Reports

show that VRAC represents a distinct class of anion channels that also con- Identification of LRRC8 Heteromers as duct organic osmolytes. To identify VRAC, we opted for a an Essential Component of the genome-wide RNA interference screen that could identify non-redundant VRAC components. Swelling-induced Volume-Regulated Anion Channel iodide influx into HEK cells expressing the iodide-sensitive yellow fluorescent VRAC protein YFP(H148Q/I152L) (17) was used as read-out in a fluorometric imag- Felizia K. Voss,1,2,3 Florian Ullrich,1,2,3 Jonas Münch,1,2,3 Katina Lazarow,1 Darius ing plate reader (Fig. 1A). Exposure to Lutter,1,2,3 Nancy Mah,2 Miguel A. Andrade-Navarro,2 Jens P. von Kries,1 Tobias saline containing 50 mM iodide en- Stauber,1,2* Thomas J. Jentsch1,2,4* tailed a slow fluorescence decay under isotonic conditions, whereas hypotonic- 1Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin. 2Max-Delbrück-Centrum für Molekulare ity induced a delayed increase in YFP 3 4 Medizin (MDC), Berlin. Graduate program of the Freie Universität Berlin. Neurocure, Charité quenching (Fig. 1B) that could be re- Universitätsmedizin, Berlin. duced by VRAC inhibitors like carbe- *Corresponding author. E-mail: [email protected] (T.J.J.); [email protected] (T.S.) noxolone (18) (fig. S1). In a prescreen targeting 21 anion transporters (table - - Regulation of cell volume is critical for many cellular and organismal functions, yet S1), only siRNAs against the Cl /HCO3 the molecular identity of a key player, the volume-regulated anion channel VRAC, exchanger AE2 gave significant effects has remained unknown. A genome-wide siRNA screen in mammalian cells identified (Fig. 1B). They decreased iodide influx LRRC8A as a VRAC component. LRRC8A formed heteromers with other LRRC8 under both isotonic and hypotonic con- multispan membrane proteins. Genomic disruption of LRRC8A ablated VRAC ditions. currents. Cells with disruption of all five LRRC8 genes required LRRC8A co- Our genome-wide screen utilized transfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform three separately transfected siRNAs per combination determined VRAC inactivation kinetics. Taurine flux and regulatory gene (fig. S2). Offline data analysis volume decrease also depended on LRRC8 proteins. Our work shows that VRAC (fig. S3, A and B) yielded the maximal defines a class of anion channels, suggests that VRAC is identical to the volume- slope of fluorescence quenching that sensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity was used to define hits. Further criteria of native VRAC currents. included the presence of predicted transmembrane domains and a wide expression pattern. 87 genes (table S2) Cells regulate their volume to counteract swelling or shrinkage caused were taken into a secondary screen with independent siRNAs. Of these, by osmotic challenges and during processes like cell growth, division, only suppression of LRRC8A robustly slowed hypotonicity-induced and migration. As water transport across cellular membranes is driven by YFP quenching (Fig. 1C). LRRC8A knock-down also strongly sup- osmotic gradients, cell volume regulation requires appropriate changes pressed ICl(swell) in patch-clamp experiments (Fig. 1, D to F), suggesting of intracellular concentrations of ions or organic osmolytes like taurine that the multispan membrane protein