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

Genetic Screening and Analysis.

To identify genes that are essential for hypertonic stress survival, an unbiased loss-of-function genetic screening was performed in HeLa cells using the CRISPR-Cas9 system (28). Cells expressing a constitutive hCas9 were infected with one of two lentiviral gRNA libraries ([Toronto KnockOut Library] TKO-1 or TKO-2) (28). Cells were selected by adding puromycin (1.5 µg/mL) at 24-h postinfection, and multiplicity of infection (MOI) was determined after 48 h (T0) by comparison with nonselected infected cells (TKO-1, MOI = 0.34; TKO-2, MOI = 0.5). At this point, 20 × 10⁶ cells were collected for subsequent genomic DNA (gDNA) extraction and sequencing to ensure good library representation (T0). The rest of the cells were split into three replicates (A, B, and C), each covering a 200-fold representation of each library (20 × 10⁶ cells). Cells were then split every 3 d, always keeping a 200-fold representation of each library. At T6, cells were seeded and 24 h later were challenged with hypertonic stress (150 mM NaCl) or kept in regular medium. One day after, they were counted and frozen (20 × 10⁶ cells) for subsequent gDNA extraction, library preparation, and next generation sequencing, which were carried out as in ref. 28. Briefly, genomic DNA was extracted from cell pellets using the QIAamp Blood Maxi Kit (Qiagen). gRNA inserts were amplified via PCR using primers harboring Illumina TruSeq adapters with i5 and i7 barcodes, and the resulting libraries were sequenced on an Illumina HiSeq2500. Each read was completed with standard primers for dual indexing with Rapid Run V1 reagents. The first 20 cycles of sequencing were “dark cycles” or base additions without imaging. The actual 26-base pairs read began after the dark cycles and contained two index reads, reading the i7 sequences first, followed by i5.

Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) (56) was used to evaluate the quality of screening data and to identify osmostress fitness genes. MAGeCK is specifically designed to analyze genome-wide CRISPR-Cas9 screenings, and it computes a per-gene fitness effect score by integrating information from all sgRNAs targeting a given gene. This so-called robust rank aggregation (RRA) score indicates the degree of selection of a gene in a screening. Correct establishment of the KO library is a proxy for good quality of the screening. An efficiently established library should be depleted of genes essential for cell survival. For this reason, both day 8 control cells and NaCl-treated cells were compared against day 0 libraries using MAGeCK. Genes were then ranked by RRA, and a gene set enrichment analysis (GSEA) (57) was performed against the entire Kyoto Encyclopedia of Genes and Genomes (KEGG) database (SI Appendix, Table S3). As expected, essential terms were enriched among the top depleted genes (false discovery rate [FDR] < 0.01) (including RNA polymerase, proteasome, spliceosome, cell cycle, and ribosome), thereby proving the correct establishment of our KO libraries. Next, day 8 control and NaCl-treated cells were compared using MAGeCK to identify genes whose KO causes defects in cell proliferation upon hyperosmotic stress (SI Appendix, Table S4). After multiple testing correction using the Benjamini–Hochberg procedure, LRRC8A/SWELL1 emerged as the top scoring candidate (Fig. 1B; rank 1, FDR = 0.064).

Cell Lines.

Cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco) containing 10% fetal calf serum (Sigma) supplemented with 1 mM sodium pyruvate and 100 U/mL penicillin (Gibco) and cultured in a 5% CO₂ humidified incubator at 37 C. HeLa cells (human, cervical epithelial, female) were purchased from the American Type Culture Collection and HeLa cells stably expressing Cas9, from ref. 28. HeLa-shLRRC8A cells (KD) were kindly provided by Dr. A. Patapoutian, Department of Neuroscience, The Scripps Research Institute, La Jolla, CA (29).

