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Leucine-rich repeat containing 8A (LRRC8A)-dependent volume-regulated anion channel activity is dispensable for T-cell development and function.

ABSTRACT: BACKGROUND:Leucine-rich repeat containing 8A (LRRC8A) is an ubiquitously expressed transmembrane protein with 17 leucine-rich repeats (LRRs) at its C-terminal end and is an essential component of the volume-regulated anion channel (VRAC), which controls cellular volume. A heterozygous mutation in LRRC8A that truncates the 2 terminal LRRs was reported in a patient with agammaglobulinemia and absent B cells and was demonstrated to exert a dominant negative effect on T- and B-cell development in mice. Lrrc8a-/- mice have severely defective T-cell development and function. It is not known whether the T- and B-cell defects caused by LRRC8A deficiency are caused by loss of VRAC activity. OBJECTIVE:We sought to determine whether VRAC activity is required for normal T-cell development and function. METHODS:VRAC activity was examined by using patch-clamp analysis. Flow cytometry was used to examine T-cell development. T-cell proliferation, cytokine secretion, and antibody titers were measured by using standard techniques. RESULTS:We demonstrate that the spontaneous mouse mutant ébouriffé (ebo/ebo) harbors a homozygous 2-bp frameshift mutation in Lrrc8a that truncates the 15 terminal LRRs of LRRC8A. The Lrrc8aebo mutation does not affect protein expression but drastically diminishes VRAC activity in T cells. ebo/ebo mice share features with Lrrc8a-/- mice that include curly hair, infertility, reduced longevity, and kidney abnormalities. However, in contrast to Lrrc8a-/- mice, ebo/ebo mice have normal T-cell development and function and intact antibody response to T-dependent antigen. CONCLUSION:LRRC8A-dependent VRAC activity is dispensable for T-cell development and function.

SUBMITTER: Platt CD

PROVIDER: S-EPMC6170198 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Leucine-rich repeat containing 8A (LRRC8A)-dependent volume-regulated anion channel activity is dispensable for T-cell development and function.

Platt Craig D CD Chou Janet J Houlihan Patrick P Badran Yousef R YR Kumar Lalit L Bainter Wayne W Poliani P Luigi PL Perez Carlos J CJ Dent Sharon Y R SYR Clapham David E DE Benavides Fernando F Geha Raif S RS

The Journal of allergy and clinical immunology 20170210 6

<h4>Background</h4>Leucine-rich repeat containing 8A (LRRC8A) is an ubiquitously expressed transmembrane protein with 17 leucine-rich repeats (LRRs) at its C-terminal end and is an essential component of the volume-regulated anion channel (VRAC), which controls cellular volume. A heterozygous mutation in LRRC8A that truncates the 2 terminal LRRs was reported in a patient with agammaglobulinemia and absent B cells and was demonstrated to exert a dominant negative effect on T- and B-cell developme ...[more]

PMID: 28192143

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Project description:BackgroundGlioblastoma (GBM) is the most common primary malignant brain tumor in adults. Ubiquitously expressed volume-regulated anion channels (VRAC) are thought to play a role in cell proliferation, migration, and apoptosis. VRAC are heteromeric channel complexes assembled from proteins belonging to the leucine-rich repeat-containing 8A (LRRC8A through E), among which LRRC8A plays an indispensable role. In the present work, we used an RNAi approach to test potential significance of VRAC and LRRC8A in GBM survival and sensitivity to chemotherapeutic agents.MethodsPrimary GBM cells were derived from a human surgical tissue sample. LRRC8A expression was determined with quantitative RT-PCR and downregulated using siRNA. The effects of LRRC8A knockdown on GBM cell viability, proliferation, and sensitivity to chemotherapeutic agents were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and Coulter counter assays. Cell cycle progression was further explored using fluorescence-activated cell sorting analysis of propidium iodide-stained cells.ResultsTemozolomide (TMZ), carmustine, and cisplatin reduced GBM cell survival with the IC50 values of ~1,250, 320, and 30?µM, respectively. Two of three tested gene-specific siRNA constructs, siLRRC8A_3 and siLRRC8A_6, downregulated LRRC8A expression by >80% and significantly reduced GBM cell numbers. The most potent siLRRC8A_3 itself reduced viable cell numbers by ?50%, and significantly increased toxicity of the sub-IC50 concentrations of TMZ (570?µM) and carmustine (167?µM). In contrast, the effects of siLRRC8A_3 and cisplatin (32?µM) were not additive, most likely because cisplatin uptake is VRAC-dependent. The results obtained in primary GBM cells were qualitatively recapitulated in U251 human GBM cell line.ConclusionDownregulation of LRRC8A expression reduces GBM cell proliferation and increases sensitivity to the clinically used TMZ and carmustine. These findings indicate that VRAC represents a potential target for the treatment of GBM, alone or in combination with the current standard-of-care.

Dataset Information

Leucine-rich repeat containing 8A (LRRC8A)-dependent volume-regulated anion channel activity is dispensable for T-cell development and function.

Publications

Leucine-rich repeat containing 8A (LRRC8A)-dependent volume-regulated anion channel activity is dispensable for T-cell development and function.

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