Volume-regulated Anion Channels Research Articles

VRAC Complex Modulates Mechano-Electrical Signal Responses in Human Airway Smooth Muscle Shortening.

Leucine-rich repeat containing 8A (LRRC8A) is an obligatory constituent of the volume-regulated anion channel (VRAC) that is fundamental to a wide range of biological processes, including regulating cell size, proliferation, and migration. Here we explored the physiological role for VRAC in excitation-contraction (E-C) coupling and shortening of human airway smooth muscle (HASM). In HASM cells, pharmacological inhibition of VRAC with DCPIB (0.1-10 μM) markedly attenuated swell-activated Cl- conductance and contractile agonist (histamine or carbachol)-induced cellular stiffening as measured by single-cell patch clamp and optical magnetic twisting cytometry, respectively. In addition, HASM cells treated with DCPIB or transfected with LRRC8A-targeting siRNA showed reduced agonist-induced phosphorylation of protein kinase B (AKT), paxillin, myosin phosphatase target subunit 1 (MYPT1), and myosin light chain (MLC). Consistent with the changes of these E-C coupling effectors, DCPIB appreciably decreased agonist-induced small airways narrowing in human precision-cut lung slices (hPCLS). Taken together, our findings shed a new light on the mechanistic link between HASM shortening and regulatory volume decrease via LRRC8A, revealing a previously unrecognized nodal point for modulation of the E-C coupling and acute airways constriction.

Separate mechanisms regulating accumbal taurine levels during baseline conditions and following ethanol exposure in the rat

Ethanol-induced dopamine release in the nucleus accumbens (nAc) is associated with reward and reinforcement, and for ethanol to elevate nAc dopamine levels, a simultaneous increase in endogenous taurine is required within the same brain region. By employing in vivo microdialysis in male Wistar rats combined with pharmacological, chemogenetic and metabolic approaches, our aim with this study was to identify mechanisms underlying ethanol-induced taurine release. Our results demonstrate that the taurine elevation, elicited by either systemic or local ethanol administration, occurs both in presence and absence of action potential firing or NMDA receptor blockade. Inhibition of volume regulated anion channels did not alter the ethanol-induced taurine levels, while inhibition of the taurine transporter occluded the ethanol-induced taurine increase, putatively due to a ceiling effect. Selective manipulation of nAc astrocytes using Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) did not affect ethanol-induced taurine release. However, activation of Gi-coupled DREADDs, or metabolic inhibition using fluorocitrate, rather enhanced than depressed taurine elevation. Finally, ethanol-induced taurine increase was fully blocked in rats pre-treated with the L-type Ca2+-channel blocker nicardipine, suggesting that the release is Ca2+ dependent. In conclusion, while astrocytes appear to be important regulators of basal taurine levels in the nAc, they do not appear to be the main cells underlying ethanol-induced taurine release.

LRRC8 complexes are adenosine nucleotide release channels regulating platelet activation and arterial thrombosis.

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A cKO mice have reduced arteriolar thrombus formation and prolonged arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP/ADP release to amplify agonist-stimulated calcium and PI3K-AKT signaling via P2X1, P2Y 1 and P2Y 12 receptors. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo . These studies identify the mechanoresponsive LRRC8 channel complex as an ATP/ADP release channel in platelets which regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Targeting harmful effects of non-excitatory amino acids as an alternative therapeutic strategy to reduce ischemic damage.

The involvement of the excitatory amino acids glutamate and aspartate in cerebral ischemia and excitotoxicity is well-documented. Nevertheless, the role of non-excitatory amino acids in brain damage following a stroke or brain trauma remains largely understudied. The release of amino acids by necrotic cells in the ischemic core may contribute to the expansion of the penumbra. Our findings indicated that the reversible loss of field excitatory postsynaptic potentials caused by transient hypoxia became irreversible when exposed to a mixture of just four non-excitatory amino acids (L-alanine, glycine, L-glutamine, and L-serine) at their plasma concentrations. These amino acids induce swelling in the somas of neurons and astrocytes during hypoxia, along with permanent dendritic damage mediated by N-methyl-D-aspartate receptors. Blocking N-methyl-D-aspartate receptors prevented neuronal damage in the presence of these amino acids during hypoxia. It is likely that astroglial swelling caused by the accumulation of these amino acids via the alanine-serine-cysteine transporter 2 exchanger and system N transporters activates volume-regulated anion channels, leading to the release of excitotoxins and subsequent neuronal damage through N-methyl-D-aspartate receptor activation. Thus, previously unrecognized mechanisms involving non-excitatory amino acids may contribute to the progression and expansion of brain injury in neurological emergencies such as stroke and traumatic brain injury. Understanding these pathways could highlight new therapeutic targets to mitigate brain injury.

Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2-Nrf2-CEBPB Transcriptional Axis in THP-1-Derived M2 Macrophages.