Control HeLa Cas9 LZ cells, LRRC8A-KO (KO) cells, p38-KO cells, and HeLa Cas9 LRRC8A-KO clone 6 cells expressing LRRC8A-WT (KO-iWT) or LRRC8A-S217A (KO-iS217A) under an inducible promoter were generated as described below. gRNA targeting LacZ or KOs from the TKO-1 library (see gRNAs sequences; SI Appendix, Table S2) were cloned into the pLCKO vector. To produce lentiviruses, guidelines of the “The RNAi Consortium” at the Broad Institute(https://portals.broadinstitute.org/gpp/public/resources/protocols) were followed. Briefly, 60 to 70% confluent 293T cells were transfected with pLCKO together with packing vectors (pMDG2 and psPAX2) and switched to inactivated high-serum (30%) medium. At 24- and 48-h posttransfection, supernatants were collected, pooled, spun, and filtered to eliminate residual cells. Cas9-expressing HeLa cells were then infected with 200 to 300 μL virus together with polybrene; at 24-h postinfection, they were selected with puromycin (1.5 µg/mL) for at least 48 h. LRRC8A KO clones were obtained by single-cell sorting in 96-well plates on FACSAria Fusion (Becton Dickinson). After clonal expansion, clones were checked by Western blot for the presence of LRRC8A protein. LRRC8A KO clone 6 was used to generate cell lines expressing LRRC8A-WT and LRRC8A-S217A under an inducible promoter following the transfection protocol described by Kowarz and colleagues (60). Briefly, cells were seeded in 6-well plates and transfected the following day with Lipofectamine 3000 (Invitrogene) using pSB100X transposase plasmid and pSBtet-BN constructs with the LRRC8A WT and mutants described in this work. At 24-h posttransfection, cells were selected with up to 5,000 μg/mL G418 (Sigma) for up to 1 wk. Induction was determined using a range of doxycycline (Sigma) concentrations (from 10 to 250 ng/mL), and protein levels were analyzed from cell extracts by Western blot.

Cell Transfection.

Cells were transfected with 1 mg/mL polyethylenimine (PEI, Polysciences, 23966) (PEI:DNA ratio of 5:1) diluted in 150 mM NaCl (for patch-clamp studies of MSK1 activation of LRRC8A, YFP quenching, and MSK1 imaging) or with Lipofectamine 3000 (Invitrogen) when transfected with WNK1 mutants, siLRRC8A, or siRNA-resistant LRRC8A-WT or LRRC8A-S217A in KD cells. A ratio of 1:1 LIPO:DNA diluted in Opti-MEM (Gibco) was used, following manufacturer’s instructions. All experiments were performed between 24- and 36-h posttransfection, except for patch-clamp experiments, which were carried out in KD cells overexpressing an siRNA-resistant channel, which were performed between 48- and 72-h posttransfection.

Generation of Constructs.

MSK1 putative phosphorylation sites in LRRC8A were mutated on the pIRES2-EGFP construct (gift from the Patapoutian laboratory; note that the LRRC8A sequence has two siRNA resistance regions) using the Q5 site-directed mutagenesis kit (NEB), following the manufacturer’s instructions. NEBaseChanger software was used for oligonucleotide design (SI Appendix, Table S2). The ICL fragments (amino acids F144 to D258) from WT or mutated LRRC8A pIRES2-EGFP plasmids were cloned into EcoRI/NotI sites of the pGEX-6P-1 plasmid for bacterial expression by PCR amplification. Full-length WT LRRC8A or S217A mutant were cloned into EcoRI/NotI sites of pGEX-6P-1 plasmid by PCR amplification with a forward oligonucleotide containing the myc tag after the EcoRI restriction site. MSK1 was subcloned from MSK1-BioID2-HA plasmid (a gift from R. Gomis, Institute for Research in Biomedicine [IRB], Barcelona) into the EcoRI site of pGEX-6P-1 plasmid for GST purification or PCR amplified and cloned into NotI/NcoI sites of the pETM11 plasmid for His-tag purification. Constructs with LRRC8A under an inducible promoter were generated by PCR amplification from the corresponding pIRES-EGFP plasmids with a reverse oligonucleotide carrying the CRISPR resistance nucleotide changes against the guide SC GD2 and cloned into the SfiI site of the pSB-BN plasmid. The pEBG-FLAG-WNK1 L369F L371F construct was generated by substituting the MreI-Bsu36I region from the pcDNA5 FRT/TO FLAG WNK1 L369F L371F into the pEBG-FLAG- WNK1 Wt plasmid.

