Disruption of mitochondrial respiration inhibits volume-regulated anion channels and provokes neuronal cell swelling.
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ABSTRACT:
SUBMITTER: Patel AJ
PROVIDER: S-EPMC6792668 | biostudies-literature | 1998 May
REPOSITORIES: biostudies-literature
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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:Volume-regulated anion channels (VRACs) are hexamers of LRRC8 proteins that are crucial for cell volume regulation. N termini (NTs) of the obligatory LRRC8A subunit modulate VRACs activation and ion selectivity, but the underlying mechanisms remain poorly understood. Here, we report a 2.8-Å cryo-electron microscopy structure of human LRRC8A that displays well-resolved NTs. Amino-terminal halves of NTs fold back into the pore and constrict the permeation path, thereby determining ion selectivity together with an extracellular selectivity filter with which it works in series. They also interact with pore-surrounding helices and support their compact arrangement. The C-terminal halves of NTs interact with intracellular loops that are crucial for channel activation. Molecular dynamics simulations indicate that low ionic strength increases NT mobility and expands the radial distance between pore-surrounding helices. Our work suggests an unusual pore architecture with two selectivity filters in series and a mechanism for VRAC activation by cell swelling.
| S-EPMC10480491 | biostudies-literature
Project description:The volume-regulated anion channel (VRAC) is activated when a cell swells, and it plays a central role in maintaining cell volume in response to osmotic challenges. SWELL1 (LRRC8A) was recently identified as an essential component of VRAC. However, the identity of the pore-forming subunits of VRAC and how the channel is gated by cell swelling are unknown. Here, we show that SWELL1 and up to four other LRRC8 subunits assemble into heterogeneous complexes of ?800 kDa. When reconstituted into bilayers, LRRC8 complexes are sufficient to form anion channels activated by osmolality gradients. In bilayers, as well as in cells, the single-channel conductance of the complexes depends on the LRRC8 composition. Finally, low ionic strength (?) in the absence of an osmotic gradient activates the complexes in bilayers. These data demonstrate that LRRC8 proteins together constitute the VRAC pore and that hypotonic stress can activate VRAC through a decrease in cytoplasmic ?.
| S-EPMC4733249 | biostudies-literature
Project description:4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB) was identified as the selective blocker of volume-regulated anion channels (VRAC). VRAC are permeable to small inorganic and organic anions, including the excitatory neurotransmitter glutamate. In recent years DCPIB has been increasingly used for probing the physiologic and pathologic roles of VRAC and was found to potently suppress pathologic glutamate release in cerebral ischemia. Because ischemic glutamate release can be mediated by a plethora of mechanisms, in this study we explored the selectivity of DCPIB toward the majority of previously identified glutamate transporters and permeability pathways. l-[(3)H]glutamate, d-[(3)H]aspartate, and l-[(14)C]cystine were used to trace amino acid release and uptake. We found that in addition to its well-characterized effect on VRAC, DCPIB potently inhibited glutamate release via connexin hemichannels and glutamate uptake via the glutamate transporter GLT-1 in rat glial cells. In contrast, DCPIB had no direct effect on vesicular glutamate release from rat brain synaptosomes or the cystine/glutamate exchange in astrocytes. The compound did not affect the astrocytic glutamate transporter GLAST, nor did it block glutamate release via the P2X(7)/pannexin permeability pathway. The ability of DCPIB to directly block connexin hemichannels was confirmed using a gene-specific siRNA knockdown approach. Overall, our data demonstrate that DCPIB influences several glutamate transport pathways and that its effects on VRAC in vivo should be verified using additional pharmacological controls.
| S-EPMC3533478 | biostudies-literature
Project description:Background and purposeThe ethacrynic acid derivative, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume-regulated anion channels (VRACs). In the CNS, DCPIB was shown to be neuroprotective through mechanisms principally associated to its action on VRACs. We hypothesized that DCPIB could also regulate the activity of other astroglial channels involved in cell volume homeostasis.Experimental approachExperiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytes in situ. The effect of DCPIB was evaluated by patch-clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed in COS-7 cells.Key resultsIn cultured astrocytes, DCPIB promoted the activation of a K(+) conductance mediated by two-pore-domain K(+) (K(2P) ) channels. The DCPIB effect occluded that of arachidonic acid, which activates K(2P) channels K(2P) 2.1 (TREK-1) and K(2P) 10.1 (TREK-2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes express K(2P) 2.1 and K(2P) 10.1 proteins. Moreover, DCPIB opened recombinant K(2P) 2.1 and K(2P) 10.1 expressed in heterologous system. In brain slices, DCPIB did not augment the large background K(+) conductance in hippocampal astrocytes, but caused an increment in basal K(+) current of neurons.Conclusion and implicationsOur results indicate that the neuroprotective effect of DCPIB could be due, at least in part, to activation of TREK channels. DCPIB could be used as template to build new pharmacological tools able to increase background K(+) conductance in astroglia and neuronal cells.
