alone or together with LRRC8D or LRRC8E in KCP-4 cells failed to restore VSOR activity. These results show that deficiency of VSOR currents in KCP-4 cells is not due to insufficient expression of the LRRC8A/D/E gene, suggesting an essential involvement of some other factor(s), and indicate that further study is required to better understand the complexities of the molecular determinants of VSOR, including the precise role of LRRC8 proteins.

Keywords: LRRC8; VSOR; anion channel; cisplatin resistance; volume-sensitive.

Figure 1.

Suppressive effects of siRNA for LRRC8A on VSOR currents in murine C127 cells. (A) RT-PCR data confirming a knockdown effect of siRNA for LRRC8A. Data represent duplicate experiments. GAPDH was used as an internal control. (B) Whole-cell VSOR current responses to voltage steps in mock-transfected control cells after maximal activation by hypoosmotic stimulation (244 mosmol/kg-H₂O). The holding potential was 0 mV. After a pre-pulse to −100 mV (500 ms), currents were elicited by application of step pulses (1000 ms) from −100 to +100 mV in 20-mV increments followed by 500 ms at −100 mV. (C) Instantaneous current-to-voltage relationships of VSOR in cells treated with non-targeting siRNA (Mock control; open circles) and in cells treated with siRNA against LRRC8A (filled circles). The current density (normalized by cell capacitance) was measured at the beginning of test pulses from current recordings similar to those shown in (B). *Significantly different from the mock control at P < 0.05. (D) Mean values of current density recorded at +40 mV in mock-transfected and LRRC8A-siRNA-transfected cells.

Figure 2.

No significant effects of siRNA for LRRC8A on 4 other Cl⁻ channel currents in C127/CFTR cells. (A) Effects of siRNA for LRRC8A on the acid-sensitive outwardly rectifying (ASOR) anion channel currents. Left panel: Representative whole-cell ASOR current responses to voltage steps. ASOR currents were evoked by a low-pH stimulation (pH 4.5). Whole–cell currents were elicited by a pulse-protocol same as in Figure 1B. Right panel: Current-to-voltage relationships in cells treated with non-targeting siRNA (Mock control; open circles) and in cells treated with siRNA against LRRC8A (filled circles). Currents were measured at the end of test pulses from current recordings similar to those shown on the left panel. (B) Effects of siRNA for LRRC8A on the maxi-conductance Cl⁻ channel (Maxi-Cl) currents. Left panel: Representative Maxi-Cl current responses to voltage steps recorded after full activation upon patch excision (inside-out mode) from the cells transfected with non-targeting siRNA (Mock control). The holding potential was 0 mV. Currents were elicited by application of step pulses (500 ms) from −50 to +50 mV in 10-mV increments. Right panel: Mean values of macropatch Maxi-Cl current measured at +25 mV after full activation upon patch excision from mock-transfected (open column) and LRRC8A-siRNA transfected cells (hatched column). (C) Effects of siRNA for LRRC8A on the Ca²⁺-activated Cl⁻ channel (CaCC) currents. Left panel: Representative whole-cell CaCC current responses to voltage steps (a pulse-protocol same as in Figure 1B) in non-transfected control cells. Right panel: Current-to-voltage relationships in cells treated with non-targeting siRNA (Mock control: open circles) and with siRNA against LRRC8A (filled circles). Currents were measured at the end of test pulses from current recordings similar to those shown on left panel. (D) Effects of siRNA for LRRC8A on the cAMP-activated anion channel (CFTR) currents. Left panel: Representative whole-cell CFTR current responses to voltage steps in the cells transfected with non-targeting (Mock control) siRNA. The holding potential was 0 mV. Currents were elicited by application of step pulses (1000 ms) from −100 to +100 mV in 20-mV increments. CFTR currents were activated by bath-application of a cocktail containing forskolin (5 µM) + dibutyryl-cAMP (dbcAMP: 1 mM) in standard isotonic Ringer solution. Right panel: Mean values of CFTR current density recorded at +40 mV in mock-transfected (open column) and LRRC8A-siRNA-transfected cells (hatched column). No statistically significant difference was observed between anion channel currents elicited from mock-transfected and LRRC8A-siRNA-transfected cells.

Figure 3.

Expression profiles of LRRC8A in KB, KCP-4, HEK293T and HeLa cells. (A) RT-PCR data. The expression level of mRNA of LRRC8A was almost identical between the VSOR-deficient KCP-4 cell line and its parental VSOR-rich KB cell line. Data represent triplicate experiments. GAPDH was used as an internal control. MM: molecular marker. (B) Western blotting data. Expression of LRRC8A protein in KCP-4 was almost identical when compared with 3 other human cell lines, KB, HEK293T (HEK) and HeLa. We used protein samples isolated from LRRC8A-transfected HEK293T cells as a positive control where LRRC8A was overexpressed (HEK/LRRC8A-OE). Data represent duplicate experiments.

Figure 4.

Expression of exogenous LRRC8A and its suppressing, but not augmenting, effect on VSOR currents in KCP-4 cells. (A) GFP-tagged LRRC8A overexpressed in KB cells (upper row) and KCP-4 cells (lower row) was observed under a confocal microscope at 1 day after transfection. The plasma membrane was stained by Cell Mask™ Orange Plasma Membrane Stain (red) and nuclei by Hoechst stain (blue). (B) Current-to-voltage relationships of VSOR in KB cells (triangles) and KCP-4 cells (circles) transfected with mock (open circles) and with LRRC8A-IRES-EGFP vector (filled circles). Current recordings were performed as described in Figure 1B. (C) Mean values of VSOR current density recorded at +40 mV in KB cells (open column), mock-transfected KCP-4 cells (hatched column) and LRRC8A-IRES-EGFP vector-transfected KCP-4 cells (filled column). Effective transfection of LRRC8A was confirmed by GFP fluorescence (Inset). (D) Mean values of VSOR current density recorded at +40 mV in mock-transfected KCP-4 cells (open column) and in KCP-4 cells transfected with a combination of LRRC8A-IRES-EGFP and LRRC8E-IRES-DsRed2 vectors (hatched column) or in KCP-4 cells with LRRC8E-IRES-DsRed2 vector alone (filled column). No significant difference was observed compared to Mock control, while effective transfections of LRRC8A and LRRC8E were confirmed by GFP and DsRed2 fluorescence (Inset). (E) Mean values of VSOR current density recorded at +40 mV in KCP-4 cells transfected with mock (open column) and with a combination of LRRC8A-IRES-DsRed2 and LRRC8D-IRES-EGFP vectors (hatched column) or with LRRC8D-IRES-EGFP vector alone (filled column). No significant difference was observed as compared with mock control, while effective transfections of LRRC8A and LRRC8D were confirmed by DsRed2 and GFP fluorescence (Inset).

Figure 5.

Hypothetical model of the VSOR pore domain depicted by presuming a heteromeric hexamer structure before (A) and after (B, C) repletion of LRRC8A. A and non-A represent LRRC8A and LRRC8B/C/D/E, respectively, and X represents an as-yet-unidentified essential component. Here, we assume that incorporation of the A-X-A complex into the VSOR pore domain results in closure (B) or disruption of the VSOR pore (C).