LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions
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ABSTRACT:
PROVIDER: PRJNA664795 | ENA |
REPOSITORIES: ENA
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Project description:Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such process was unknown to date. Using a genome wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na+ /K+ /2Cl cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.
2021-05-29 | GSE158316 | GEO
Project description:Taste substances are received by taste receptors expressed in taste cells. “Salty taste” sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. This channel could be activated without an increase in intracellular calcium ion. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.
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Project description:Methylene diphenyl diisocyanate is a chemical known to cause asthma. The present study uses mice to investigate exposure-induced changes in lung gene expression and effects of a chloride channel inhibitor We used microarrays to detail global whole lung gene expression following respiratory tract exposure to methylene diphenyl diisocyanate (MDI) vs. control exposure in mice immune-sensitized to MDI by prior skin exposure. In some studies mice were given a chloride channel inhibitor (crofelemer) via the respiratory tract before MDI.
2020-03-02 | GSE136146 | GEO
Project description:The volume-regulated anion channel (VRAC) is a hexameric complex formed by LRRC8 proteins. VRACs are responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling by mediating the efflux of chloride. Besides chloride, LRRC8 proteins transport other molecules including immunomodulatory cyclic dinucleotides (CDNs) such as 2’3’cGAMP. Here we identify LRRC8C as a critical component of VRACs in T cells, where its deletion abolishes VRAC currents and RVD. We find that LRRC8C mediates the transport of 2’3’cGAMP in T cells. T cells of Lrrc8c-/- mice have increased cell cycle progression, proliferation, survival, Ca2+ influx and cytokine production in vitro and enhanced T cell mediated immunity in vivo. We used RNA sequencing of CD4+ T cells from wild-type (WT) and Lrrc8c-/- mice to determine the effects of impaired cGAMP signaling in T cells. T cells of Lrrc8c-/- mice had impaired p53 signaling which was associated with decreased p53 expression. We found that uptake of 2’3’cGAMP and other CDNs via LRRC8C results in STING activation and p53 accumulation. Inhibition of STING in WT T cells recapitulates the phenotype of LRRC8C-deficient T cells whereas overexpression of p53 inhibits enhanced T cell function in the absence of LRRC8C. These findings establish cGAMP uptake through LRRC8C and subsequent STING-p53 signaling as a novel inhibitory signaling pathway in T cells and adaptive immunity.
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Project description:CLCN2 Chloride Channel Mutations in Familial Hyperaldosteronism Type II
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Project description:Primary aldosteronism (PA), a common cause of severe hypertension, features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II) and 80 additional probands with unsolved early-onset PA. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of the identical p.Arg172Gln mutation; all relatives with early-onset PA carried the CLCN2 variant found in probands. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. In this data set, we examined RNA expression in H295R cells transfected with empty vector, WT and p.Arg172Gln CLCN2. Expression of CLCN2 led to increased expression of CYP11B2 and its upstream regulator NR4A2.
2017-12-08 | GSE107030 | GEO
Project description:Volume-regulated anion channels participate in the cellular response to osmotic swelling. These membrane proteins are heteromers of LRRC8 family members whose composition determines permeation properties. Although structures of the obligatory LRRC8A subunit have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have closed this gap by the structural characterization of channels consisting of LRRC8A and C. Like homomeric LRRC8A, these proteins assemble as hexamers. Despite the twelve possible arrangements, we find a single predominant species with an A/C ratio of two. In this assembly, the four LRRCA subunits cluster in their preferred conformation observed in homomers as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C-subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein.
2023-01-03 | PXD035350 | Pride
Project description:The Cl- intracellular channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs’ function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose a novel function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs’ transition to a membrane-associated conformation and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal formation in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.
2024-03-10 | PXD045186 | Pride
Project description:TMC4 is a novel chloride channel involved in high-concentration salt taste sensation
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