| S-EPMC3594680 | biostudies-literature
Project description:Volume-regulated anion channels (VRACs) are key players in regulatory volume decrease of vertebrate cells by mediating the extrusion of chloride and organic osmolytes. They play additional roles in various physiological processes beyond their role in osmotic volume regulation. VRACs are formed by heteromers of LRRC8 proteins; LRRC8A (also called SWELL1) is an essential subunit that combines with any of its paralogs, LRRC8B-E, to form hexameric VRAC complexes. The subunit composition of VRACs determines electrophysiological characteristics of their anion transport such as single-channel conductance, outward rectification, and depolarization-dependent inactivation kinetics. In addition, differently composed VRACs conduct diverse substrates, such as LRRC8D enhancing VRAC permeability to organic substances like taurine or cisplatin. Here, after a recapitulation of the biophysical properties of VRAC-mediated ion and osmolyte transport, we summarize the insights gathered since the molecular identification of VRACs. We describe the recently solved structures of LRRC8 complexes and discuss them in terms of their structure-function relationships. These studies open up many potential avenues for future research.
| S-EPMC6451053 | biostudies-literature
Project description:Biosynthesis drives the cell volume increase during T cell activation. However, the contribution of cell volume regulation in TCR signaling during T lymphoblast formation and its underlying mechanisms remain unclear. Here we show that cell volume regulation is required for optimal T cell activation. Inhibition of VRACs (volume-regulated anion channels) and deletion of leucine-rich repeat-containing protein 8A (LRRC8A) channel components impair T cell activation and function, particularly under weak TCR stimulation. Additionally, LRRC8A has distinct influences on mRNA transcriptional profiles, indicating the prominent effects of cell volume regulation for T cell functions. Moreover, cell volume regulation via LRRC8A controls T cell-mediated antiviral immunity and shapes the TCR repertoire in the thymus. Mechanistically, LRRC8A governs stringent cell volume increase via regulated volume decrease (RVD) during T cell blast formation to keep the TCR signaling molecules at an adequate density. Together, our results show a further layer of T cell activation regulation that LRRC8A functions as a cell volume controlling "valve" to facilitate T cell activation.
| S-EPMC10625614 | biostudies-literature
Project description:Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology. VRACs were recently discovered to be formed by heteromers of leucine-rich repeat-containing 8 (LRRC8) proteins. However, the structural determinants of VRAC permeation and gating remain largely unknown. We show here that the short stretch preceding the first LRRC8 transmembrane domain determines VRAC conductance, ion permeability, and inactivation gating. Substituted-cysteine accessibility studies revealed that several of the first 15 LRRC8 residues are functionally important and exposed to a hydrophilic environment. Substituting glutamate 6 with cysteine decreased the amplitudes of swelling-activated ICl,vol currents, strongly increased iodide-over-chloride permeability, and markedly shifted the voltage dependence of channel inactivation. Importantly, these effects were reversed by 2-sulfonatoethyl methanethiosulfonate, which restores the negative charge at this amino acid position. Cd2+-mediated blocking of ICl,vol in cysteine variants suggested that the LRRC8 N termini come close together in the multimeric channel complex and might form part of the pore. We propose a model in which the N termini of the LRRC8 subunits line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway.
| S-EPMC6120214 | biostudies-literature
Project description:Volume-regulated anion channels (VRACs) are involved in cellular functions such as regulation of cell volume, proliferation, migration, and cell death. Although leucine-rich repeat-containing 8A (LRRC8A) has been characterized as a molecular component of VRACs, here we show that Drosophila melanogaster tweety homologue 1 and 2 (TTYH1 and TTYH2) are critical for VRAC currents in cancer cells. LRRC8A-independent VRAC currents were present in the gastric cancer cell line SNU-601, but almost completely absent in its cisplatin-resistant derivative SNU-601-R10 (R10). The VRAC current in R10 was partially restored by treatment with trichostatin A (TSA), a histone deacetylase inhibitor. Based on microarray expression profiling of these cells, we selected two chloride channels, TTYH1 and TTYH2, as VRAC candidates. VRAC currents were completely absent from TTYH1- and TTYH2-deficient SNU-601 cells, and were clearly restored by expression of TTYH1 or TTYH2. In addition, we examined the expression of TTYH1 or TTYH2 in several cancer cell lines and found that VRAC currents of these cells were abolished by gene silencing of TTYH1 or TTYH2. Taken together, our data clearly show that TTYH1 and TTYH2 can act as LRRC8A-independent VRACs, suggesting novel therapeutic approaches for VRACs in cancer cells.
| S-EPMC6628158 | biostudies-literature
Project description:High interstitial level of ATP and its lysate adenosine in the cancer microenvironment are considered a halo mark of cancer. Adenosine acts as a strong immune suppressor. However, the source of ATP release is unclear. We clarified the release of ATP via volume-regulated anion channels (VRACs) in breast cell lines using an ATP luminescence imaging system. We detected a slowly rising diffuse pattern of ATP release that was only observed in undifferentiated cells, not in differentiated primary cultured cells. This was confirmed by suppression with DCPIB, a blocker of VRACs, and shRNA for LRRC8A, an indispensable subunit of VRACs. We herein demonstrated that the inflammatory mediator sphingosine-1-phosphate (S1P), which exists abundantly in the cancer microenvironment, induced a diffuse pattern of ATP release isovolumetrically. The response was dose-dependent and suppressed by the knock-down of LRRC8A. It was also suppressed by blockers of S1P receptor 1 and 2 (W146 and JTE013, respectively). RTqPCR demonstrated the prominent presence of S1PR1 and S1PR2 mRNAs. We discussed the roles of S1P-induced ATP release in the cancer microenvironment.
| S-EPMC8401269 | biostudies-literature