Cell Viability Assays.

Cells were seeded into 6-well plates and then challenged with the indicated NaCl concentration on the following day. After 24 h, supernatants and cells were collected, pelleted, washed in phosphate-buffered saline (PBS), and then stained with 1 μg/mL PI (Sigma). PI staining was assessed by flow cytometry in FACSCalibur using CellQuest software (Becton Dickinson). LRRC8A KO cells expressing WT or mutant LRRC8A under an inducible promoter were seeded in 12-well plates with doxycycline and were challenged with the indicated NaCl concentration on the following day. After 24 h, supernatants and cells were collected, pelleted, washed with PBS, and stained with 1 μg/mL PI. PI staining was assessed by flow cytometry in Gallios (Beckman Coulter), and profiles were generated by using FlowJo software. Alternatively, after 24 to 48 h of stress, plates were washed with PBS, fixed, and stained with crystal violet solution for 20 min at room temperature (RT), rinsed with water, and air dried. Viable cells attached to the plate stained with crystal violet, making the intensity of the staining directly proportional to cell viability.

Protein Purification.

Recombinant GST proteins were expressed in Escherichia coli BL21 cells grown at 37 °C to an optical density (wavelength of 600nm) (OD600) of 0.5 for ICL-LRRC8A and MSK1 and of 0.8 for full-length LRRC8A proteins. GST-tagged proteins were induced for 3 h by adding 1 mM IPTG and switching the culture temperature to 25 °C. After induction, cells were collected by centrifugation and resuspended in a 1/50 volume of STET 1× buffer (100 mM NaCl, 10 mM Tris ⋅ HCl pH 8.0, 10 mM ethylenediaminetetraacetic acid [EDTA] pH 8.0, 5% Triton X-100 supplemented with 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM benzamidine, 200 mg/mL leupeptin, and 200 mg/mL pepstatin). Cells were lysed by brief ice-cold sonication and cleared by high-speed centrifugation. GST-fused proteins were pulled down from supernatants with 300 μL Glutathione-Sepharose beads (GE Healthcare, 50% slurry equilibrated with STET) by mixing for 45 min at 4 °C. The Glutathione-Sepharose beads were collected by brief centrifugation and washed first four times in STET buffer and then four times in 50 mM Tris ⋅ HCl pH 8.0 buffer supplemented with 2 mM DTT. GST-fused proteins were eluted in 500 μL (for ICL-LRRC8A and MSK1) or 200 μL (for full-length LRRC8A) 50 mM Tris ⋅ HCl pH 8.0 buffer supplemented with 2 mM DTT and 10 mM reduced glutathione (Sigma) by mixing for 30 min at 4 °C.

His-MSK1 was expressed in E. coli BL21 cells grown at 37 °C until they reached an OD600 of 0.5, followed by 3 h of induction with 1 mM IPTG at 25 °C. After induction, cells were collected by centrifugation and resuspended in sonication buffer (20 mM Tris pH 8, 100 mM NaCl, 1 mM PMSF, 1 mM benzamidine, 2 μg/mL leupeptin, and 2 μg/mL pepstatin). Cells were lysed by brief ice-cold sonication and cleared by high-speed centrifugation. His-MSK1 was pulled down from the supernatant with 300 μL TALON Metal Affinity resin (Clontech) and equilibrated with sonication buffer by mixing for 45 min at 4 °C. The TALON Metal Affinity resins were collected by brief centrifugation and washed six times with sonication buffer. The His-MSK1 was eluted in 300 μL elution buffer (20 mM Tris ⋅ HCl pH 8.0, 100 mM NaCl, and 50 mM imidazole) by mixing for 30 min at 4 °C.

In Vitro Kinase Assay.