LRRC8A is an indispensable com- (1, 2). Regulatory volume decrease (RVD) follows the extrusion of in- ponent of VRAC or is needed for its activation. - + tracellular Cl and K and other osmolytes across the plasma membrane. Although LRRC8A reached the plasma membrane (fig. S4A for A key player is the volume-regulated anion channel VRAC that mediates HeLa cells), its transfection into HEK cells rather decreased ICl(swell) - characteristic swelling-activated Cl -currents (ICl(swell)) and is ubiquitous- (Fig. 1F). We hypothesized that VRAC contains LRRC8A as part of a ly expressed in vertebrate cells (3–5). Nearly inactive under resting con- heteromer and that LRRC8A overexpression led to a subunit stoichiome- ditions, VRAC slowly opens upon hypotonic swelling. The mechanism try that was incompatible with channel activity. LRRC8A has four close- behind VRAC opening remains enigmatic. VRAC currents are outward- ly related homologs (LRRC8B - LRRC8E) which all have four predicted ly rectifying (hence the alternative name VSOR for volume-stimulated transmembrane domains (19, 20). EST databases suggested that all hom- outward rectifier (4, 5)) and show variable inactivation at inside-positive ologs were widely expressed. Immunocytochemistry of transfected HeLa voltages. VRAC conducts iodide better than chloride and might also cells (fig. S4A) and of native HEK cells (Fig. 1, G and H) detected conduct organic osmolytes like taurine (6) (hence VSOAC, volume- LRRC8A at the plasma membrane. Truncation of its C terminus as in a stimulated organic osmolyte/anion channel (7)), but this notion is con- patient with agammaglobulinemia (21) led to cytoplasmic retention (fig. troversial (8–10). VRAC is believed to be important for cell volume S4B). LRRC8B through LRRC8E remained intracellular when transfect- regulation and swelling-induced exocytosis (11), and also for cell cycle ed alone, but reached the plasma membrane when co-transfected with regulation, proliferation and migration (1, 3, 4). It may play a role in LRRC8A (Fig. 1, I and J, fig. S4, C to H). Unlike LRRC8A transfection, apoptosis and various pathological states including ischemic brain ede- LRRC8A/LRRC8C co-expression did not suppress ICl(swell) (Fig. 1F). ma and cancer (4, 12). Progress in the characterization of VRAC and its However, neither this co-expression, nor any other combination tested, biological roles has been limited by the failure to identify the underlying significantly increased current amplitudes above WT levels. - protein(s) despite efforts for decades (1, 5). ClC-2 Cl -channels activate We used zinc-finger nuclease and CRISPR/Cas (22) technologies to - - upon cell swelling, but their inward rectification and Cl over I selectivi- constitutively disrupt LRRC8 genes. Besides polyploid HEK cells we ty deviate from VRAC (13) Drosophila dBest1, a member of a family of used stably diploid human HCT116 cells for increased disruption effi- 2+ - - Ca -activated Cl -channels, mediates swelling-activated Cl -currents in ciency. Gene disruption was confirmed by sequencing and Western blots insect cells (14, 15), but their characteristics differ from VRAC currents (Fig. 2A, table S3). To exclude off-target effects, we generated two HEK and the mammalian homolog of dBest1 is swelling-insensitive (16). We and three HCT116 lines in which LRRC8A was disrupted at different

