Fig. 5: Characterization of cells stably expressing LRRC8A-BioID fusion protein.

A Schematic representation of the BioID technique, a method for exploring protein complexes in live cells [114, 120]. Within the BioID methodology, the target protein is expressed in cells as a fusion with a specialized tagging enzyme, BirA R118G (a promiscuous mutant biotin ligase, hereinafter referred to as BirA*). This enzyme utilizes exogenous biotin to catalyse the formation of biotinoyl-5’-AMP, a highly reactive molecule that biotinylates primary amines, such as the lysine side chain, within a proximity of approximately 10 nm [121]. Subsequently, cells are lysed, and the labeled proteins are subjected to affinity purification, followed by detection through MS. The identification of pertinent biotinylated proteins is then accomplished through quantitative and statistical methodologies B Verification of BirA* activity in cells expressing LRRC8A-BirA*-HA by Western blot. The expression of the LRRC8A-BirA*-HA fusion protein was assessed using an anti-HA antibody in the absence and presence of biotin. BirA* activity in cells expressing LRRC8A-BirA*-HA was assessed by WB using streptavidin-HRP for the detection of biotinylated proteins. Activity was assessed in the presence and absence of biotin (50 μM), using HEK293 cells and cells transfected with pcDNA3.1 MCS-BirA(R118G)-HA (in the blot indicated as BirA*) as controls. Actin was used as a loading control. For each sample, 50 μg of total protein extract was loaded. C Validation of the subcellular localization of the LRRC8A-BirA*-HA protein by immunofluorescence. Nuclei were visualized by DAPI staining; α-HA antibody and Alexa Fluor 586-conjugated red-emitting secondary antibody were used to visualize LRRC8A-BirA*-HA; streptavidin-HRP was used to visualize biotinylated proteins. D Validation of LRRC8A-BirA*-HA protein channel activity by patch clamp: Representative time course of current activation upon perfusion with a hypotonic solution of cells co-expressing the 8A-BirA*-8E heteromers. E Validation of the subcellular localization of the LRRC8A-BirA*-HA protein by Western Blotting. The localization of the LRRC8A-BirA*-HA protein was assessed in the two clones and in HEK293 cells and cells transfected with pcDNA3.1 MCS-BirA(R118G)-HA using the α-LRRC8A antibody. For each sample, 50 μg of protein extract enriched with the cytoplasmic (S) or membrane (M) protein fraction was loaded. The α-PMCA antibody was used to exclude contamination of the S-fraction by proteins from the M-fraction.