GST-MSK1 or His-MSK1 was activated in vitro in 1× kinase assay buffer (50 mM Tris ⋅ HCl pH 7.5, 10 mM MgCl₂, and 2 mM DTT) with 100 μM cold ATP for 30 min at 37 °C. This activated GST-MSK1 or His-MSK1 was used to phosphorylate eluted GST-fused proteins in vitro. The reactions were carried out in 1× kinase assay buffer in the presence of 1 μCi/assay of radiolabeled 32P-γ-ATP (3000 Ci/mmol, Perkin-Elmer) in a final volume of 40 μL/assay for 30 min at 37 °C. Reactions were stopped by adding SB 5× (250 mM Tris ⋅ HCl pH 6.8, 0.5 M DTT, 10% sodium dodecyl sulfate [SDS], 20% glycerol, and 0.5% bromophenol blue) and boiling at 95 °C for 5 min. Phosphorylated proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and blotted onto a polyvinylidene difluoride (PVDF) membrane, which was exposed to KODAK BIOMAX XAR films or a phosphorimager.

In Vivo LRRC8A Phosphorylation Assays.

HeLa cells were seeded in 10-cm dishes. After 1 d, they were stressed with 100 mM NaCl for 15 min with or without a 1-h pretreatment with 10 μM MSK1 inhibitor SB747651A (Tocris) dissolved in DMSO. Cell extracts were collected with 1× Laemmli buffer (2% SDS) supplemented with PhosSTOP (Roche) and protease inhibitors and briefly sonicated to reduce viscosity. Samples were run on an 8% PAGE gel supplemented with 50 μM Phos-tag (Wako) and 20 μM MnCl₂ for 2 h at 30 mA in the cold. Proteins were transferred onto a PVDF membrane for 2 h at 300 mA on ice. LRRC8A mobility shift was detected by incubating the membranes with LRRC8A monoclonal antibody (Sigma), and signals were detected with ECL Clarity reagent (Bio Rad).

Western Blotting.

After the indicated treatments, cells were washed with ice-cold PBS and scraped into 300 μL lysis buffer (10 mM Tris ⋅ HCL pH 7.5, 1% Nonidet P-40, 2 mM EDTA, 50 mM NaF, 50 mM b-glycerophosphate, 1 mM sodium vanadate, supplemented with the protease inhibitors 1 mM PMSF, 1 mM benzamidine, 200 μg/mL leupeptin, and 200 μg/mL pepstatin). Lysates were cleared by centrifugation. Samples were resolved using SDS-PAGE and then blotted onto a PDVF membrane. Following incubation of the blots with the indicated antibodies, signals were detected using the ECL detection reagent (Amersham).

Patch-Clamp Experiments.

Whole-cell recordings were obtained as previously described (50) using an Axon 200A amplifier (Axon Instruments). Currents were acquired at 33 kHz and filtered at 1 kHz. The pClamp8 software (Axon Instruments) was used for pulse generation, data acquisition, and subsequent analysis. LRRC8A-like chloride currents were measured in cells clamped at 0 mV and pulsed for 400 ms from −100 mV to +100 mV in 50-mV steps every 30 s. I_Cl⁻ whole-cell currents were measured using pipettes (2 to 3 MΩ) filled with a solution containing 100 mM N-methyl-D-glucamine chloride (NMDGCl^–), 1.2 mM MgCl₂, 1 mM EGTA, 10 mM Hepes, 2 mM Na₂ATP, and 0.5 mM Na₃GTP (pH 7.3 and 300 mOsm/l). To achieve a controlled intracellular IS of 0.08, 78.2 mM NaCl was used instead of NMDGCl^– and adjusted osmolarity to ∼410 mOsm/l. The external solution contained NMDGCl^– at 100 mM (for iso- and hypotonic conditions) or 185 mM (hypertonic conditions), 0.5 mM MgCl₂, 5 mM KCl, 1.8 mM CaCl₂, 5 mM glucose, and 10 mM Hepes, pH 7.4. Osmolarity was adjusted to 310 (isotonic), 220 (hypotonic), or 415 (hypertonic) mOsm/l with mannitol.

YFP and Chloride Imaging.

