LRRC8 Proteins Form Volume-Regulated Anion Channels that Sense Ionic Strength

Abstract

The volume-regulated anion channel (VRAC) is activated when a cell swells, and it plays a central role in maintaining cell volume in response to osmotic challenges. SWELL1 (LRRC8A) was recently identified as an essential component of VRAC. However, the identity of the pore-forming subunits of VRAC and how the channel is gated by cell swelling are unknown. Here, we show that SWELL1 and up to four other LRRC8 subunits assemble into heterogeneous complexes of ∼800 kDa. When reconstituted into bilayers, LRRC8 complexes are sufficient to form anion channels activated by osmolality gradients. In bilayers, as well as in cells, the single-channel conductance of the complexes depends on the LRRC8 composition. Finally, low ionic strength (Γ) in the absence of an osmotic gradient activates the complexes in bilayers. These data demonstrate that LRRC8 proteins together constitute the VRAC pore and that hypotonic stress can activate VRAC through a decrease in cytoplasmic Γ.

Figure 1. Purification and reconstitution of SWELL1-containing protein complexes

(A) Western blot showing SWELL1-FLAG expression in inducible HeLa cells at low TET concentration (5 ng/ml). (B–C) Purified protein samples separated on native gels and visualized by Coomassie staining or western blotting with anti-SWELL1 antibody. (D) Western blot of native gel separating proteins purified from paraformaldehyde (PFA)-treated cells. (E) A representative mass spectrometry result identifying SWELL1 and its four homologs in the indicated purified protein samples. Relative protein concentrations observed in SWELL1-inducible HeLa cells were expressed as mean ± SEM (n = 3). (F) Purified complexes treated with or without formaldehyde (FA) for the indicated time, separated on a denaturing gel and detected with anti-FLAG antibody. Ctr, control. (G–J) Single-channel currents of reconstituted complexes in lipid bilayers at −100 mV and all-point current histograms for (G) isotonic (n=15) and (H–J) hypotonic conditions generated by asymmetric mannitol (n>30) as illustrated (right). (K) Blockade of currents after injection of DCPIB (40 μM final) into cis droplet. c and o indicate closed and open states. Single-channel conductance (γ) in panels H–J are indicated below o. See Figures S1–S3, Table S1–S2.

Figure 2. Purified LRRC8 subunit combinations form ion channels with distinct channel conductance in droplet lipid bilayers

(A) Purified protein samples from WT and LRRC8 triple (BDE, BCE, BCD)- and quadruple (BCDE)-KO SWELL1-inducible HeLa cells separated on a native gel and visualized by Coomassie staining. (B) Abundance of each LRRC8 protein normalized to WT (mean±SEM, n=2) estimated by mass spectrometry. (C) Whole-cell current densities of maximally activated I_Cl,swell in WT and KO HeLa cells. (D–H) Hypotonicity-induced γ of reconstituted complexes after purification from (D) WT HeLa, (E–G) triple KO HeLa, and (H) quadruple KO HeLa cells. At −100 mV, large conductance events were observed in samples from triple-KO cells that differed significantly from the small conductance events apparent in all samples including those from quadruple-KO cells (brown bars) (** p<0.01, *** p<0.001; Student’s t-test). See Figure S4, Table S3.

Figure 3. Single-channel currents in cell-attached patches from swollen HeLa cells reveal conductance depends on subunit composition

(A–C) Representative single-channel traces (10 s, left) and all-point current histograms (middle) from WT (A), BDE KO (B), and BCE KO (C) cells recorded near +100mV (pipette potential −100mV). Right: Frequency distribution of current amplitudes observed in single patches from separate cells. (A) A wide range of single-channel current amplitudes was observed from WT HeLa cells (n=19, right) (Example 1: 3.0 pA; Example 2: 2.5 pA and 3.8 pA). Background channel activity was present in Example 1 (0.9 pA). Voltage protocol indicated above. (B) Current amplitudes of channels composed of SWELL1 and LRRC8C were 4.6±0.2 pA (mean (arrow) ±SEM, n=22 from 11 patches). (C) Current amplitudes of channels composed of SWELL1 and LRRC8D were 2.5±0.2 pA (n=7 from 5 patches). See Figure S5, Table S4.

Figure 4. LRRC8 subunit combinations determine VRAC outward rectification

(A–C) Single-channel recordings and all-point current histograms of SWELL1-containing complexes purified from the indicated LRRC8 KO HeLa cells. Inset: block by DCPIB injection (40 μM final) into the cis droplet. (D–F) The γ of purified SWELL1-containing complexes and γ ratio (+V:−V). (G–I) P_o and P_o ratio (+V:−V); P<0.01 for A+C vs. A+D and P<0.001 for A+E vs. A+D heteromers. P_o ratio for A+C vs. A+E heteromers was not significantly different. (J) Representative leak-subtracted whole-cell hypotonicity-induced currents for LRRC8(B/D/E)^−/− (red), LRRC8(B/C/E)^−/− (blue), and LRRC8(B/C/D)^−/− (green) normalized to the maximum current at +100 mV and centered at V_rev. (K) Ratio of whole-cell current at ±80mV from V_rev for the indicated cells reveals stronger outward rectification for I_Cl,swell in BCE KO compared to other KO cells. Error bars indicate SEM. Significant differences (1- way ANOVA test) are shown: *** (p<0.001), ** (p<0.01). See Figure S6.

Figure 5. T44C mutations in LRRC8 subunits alter anion selectivity

(A) V_rev for I_Cl,swell recorded from parental or LRRC8(B/C/D/E)^−/− HeLa cells expressing WT or T44C LRRC8 subunits was determined in the presence of I⁻ or Cl⁻ as the only extracellular anion. Bars represent mean±SEM (*P<0.05; **P<0.01; ***P<0.001; n.s., not significant; unpaired t-test). (B) Representative leak-subtracted currents recorded in I⁻ (black) and Cl⁻ (green). More negative V_rev (arrows) in I⁻ vs. Cl⁻ solutions indicates selectivity for I⁻ over Cl⁻. (C) P_I/P_Cl calculations based on data shown in (A); values in parentheses indicate the upper and lower compound SEM.

Figure 6. LRRC8 proteins are activated by low ionic strength

(A–C) Single-channel current recordings of the indicated SWELL1-containing complexes after reconstitution in lipid bilayers under different salt conditions (in mM): (A) 500 KCl (n=6), (B) 150 KCl (n=8), and (C) 70 KCl (n=7). (D–E) Single-channel currents in 70 mM KCl and mannitol to adjust to physiological osmolality (~300 mOsm/kg) (n=6 and 8 for panels D and E). (F) Maximum currents from multi-channel recordings of indicated complexes in low Γ (n>15). All data were acquired at +100 mV except panel E (+150 mV). Significant differences were observed in low Γ for all groups (1-way ANOVA); ** P<0.01. γ and P_o presented in C, D and E are mean±SEM.