/ http://www.sciencemag.org/content/early/recent / 10 April 2014 / Page 1 / 10.1126/science.1252826 positions (tables S3 and S4). ICl(swell) was abolished in all five lines and We ascertained the pannexin- and connexin-like transmembrane to- could be rescued by LRRC8A transfection (Fig. 2, B and C, and fig. S5), pology of LRRC8A. Mutating potential N-linked glycosylation sites proving that LRRC8A is essential for ICl(swell). We also produced between TMD1 and TMD2 abolished the size shift upon PNGaseF HCT116 cells in which other LRRC8 genes were disrupted singly or in treatment (Fig. 3B), demonstrating that this loop is extracellular. Immu- combinations, including a line with disruption of all five LRRC8 genes nofluorescence of cells transfected with HA-tagged LRRC8A constructs (henceforth called LRRC8−/− cells). Except for LRRC8A, disruption of showed that the TMD3-4 segment is extracellular and the C terminus single LRRC8 genes did not abolish VRAC currents (Fig. 2, B and C). cytoplasmic (Fig. 3C). −/− However, ICl(swell) amplitudes were robustly reduced in LRRC8E and in The formation of LRRC8 heteromers was confirmed by co- LRRC8(C/E)−/− double and LRRC8(C/D/E)−/− triple knock-out (KO) immunoprecipitation from HEK cells transfected with LRRC8A and −/− cells. ICl(swell) was abolished in LRRC8(B/C/D/E) cells (Fig. 2, B and epitope-tagged versions of either LRRC8B, C, D, or E. LRRC8A co- −/− - C). ICl(swell) inactivated faster and at less positive potentials in LRRC8C precipitated each of the other isoforms, but not the Cl -channel ClC-1 and LRRC8(C/E)−/− cells compared to wild-type (WT) HCT116, used as control (Fig. 3, D and E). Conversely, precipitation of epitope- −/− −/− −/− LRRC8B , LRRC8D or LRRC8E cells. By contrast, ICl(swell) inacti- tagged versions of LRRC8B through LRRC8E brought down LRRC8A vated more slowly and at more positive voltages in LRRC8(D/E)−/− (Fig. 3F). Co-precipitation of LRRC8 isoforms was also observed for HTC116 cells (Fig. 2, B and D, and fig. S6D) and in WT HEK cells native HEK cells (fig. S8). (Fig. 1E). ICl(swell) of these mutant cell lines retained the ion selectivity of Hypotonicity induced a robust taurine efflux from HEK and - - - −/− WT VRAC with the characteristic I >NO3 >Cl >>gluconate permeability HCT116 cell lines, but not from their LRRC8A derivates (Fig. 4A and sequence (fig S6A). fig. S9A) where it could be rescued by LRRC8A/LRRC8C co- LRRC8A transfection into quintuple KO LRRC8−/− cells failed to transfection (fig. S9B for HEK). Taurine efflux was also abolished in −/− rescue ICl(swell) (Fig. 2, E and F), agreeing with the absence of ICl(swell) in LRRC8(B/C/D/E) HCT116 cells (Fig. 4A). Since both ICl(swell) and LRRC8(B/C/D/E)−/− cells (Fig. 2, B and C). Co-transfecting LRRC8−/− swelling-induced taurine efflux similarly depended on LRRC8 heterom- cells with LRRC8A and either LRRC8C or LRRC8E yielded ICl(swell) ers, VRAC is most likely identical to VSOAC, the volume-stimulated with current densities similar to native cells (Fig. 2F). Co-expressing organic osmolyte/anion channel (7). Accordingly, LRRC8A−/− HEK cells LRRC8A with LRRC8D yielded lower currents (Fig. 2, E and F). No showed severely impaired volume regulation. After initial swelling, WT, current was observed upon LRRC8A+B co-expression, which may relate but not LRCC8A−/− cells slowly reduced their cell volume in the continu- to the poor expressibility of LRRC8B (Fig. 3, D and F). These findings ous presence of extracellular hypotonicity (Fig. 4B). Hence, LRRC8- fit to the low currents of LRRC8(C/E)−/− cells (Fig. 2C) where LRRC8A containing VSOAC is a major player in RVD. can only interact with poorly expressible LRRC8B and/or LRRC8D. The identification of LRRC8 proteins as crucial VRAC constituents Reconstituted ICl(swell) activated like WT VRAC upon swelling (Fig. 2E) ends a decades-long hunt for the elusive molecular identity of this im- and displayed its typical anion permeability sequence (fig. S6, B and C). portant channel. The absence of ICl(swell) upon genomic disruption of ICl(swell) inactivated more slowly and at more positive voltages when LRRC8A and its rescue by transient re-expression identified LRRC8A as