Cells grown in coverslips and transiently transfected with halide-sensitive YFP constructs (37) were washed thoroughly with isotonic solution containing 110 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 5 mM glucose, 10 mM Hepes, pH 7.4. Osmolarity was adjusted to 310 mOsm/l with 80 mM mannitol. The hypotonic solution (220 mOsm/l) contained 85 mM NaCl and no mannitol, while the hypertonic solution (410 mOsm/l) contained 185 mM NaCl or NaI. For YFP-NKCC1 imaging experiments, hypertonic solutions were prepared by adding 100 mOsm of mannitol (410 mOsm/l) to an isotonic solution in order to avoid changes in ionic concentrations that could affect the sensor. Video microscopic measurements of YFP fluorescence were obtained using an Olympus IX70 inverted microscope with a 20× or 40× oil-immersion objective. The excitation light (488 nm) was supplied by a Polychrome IV monochromator (Till Photonics) and directed toward the cells under study by a 505DR dichromatic mirror (Omega Optical). Fluorescence images were collected by a digital charge-coupled device camera (Hamamatsu Photonics) after passing through a 535DF emission filter (Omega Optical) using AquaCosmos software (Hamamatsu Photonics). Images of basal fluorescence levels in isotonic solution were computed every 5 s and recorded for 2 min, followed by exposure to either 30% hypo- or hypertonic solutions for 5 or 10 min, respectively. After this, 80 μL 200 mM Nal was added to induce YFP quenching (adapted from ref. 23). Alternatively, instead of a NaI buffer, a hypertonic NaCl solution was substituted for an equivalent solution containing an equimolar concentration of NaI. The YFP signal was normalized to 1 (set at 100% of YFP fluorescence) at 5 s before NaI addition. The percentage of fluorescence decay after NaI addition was calculated as the YFP quenching response.

Recordings of relative changes in intracellular [Cl⁻] were carried out in cells loaded with 5 mM N-(6-methoxyquinolyl)acetoethyl ester (MQAE) (61) for 60 min at 37 °C or in cells transfected with the ratiometric Cl⁻ sensor ClopHensor. For MQAE experiments, cells were excited at 360 nm and emission was collected at >515 nm. Photobleaching correction of the normalized MQAE signal was performed when necessary. For experiments using the ClopHensor, fluorescent images were collected using a Leica laser-scanning confocal inverted microscope TCS Sp5 with a 40× oil-immersion objective. Cyan channel fluorescence was excited at 458 nm using an Argon laser and detected from 500 to 550 nm, and the red channel was excited using an He-Ne laser at 561 nm and detected from 600 to 700 nm. Laser scanning was performed using 400 Hz line frequency, 512 × 512 pixel format, and pinhole aperture set at 2 AU. Images were taken every 30 s using 3-line average. HeLa cells were monitored in iso for 2.5 min, treated with hypertonic solution for 10 min, and calibrated with 0 and 120 mM [Cl⁻] in the presence of the Cl⁻/OH⁻ ionophore exchanger tributyltinchloride (10 μM) and the K⁺/H⁺ exchanger nigericin (5 μM) (TBTN). Calibration of ClopH sensor was performed in vivo. Cells (n = 7) were monitored in isotonic solution for 2.5 min and then perfused with standard solutions at 0, 15, 30, 50, 80, 110, and 120 mM [Cl⁻] for 4 min in the presence of TBTN. All calibration solutions contained 120 mM [K⁺] 20 mM [Na⁺], 1 mM Mg gluconate, 1 mM Ca gluconate, 5 mM glucose and 10 mM Hepes, pH 7.3 and 300 mOsm/l. The desired [Cl⁻] in each standard solution was achieved by a combination of Na and K gluconate replacing NaCl and KCl. Cyan-to-red ratio of fluorescence intensity was normalized to isotonic treatment and expressed as mean ± SEM.

Cell Volume Measurements.

One confluent T75 flask was used for each volume measurement experiment. When needed, drug preincubations were performed on detached cells inside the cell incubator for the indicated time. All volume measurements were performed at RT, keeping the cells in warm complete medium. A Z2 Coulter Counter Analyzer (Beckmann) was used to measure the volumes of the cell populations. Each sample taken by the counter contained between 20,000 and 100,000 cells in a volume of 100 or 500 μL. Three baseline measures were taken in normal (isotonic) medium before adding the hypertonic or hypotonic medium. Changes in cell volume were normalized to basal volumes. To calculate the percentage of RVD or RVI, the Δ of mean basal volume was calculated to the volume at each time point. Maximum (swollen cells) or minimum (shrunken cells) cell volume changes were set as 100% and subsequent values were referred to this maximum/minimun to calculate the percentage of recovery (%RVI) (62).

