Journal: bioRxiv

Article Title: Cryo-EM structure of the volume-regulated anion channel LRRC8

doi: 10.1101/331207

Figure Lengend Snippet: ( a ) Close-up view of the neighboring EL1 helices forming the pore constriction site. The side chains are depicted by stick models. ( b ) Sequence alignment around the pore constriction site, depicted according to Extended Data Fig. 2. ( c ) VRAC activity in the LRRC8A-rescued LRRC8A -knockout HEK293A cells. Each line indicates the mean ± s.e.m. of the normalized YFP fluorescence ( n = 4). Hypotonicity after the addition of iodide: 231 mOsm. WT (+) and WT (++++) represent the different expression levels of the LRRC8A wild-type protein detected by western blotting, according to Extended Data Fig. 6c. ( d ) Maximum speed of hypotonicity-induced YFP quenching. Data are presented as the mean ± s.e.m. with each individual light grey point (n = 4). ** P < 0.01, *** P < 0.001 by Dunnett’s test.

Article Snippet: The human LRRC8A WT protein (NCBI Reference sequence number: NP_062540.2) was cloned from human brain cDNA (ZYAGEN) into the pEG BacMam vector, with an C-terminal GFP-His8 tag and a tobacco etch virus (TEV) cleavage site, and was expressed in HEK293S GnTI − (N-acetylglucosaminyl-transferase I-negative) cells (ATCC, cat. no. CRL-3022) .

Techniques: Sequencing, Activity Assay, Knock-Out, Fluorescence, Expressing, Western Blot

Journal: bioRxiv

Article Title: Cryo-EM structure of the volume-regulated anion channel LRRC8

doi: 10.1101/331207

Figure Lengend Snippet: ( a ) Subcellular localization of LRRC8A mutants rescued in LRRC8A -knockout HEK293A cells. The white dashed square indicates a region around the cell membrane; the magnified image of the LRRC8A-derived fluorescent channel is located in the corner. The white scale bar indicates 25 μm. ( b ) FSEC profiles on a Superose 6 Increase 10/300 GL column (GE Healthcare) for the EGFP-fused HsLRRC8A mutants expressed in HEK293S GnTI − cells. The arrows indicate the estimated elution positions of the void volume, the EGFP-fused HsLRRC8A, and the free EGFP. The analysis of the mutants showed symmetric and monodisperse peaks, similar to those of the wild-type channel, confirming the proper structural integrities of these mutant channels. ( c ) Amounts of HsLRRC8A expression in rescued LRRC8A -knockout HEK293A cells. † indicates non-specific bands. ( d ) Comparison between individual time courses and the fitting curve of the normalized YFP fluorescence. Grey lines and light blue lines indicate individual time courses of normalized YFP fluorescence ( n = 4). Blue lines indicate the mean of one-phase exponential decay-fitting curves. I − -iso: 332 mOsm, I − -hypo: 231 mOsm.

Article Snippet: The human LRRC8A WT protein (NCBI Reference sequence number: NP_062540.2) was cloned from human brain cDNA (ZYAGEN) into the pEG BacMam vector, with an C-terminal GFP-His8 tag and a tobacco etch virus (TEV) cleavage site, and was expressed in HEK293S GnTI − (N-acetylglucosaminyl-transferase I-negative) cells (ATCC, cat. no. CRL-3022) .

Techniques: Knock-Out, Derivative Assay, Mutagenesis, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Cryo-EM structure of the volume-regulated anion channel LRRC8

doi: 10.1101/331207

Figure Lengend Snippet: ( a ) The conservation score mapped on the molecular surface of HsLRRC8A. The score was calculated by the program CONSURF , based on the multiple-sequence alignment of the A-E isomers in . The molecular surface is colored according to the normalized conservation score, from 0.752 (pink) to −0.983 (purple). ( b ) The model structure of the (LRRC8A) 5 ·LRRC8D heterohexamer, viewed parallel to the membrane (left), and from the extracellular side (right). The homology model of human LRRC8D was generated by the program MODELLER , using the alignment in , and then the α subunit of the present structure was replaced by this model. In the inset, a close-up view of the channel pore forming regions from the extracellular side is shown, and the side chains of pore-lining amino residues are depicted in stick representations. The five LRRC8A subunits are colored light blue, while the LRRC8D isoform is colored light brown.

Article Snippet: The human LRRC8A WT protein (NCBI Reference sequence number: NP_062540.2) was cloned from human brain cDNA (ZYAGEN) into the pEG BacMam vector, with an C-terminal GFP-His8 tag and a tobacco etch virus (TEV) cleavage site, and was expressed in HEK293S GnTI − (N-acetylglucosaminyl-transferase I-negative) cells (ATCC, cat. no. CRL-3022) .

Techniques: Sequencing, Generated