LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions

Abstract

Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl^- efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na⁺/K⁺/2Cl^- cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.

Keywords: LRRC8A chloride channel; NKCC; RVI; osmostress; p38/MSK1.

Fig. 1.

LRRC8A activation is essential for cell fitness upon hypertonicity. (A) Schematic diagram of unbiased CRISPR/Cas9 genome-wide genetic screening to identify genes relevant for adaptation to high osmolarity. (B) Representation of the MAGeCK per-gene fitness effect score (–log₁₀ RRA score). A higher fitness effect indicates a higher degree of importance for cell viability upon hyperosmotic stress. Gene name of LRRC8A is indicated, together with the name of nontargeting controls (LacZ, EGFP, luciferase, blue dots). (C and D) Crystal violet staining or PI staining (to monitor cell death) of LZ, LRRC8A-KO, and HeLa cells treated with DCPIB and exposed to different doses of hypertonic stress. P values determined by one-way ANOVA followed by post hoc Dunnett’s test versus LZ control group. (E) Mean ± SEM. YFP fluorescence changes (normalized to the baseline in isotonic conditions) in LZ and KO HeLa cells transiently transfected with halide-sensitive YFP. Addition of isotonic and hypertonic bathing solutions containing NaCl or NaI is indicated by boxes at the top of the recordings. (F) Percentage of YFP quenching measured 8 min after the addition of NaI. Mean ± SEM (LZ, n = 66; KO, n = 59). P values were determined by two-tailed Student’s t test. (G) Time course of whole-cell chloride currents recorded at –100 mV and +100 mV in LZ (gray) and KO (red) HeLa cells dialyzed with hyperosmotic solutions with an IS of 0.08 and then exposed to hypertonic solutions followed by exposure to 36 μM DCPIB. (Right) Families of chloride currents measured at the points indicated in the Left. Cells here held at 0 mV and pulsed from –100 mV to +100 mV in 50-mV steps. (H) Maximal mean current densities measured in LZ and KO HeLa cells under the experimental conditions shown in G. P values determined by Student’s t test.

Fig. 2.

The p38/MSK1 pathway regulates LRRC8A activation upon hypertonicity. (A) Maximal mean current densities (±SEM) measured in LZ and KO HeLa cells dialyzed with hyperosmotic solutions of 0.08 IS and exposed to hypertonic solutions containing DMSO (0.1%), the p38 inhibitor (SB203580, 10 μM), or the MSK1 inhibitor (SB747651A, 10 μM). P values were determined by one-way ANOVA followed by post hoc Dunnett’s test versus a DMSO control group. (B) Western blot analysis of LRRC8A in a Phos-tag gel of extracts obtained from HeLa cells exposed to hypertonic (+100 mM NaCl) solutions in the absence or presence of SB747651A. (C) Schematic diagram of MSK1 phosphorylation sites in LRRC8A. (D) In vitro phosphorylation by MSK1 of the ICL (amino acids 144 to 258) of LRRC8A-WT as well as of single mutants. (E) In vitro phosphorylation of full-length LRRC8A-WT and LRRC8A-S217A by MSK1. (F) Maximal mean current densities (±SEM) measured in stable LRRC8A KD HeLa cells overexpressing shRNA-resistant LRRC8A-WT or LRRC8A-S217A. Cells were dialyzed with hyperosmotic solutions with an IS of 0.08 and exposed to hypertonic solutions. P values were determined by one-way ANOVA followed by post hoc Dunnett’s test versus KD control group. (G) Maximal mean current densities (±SEM) measured in KD HeLa cells recorded under control isotonic conditions. Cells overexpressed shRNA-resistant WT or S217A LRRC8A with or without coexpression of a constitutively active MSK1 (MSK1-T581D/T700D). P values were determined by Student’s t test comparing the effects of MSK1 expression on WT or mutant channel.

Fig. 3.

The p38/MSK1 pathway and LRRC8A regulate NKCC-mediated RVI. (A) Time course of relative changes in cell volume of LZ and KO HeLa cells (normalized to isotonic conditions) before and after exposure to a 30% hypertonic medium. Mean ± SEM (n = 6). (B) Percentage of RVI, calculated as the percentage of volume recovered following the initial cell shrinkage at the different time points after the hypertonic stress. (C) Mean RVI (%) calculated at 60 min in LZ (n = 6), KO (n = 6), or p38α-KO (n = 9) HeLa cells. (D) Mean RVI (%) calculated at 60 min in HeLa cells superfused with DMSO (n = 7), the LRRC8A channel inhibitor DCPIB (n = 6), the p38 inhibitor SB203580 (n = 6), or the MSK1 inhibitor SB747651A (n = 4). (E) Mean RVI (%) calculated at 60 min in KD (n = 4) HeLa cells overexpressing shRNA-resistant WT (n = 6) or S217A (n = 4) LRRC8A channels. (F) Mean RVI (%) calculated at 60 min after LZ or KO HeLa cells were exposed to DMSO or 50 μM bumetanide. (G) Relative changes in cell volume measured before and after superfusion of LZ or KO HeLa cells with a 30% hypotonic medium. (H) Mean RVI (%), generated after RVD, was calculated at 60 min after return to isotonic medium, as shown in G (n = 3). P values were determined by two-tailed Student’s t test (H), a Bonferroni's all pairwise comparison (F), or one-way ANOVA followed by post hoc Dunnett’s test versus control group (Left Bar) (all others).

Fig. 4.

LRRC8A triggers WNK activation to promote RVI and cell survival under hypertonicity. (A) Phosphorylation of immunoprecipitated WNK1 obtained from cell lysates of LZ and KO HeLa cells exposed to 30% hypertonic medium. (Left) Western blot of p-S382 and total WNK1; (Right) quantification of phosphorylated WNK1 (normalized to total immunoprecipitated WNK1) after 30-min exposure to hypertonic medium. P values were determined by two-tailed Student’s t test (n = 3). (B) Western blot and quantification of p-S382 WNK1 of immunoprecipitates obtained from cell lysates of LZ or KO HeLa cells (n = 3) in an isotonic medium after exposure to 30% hypotonic medium (as described in Fig. 3G). (C) Mean (±SEM; n = 4 to 7) RVI (%) calculated at 60 min in LZ or KO HeLa cells overexpressing mock, WNK1-S382A (A), WNK1-S382E (E), or WNK1-L369F/L371F (FF). P values were determined by all pairwise one-way ANOVA followed by Holm–Sidak post hoc test. P < 0.01 only when comparing KO mock or KO WNKA with any other condition. (D) PI staining (to monitor cell death) of KO cells expressing WT or S217A mutant LRRC8A under an inducible promoter (DOX). (E) Model of the mechanism that links the p38/MSK1 phosphorylation pathway with the WNK1–NKCC1 axis via LRRC8A to promote RVI and cell survival in response to hypertonic environments.