Deficient LRRC8A-dependent Volume-Regulated Anion Channel (VRAC) activity is associated with male infertility in mouse and humans
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE112730 | GEO | 2020/05/01
REPOSITORIES: GEO
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Project description:Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell-only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients.
| S-EPMC6141173 | biostudies-literature
Project description:Deficient LRRC8A-dependent Volume-Regulated Anion Channel (VRAC) activity is associated with male infertility in mouse and humans
| PRJNA448855 | ENA
Project description:To become fully functional effector T cells, naïve T cells undergo a drastic metabolic status shift from quiescence to substantial biosynthesis. The synthesis of biomass initiated by T cell receptor (TCR) signals drives the cell volume increase during T cell activation (T lymphoblast), which resembles the slowest extreme of hypotonic cell swelling. However, the fundamental role of cell volume regulation in TCR signaling during T lymphoblast and its underlying mechanisms remain a long-standing enigma. Here we show that optimal T cell activation and function require appropriate cell volume regulation by regulated volume decrease (RVD) mediated via volume-regulated anion channels (VRACs) during T cell blast. Pharmacological blockade of VRACs and genetic deletion of LRRC8A in T cells, the essential component of VRACs, impair T cell activation and function, particularly under weak TCR stimulation, such as low-affinity TCR-pMHC engagement and unsaturated anti-CD3e crosslinking. Additionally, LRRC8A has distinct influences on mRNA transcriptional profiles driven by various TCR signal strengths, revealing the prominent effects of cell volume regulation for T cell functions. More importantly, LRRC8A-deficient mice have defective antiviral immunity and obstacles in effector-memory transition of CD8+ T cells. Furthermore, LRRC8A escorts the thymic selection of T cells and shapes the T cell repertoire in the thymus. Mechanistically, LRRC8A governs stringent cell volume increase via RVD during T cell blast induced by activation-driven intracellular osmolytes accumulation to keep the proximal TCR signaling molecules at adequate density. T cell blast driven by TCR signal feedbacks to control T cell activation. Together, our results reveal a previously unappreciated layer of T cell activation regulation that LRRC8A acts as a cell volume controlling "valve" to facilitate T cell activation and function
2023-09-23 | GSE243574 | GEO
Project description:Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC), which is activated by cell swelling and transports halide anions and small organic compounds. Despite the recent availability of the cryo-EM structures of homo-hexameric LRRC8A, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. In this study, we confirmed the absence of LRRC8 isoforms in the purified HsLRRC8D using mass spectrometry, and the structure of the HsLRRC8D homomer was analyzed using the cryo-EM. This work provides structural insights into the functional diversity of the LRRC8 protein family.
2020-05-27 | PXD018780 | Pride
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:Malignancy of glioblastoma multiforme (GBM), the most common and aggressive form of human brain tumor, strongly depends on its enhanced cell invasion and death evasion which make surgery and accompanying therapies highly ineffective. Several ion channels that regulate membrane potential, cytosolic Ca2+ concentration and cell volume in GBM cells play significant roles in sustaining these processes. Among them, the volume-regulated anion channel (VRAC), which mediates the swelling-activated chloride current (IClswell) and is highly expressed in GBM cells, arguably plays a major role. VRAC is primarily involved in reestablishing the original cell volume that may be lost under several physiopathological conditions, but also in sustaining the shape and cell volume changes needed for cell migration and proliferation. While experimentally VRAC is activated by exposing cells to hypotonic solutions that cause the increase of cell volume, in vivo it is thought to be controlled by several different stimuli and modulators. In this review we focus on our recent work showing that two conditions normally occurring in pathological GBM tissues, namely high serum levels and severe hypoxia, were both able to activate VRAC, and their activation was found to promote cell migration and resistance to cell death, both features enhancing GBM malignancy. Also, the fact that the signal transduction pathway leading to VRAC activation appears to involve GBM specific intracellular components, such as diacylglicerol kinase and phosphatidic acid, reportedly not involved in the activation of VRAC in healthy tissues, is a relevant finding. Based on these observations and the impact of VRAC in the physiopathology of GBM, targeting this channel or its intracellular regulators may represent an effective strategy to contrast this lethal tumor.