LRRC8A was co-expressed with LRRC8C in LRRC8−/− cells compared an indispensable component of VRAC, or alternatively as being crucial to cells co-expressing LRRC8A with LRRC8E or LRRC8D (Fig. 2, E for its activation. The wide expression pattern of LRRC8 genes and the and G, and fig. S6E). This observation agreed with the faster ICl(swell) plasma membrane residency of LRRC8A-containing heteromers are inactivation in LRRC8C−/− cells (Fig. 2, B and D, and fig. S6D) in which fulfilled prerequisites for LRRC8 proteins forming the channel. The the ‘decelerating’ LRRC8C subunit may be replaced by LRRC8E or dependence of current properties on LRRC8 isoform combinations indi- other ‘accelerating’ subunits. cated that LRRC8 heteromers are integral components of VRAC, a no- Native ICl(swell) currents display different inactivation kinetics (3). tion buttressed by the homology of LRRC8 proteins to pannexins. Since Whereas ICl(swell) shows prominent inactivation at positive potentials in co-transfection of LRRC8 isoforms failed to significantly increase HEK (23, 24) (Fig. 1E, Fig. 2D, and fig. S6D) and even more so in ICl(swell) amplitude over WT levels other factors limit VRAC activity. HCT116 cells (Fig. 2, B and D, and fig. S6D), it inactivates much less in Such a limiting component might be an auxiliary subunit of VRAC or blood cells like promyelocytic HL-60 cells, and in vascular smooth mus- could be part of the signaling cascade leading to its activation. Indeed cle and neurons (24–26). EST databases suggest that these cells express VRAC currents seem to be highly regulated, with amplitudes differing the ‘decelerating’ subunit LRRC8C, but lack LRRC8E that potently only 2-3fold across cell types (3, 30). induces inactivation (Fig. 2, E and G, and fig. S6E). Quantitative RT- The homology between LRRC8 proteins and pannexins suggested PCR confirmed that HEK and HCT116 cells expressed LRRC8A that LRRC8 proteins form hexameric channels (20). We confirmed the through LRRC8E, whereas LRRC8E was almost absent from HL-60 pannexin-like topology of LRRC8A and propose that VRAC is formed cells (fig. S7). Moreover, HCT116 cells, whose ICl(swell) inactivates more by LRRC8 hexamers of LRRC8A and minimally one other family mem- than that of HEK cells (Fig. 1E, Fig. 2, B and D, and fig. S6D), express ber. In this model, VRAC may contain two to five different LRRC8 less ‘decelerating’ LRRC8C than HEK (fig. S7). isoforms. This could create a large variety of VRAC channels with dif- LRRC8 proteins have four predicted transmembrane domains ferent properties. The variation of ICl(swell) inactivation kinetics between (TMDs) followed by hydrophilic C-termini with up to 17 leucine-rich different tissues and cells (3) can now be ascribed to different expression repeats (27) (hence LRRC8 = leucine-rich repeat containing 8) (Fig. ratios of LRRC8 isoforms. LRRC8-dependent Cl-- and taurine-fluxes 3A). Their C-termini were originally thought to be extracellular (19, 21), indicated that VRAC is identical to VSOAC (6) and fit to a pore formed but proteome databases revealed (20) that the TMD2-TMD3 linker can by LRRC8 hexamers because hexameric pannexin channels likewise be phosphorylated and suggested that LRRC8 N- and C-termini are cy- display poor substrate specificity (28). toplasmic (Fig. 3A). LRRC8 proteins display weak homology (20) to Our work provides the basis to explore the structure-function rela- pannexins, pore-forming proteins (28) with connexin-like topology. tionship of VRAC/VSOAC, to clarify the signal transduction from cell Connexins form hexameric hemichannels and gap junctions (29). This volume increase to channel opening, and to investigate the role of the similarity suggested (20) that LRRC8 proteins form hexameric channels channel in basic cellular processes like cell division, growth, and migra- for so far unknown substrates. Just like VRAC currents (14, 15, 30), tion and in various pathological states. Interestingly, a truncating LRRC8 proteins are found in vertebrates, but not in other phyla like LRRC8A mutation has been described in a patient with agammaglobu- arthropoda (20). linemia (21) and LRRC8C may have a role in fat metabolism (31).