Immunoprecipitation of WNK1 and Phospho-WNK1.

One or two 100-mm plates of cells at maximum confluency were used for each immunoprecipitation. Medium was exchanged for isotonic solution for at least 5 min. Then, the solution was exchanged to perform the desired treatment (hypo- to isotonic or hypertonic) for the indicated time. Isotonic solutions were maintained for controls. Solutions were gently removed, and 250 to 300 μL ice-cold lysis buffer (50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% [wt/vol] Nonidet P-40, 0.27 M sucrose, 0.1% [vol/vol] 2-mercaptoethanol, and protease inhibitors [1 tablet per 50 mL]) containing 1× PhosSTOP (Sigma) were added to each plate. Cells (on ice) were scraped thoroughly. Cell lysates were clarified by a 15-min centrifugation at 26,000 g and at 4 °C. For immunoprecipitation (IP), 1 mL supernatant (∼2 mg of protein) was transferred to a new tube, and 12 μg anti-WNK1 antibody was added. For anti–phospho-WNK IP, 12 μg anti-phospho antibody plus 10 μg dephosphorylated form of the phosphopeptide antigen were added to cell lysates. IPs were incubated overnight at 4 °C under continuous rotation. Additional 25 μL aliquots cleared cell lysates were kept as inputs for Western blot analysis when needed. On the following day, 40 μL (50% slurry) protein-G-Sepharose beads (previously blocked overnight with 1% bovine serum albumin) were added to each IP and incubated for 2.5 h at 4 °C under continuous rotation. Immunoprecipitates were washed three times with 1 mL lysis buffer containing 0.15 M NaCl and 1× PhosSTOP and twice with 1 mL buffer A (50 mM Tris/HCl, pH 7.5, and 0.1 mM EGTA). Bound proteins were eluted in 40 μL 1× lithium dodecyl sulfate sample buffer containing 1× reducing agent (Invitrogen) and heated at 70 °C for 10 min. Eluted samples were subjected to electrophoresis on precast NuPAGE 3 to 8% Tris Acetate gels (Invitrogen) and transferred to nitrocellulose membranes. Membranes were blocked in 1× Tris-buffered saline 0.05% tween 20 containing 10% nonfat dried milk powder. For the total-WNK1 condition, IPs were divided and loaded into two separate wells in the same gel to obtain both total- and phospho-WNK1 bands and thus the ratio of the two signals (phospho/total). For the phospho-WNK1 condition, the ratio to total-WNK1 signal was calculated using the total-WNK1 band obtained from the inputs. Primary antibodies were incubated overnight at 4 °C and secondary antibodies for 1 h at RT. Bands were developed with ECL substrate Clarity using the ChemiDoc XRS+ system and quantified with Quantity One Software (BioRad).

Statistical Analysis.

All electrophysiological, imaging, cell volume, and biochemical data are presented as mean ± SEM. Statistical analyses were performed using SigmaPlot software. First, a normality test was run, and for the data that followed normal distributions, a Student’s paired or unpaired t test was applied between two groups and one-way ANOVA followed by Bonferroni or Dunnett post hoc tests across multiple groups. In the case of multiple groups, comparison of different conditions to a control group is run using the Dunnett’s method, whereas all pairwise comparisons were evaluated using Bonferroni’s method or one-way ANOVA followed by Holm–Sidak post hoc test, as suggested by the statistical package. For the data that did not assume Gaussian distributions, a Mann–Whitney U test was used for comparing two groups. The criterion for statistically significant difference was P < 0.05. Statistical analyses of data generated with the genetic screening were performed using bash and R scripts; visualizations were done using the ggplot2 (v3.1.0) (63) and ggrepel (v0.8.0) (64) R packages.