| S-EPMC6468384 | biostudies-literature
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:SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with four other LRRC8 family members, potentially forms a vastly heterogeneous cohort of VRAC channels with different properties; however, SWELL1 alone is also functional. Here, we report a high-resolution cryo-electron microscopy structure of full-length human homo-hexameric SWELL1. The structure reveals a trimer of dimers assembly with symmetry mismatch between the pore-forming domain and the cytosolic leucine-rich repeat (LRR) domains. Importantly, mutational analysis demonstrates that a charged residue at the narrowest constriction of the homomeric channel is an important pore determinant of heteromeric VRAC. Additionally, a mutation in the flexible N-terminal portion of SWELL1 affects pore properties, suggesting a putative link between intracellular structures and channel regulation. This structure provides a scaffold for further dissecting the heterogeneity and mechanism of activation of VRAC.
| S-EPMC6086657 | biostudies-literature
Project description:Regulation of cellular volume is an essential process to balance volume changes during cell proliferation and migration or when intracellular osmolality increases due to transepithelial transport. We previously characterized the key role of volume-regulated anion channels (VRAC) in the modulation of the volume of trabecular meshwork (TM) cells and, in turn, the aqueous humour (AH) outflow from the eye. The balance between the secretion and the drainage of AH determines the intraocular pressure (IOP) that is the major casual risk factor for glaucoma. Glaucoma is an ocular disease that causes irreversible blindness due to the degeneration of retinal ganglion cells. The recent identification of Leucine-Rich Repeat-Containing 8 (LRRC8A-E) proteins as the molecular components of VRAC opens the field to elucidate their function in the physiology of TM and glaucoma. Human TM cells derived from non-glaucomatous donors and from open-angle glaucoma patients were used to determine the expression and the functional activity of LRRC8-mediated channels. Expression levels of LRRC8A-E subunits were decreased in HTM glaucomatous cells compared to normotensive HTM cells. Consequently, the activity of VRAC currents and volume regulation of TM cells were significantly affected. Impaired cell volume regulation will likely contribute to altered aqueous outflow and intraocular pressure.
| S-EPMC6443673 | biostudies-literature
Project description:The volume-regulated anion channel (VRAC) is formed by LRRC8 subunits. Besides their role in the maintenance of cell homeostasis, VRACs are critically involved in oxidative stress mechanisms: reactive oxygen species directly modulate VRACs in a subunit-dependent manner. It was reported that LRRC8A-LRRC8E heteromeric channels are activated by oxidation, whereas LRRC8A-LRRC8C heteromers are inhibited. Here we adopted chimeric- as well as concatemeric-based strategies to identify residues responsible for the divergent effect of oxidants. We identified two cysteines in the first two leucine rich repeats of LRRC8E, C424 and C448, as the targets of oxidation. Oxidation likely results in the formation of a disulfide bond between the two cysteines, which in turn induces a conformational change leading to channel activation. Additionally, we found that LRRC8C inhibition is caused by oxidation of the first methionine. We thus identified crucial molecular elements involved in channel activation, which are conceivably relevant in determining physiological ROS effects. KEY POINTS: Volume-regulated anion channels (VRACs) are heterohexameric complexes composed of an essential LRRC8A subunit and a variable number of LRRC8B-E subunits. VRACs are directly regulated by oxidation, with LRRC8A-LRRC8E heteromers being potentiated and LRRC8A-LRRC8C heteromers being inhibited by oxidation. We identified two LRRC8E specific intracellular cysteines that form a disulfide bond upon oxidation leading to LRRC8A-LRRC8E potentiation. Inhibition of LRRC8A-LRRC8C heteromers is mediated by the oxidation of the start methionine, being additionally dependent on the identity of the LRR domain. Besides providing physiological insights concerning the outcome of reactive oxygen species modulation, the results point to key structural elements involved in VRAC activation.
| S-EPMC9540897 | biostudies-literature