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concerning the assay, Dr. H.-P. Rahn for help with FACS-sorting, Drs. A. Brockhoff and W. Meyerhof for using their FLIPRTM in a pilot experiment, J. Liebold, N. Krönke, J. Jedamzick and S. Kleissle for technical assistance. Supported by the European Research Council (ERC) Advanced Grant (FP/2007-2013) 294435 ‘Cytovolion’ and the DFG (Exc 257 ‘Neurocure’) to TJJ.

Supplementary Materials www.sciencemag.org/cgi/content/full/science.1252826/DC1 Materials and Methods Author contributions Figs. S1 to S10 Tables S1 to S4 References (33–38)

3 March 2014; accepted 2 April 2014 Published online 10 April 2014 10.1126/science.1252826

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Fig. 1. siRNA screen for volume-regulated anion channel VRAC identifies LRRC8A. (A) Principle of screen. Top, in regulatory volume decrease (RVD) VRAC releases chloride. Below, quenching of YFP fluorescence by iodide entering through VRAC used as read-out. (B) Example traces, normalized to fluorescence at ~30-50 s. Averaged from wells treated with control siRNAs (scrambled, AE2, both n=3) and no siRNA (n=2) (error bars, SEM), and individual traces from wells singly transfected with the 3 siRNAs against LRRC8A. Except for LRRC8A siRNA2 and 3, all traces are from the same plate. Arrow indicates addition of iodide-containing hypotonic (hypo; 229 mOsm) or isotonic (iso; 329 mOsm) saline. (C) Secondary screen using siRNA pools against candidate genes. Averaged control traces as above. (D) Typical time course of VRAC activation in WT or LRRC8A siRNA-treated HEK cells. Current densities at -80 mV are shown. Bar, application of hypotonic (240 mOsm) saline (hypo). (E) Current traces of fully activated ICl(swell) measured using the protocol shown below. Dotted lines indicate zero current. (F) ICl(swell) amplitudes (at -80 mV) of WT HEK cells, cells treated with LRRC8A siRNA, or transfected with indicated LRRC8 cDNAs. Error bars, SEM; number of experiments is indicated; ***, p<0.001. (G) Plasma membrane localization of endogenous LRRC8A in HEK cells. (H) No LRRC8 labeling in LRRC8A−/− HEK cells. (I) LRRC8C is intracellular when transfected into HeLa cells, but (J) reaches the plasma membrane when co-transfected with LRRC8A. Inset, magnification of boxed area showing only GFP-fluorescence. Scale bars, 10 μm.

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Fig. 2. Characterization of LRRC8 KO cells and of reconstituted ICl(swell). (A) Western blots confirm LRRC8A disruption in mutant cell lines (table S3). α-tubulin, loading control. (B) Example ICl(swell) traces (as in Fig. 1E, but 2-s pulses) of WT and mutant HCT116 cells. (C) Current densities (at -80mV) of maximally activated ICl(swell) of WT and mutant HCT116 cells. (D) ICl(swell) inactivation assessed by ratio of current at end/beginning of pulse. (E) When transfected into HCT116 LRRC8−/− cells (with all LRRC8 genes disrupted), LRRC8A rescues ICl(swell) only with LRRC8C, D or E (left panels; example traces measured as in (B)). Right panels, example ramp current traces from reconstituted ICl(swell) at isotonicity (black), 2 min after switching to hypotonicity (green) and with maximal activation (red). Ramp protocol shown at top. (F) ICl(swell) current densities at -80mV for indicated combinations. (G) Voltage-dependent inactivation of ICl(swell). Error bars, SEM. Number of cells in brackets. *, p<0.05 **, p<0.01 and ***, p<0.001 vs. WT.

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Fig. 3. Transmembrane topology and heteromerization of LRRC8A. (A) LRRC8 model (modified from (20)). Four transmembrane domains precede a C terminus with up to 17 leucine-rich repeats (27) (orange). Phosphoserines in LRRC8A (red P) and LRRC8D (blue P) according to Uniprot (32), predicted N-linked glycosylation sites (Y) and added epitopes are indicated. (B) PNGaseF treatment of endogenous LRRC8A, transfected LRRC8A, but not of LRRC8A(N66A,N83A) with disrupted glycosylation sites, decreased LRRC8A size in Western blots. The changed banding pattern of LRRC8A(N66A,N83A) suggests altered posttranslational modifications. n.t., non-transfected. (C) Immunofluorescence of non-permeabilized and permeabilized HeLa cells transfected with HA-tagged GFP-LRRC8A. Overlays of GFP (green) and HA (red) labeling. Insets show exclusively HA-staining. Scale bar, 20 μm. (D) LRRC8A co-precipitated epitope-tagged LRRC8B through LRRC8E in double-transfected HEK cells. LRRC8B and LRRC8D were poorly expressible. (E) LRRC8A did not co-precipitate the ClC-1 Cl- channel. (F) Epitope-tagged LRRC8B through LRRC8E co-precipitated LRRC8A.

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Fig. 4. LRRC8 proteins are crucial for swelling-induced taurine efflux and RVD. (A) 3[H]-taurine efflux from HCT116 cells of indicated genotypes. Cells were either in isotonic solution throughout (WT, white bars), or exposed to hypotonic solution starting at t=0 (arrows). Bars, means of 6 measurements; error bars, SEM. (B) WT and LRRC8A−/− HEK cells were shifted to hypotonic saline (96 mOsm) at t=30s and cell volume was monitored by calcein fluorescence. Mean of 6 measurements; error range, SEM. Similar results obtained in 3 experiments.

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