M2-polarized, tumor-associated macrophages (TAMs) produce pro-tumorigenic and angiogenic mediators, such as interleukin-8 (IL-8) and IL-10. Leucine-rich repeat-containing protein 8 members (LRRC8s) form volume-regulated anion channels and play an important role in macrophage functions by regulating cytokine and chemokine production. We herein examined the role of LRRC8A in IL-8 and IL-10 expression in THP-1-differentiated M2-like macrophages (M2-MACs), which are a useful tool for investigating TAMs. In M2-MACs, the pharmacological inhibition of LRRC8A led to hyperpolarizing responses after a transient depolarization phase, followed by a slight elevation in the intracellular concentration of Ca2+. Both the small interfering RNA-mediated and pharmacological inhibition of LRRC8A repressed the transcriptional expression of IL-8 and IL-10, resulting in a significant reduction in their secretion. The inhibition of LRRC8A decreased the nuclear translocation of phosphorylated nuclear factor-erythroid 2-related factor 2 (Nrf2), while the activation of Nrf2 reversed the LRRC8A inhibition-induced transcriptional repression of IL-8 and IL-10 in M2-MACs. We identified the CCAAT/enhancer-binding protein isoform B, CEBPB, as a downstream target of Nrf2 signaling in M2-MACs. Moreover, among several upstream candidates, the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) suppressed the Nrf2-CEBPB transcriptional axis in M2-MACs. Collectively, the present results indicate that the inhibition of LRRC8A repressed IL-8 and IL-10 transcription in M2-MACs through the NOX2-Nrf2-CEBPB axis and suggest that LRRC8A inhibitors suppress the IL-10-mediated evasion of tumor immune surveillance and IL-8-mediated metastasis and neovascularization in TAMs.

Abstract 09: Leucine Rich Repeat Containing 8A Anion Channels Modulate Vascular Reactivity in ApoE Knockout Mice

Volume-regulated anion channels (VRACs) are comprised of Leucine Rich Repeat Containing 8 (LRRC8) family proteins which include LRRC8A, B, C, D and E. We have proposed that these channels may allow influx of extracellular superoxide to drive inflammation and modulate cultured vascular smooth muscle cell (VSMC) inflammation. We previously demonstrated that LRRC8A knockdown or inhibition inhibits tumor necrosis factor alpha (TNFα)-induced inflammation. Mesenteric arteries from male smooth muscle-specific LRRC8A knockout (KO) mice displayed enhanced responsiveness to both endothelium-dependent (acetylcholine, ACh), and independent (sodium nitroprusside, SNP) vasodilators compared to vessels from WT mice. KO vessels were also protected from vascular dysfunction induced by TNFα in tissue culture (48hr exposure). We hypothesized that reduced VSMC inflammation in LRRC8A KO mice would protect vascular function in ApoE knockout (ApoE -/- ). We created ApoE -/- mice lacking LRRC8A only in VSMCs and assessed vascular reactivity of isolated mesenteric arteries by wire myography in female and male WT, Heterozygous (HET) and KO tissues after 14 weeks of high fat diet. WT vessels displayed enhanced in contraction compared to LRRC8A KO or HET (male PE max contraction of KCL (%), WT 177 ± 4.6 vs. KO 152 ± 3.3 or HET 153 ± 3.0, p < 0.05, n=6-7). Relaxation responses to ACh were increased in female KO or HET compared to WT (ACh LogEC 50 , WT -7.2 ± 0.09 vs. KO -7.5 ± 0.09 or HET -7.4 ± 0.07, p < 0.05, n=5-6). SNP-mediated relaxation were increased in male KO vessels compared to WT (SNP LogEC 50 , WT -8.1 ± 0.06 vs. KO -8.3 ± 0.06, p < 0.05, n=7). These results demonstrate that loss (KO) or reduction (HET) of LRRC8A expression preserves vascular function in the face of the inflammatory response induced by hypercholesterolemia in ApoE -/- mice. These channels may represent a novel therapeutic target for preservation of vascular function and this may only require partial inhibition of current.

Monitoring Leucine-Rich Repeat Containing 8 Channel (LRRC8/VRAC) Activity using Sensitized-Emission Förster Resonance Energy Transfer (SE-FRET).

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion channels (VRACs)/volume-sensitive outwardly rectifying channels (VSORs), they are activated upon osmotic cell swelling and mediate the extrusion of chloride and organic osmolytes, leading to the efflux of water and hence cell shrinkage. Beyond their role in osmotic volume regulation, VRACs have been implicated in cellular processes such as differentiation, migration, and apoptosis. Through their effect on membrane potential and their transport of various signaling molecules, leucine-rich repeat containing 8 (LRRC8) channels play roles in neuron-glia communication, insulin secretion, and immune response. The activation mechanism has remained elusive. LRRC8 channels, like other ion channels, are typically studied using electrophysiological methods. Here, we describe a method to detect LRRC8 channel activation by measuring intra-complex sensitized-emission Förster resonance energy transfer (SE-FRET) between fluorescent proteins fused to the C-terminal leucine-rich repeat domains of LRRC8 subunits. This method offers the possibility to study channel activation in situ without exchange of the cytosolic environment and during processes such as cell differentiation and apoptosis.

Abstract Tu032: LRRC8 complex regulates platelet activation and thrombosis

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

The SWELL1 Channel Promotes Ischemic Brain Damage by Mediating Neuronal Swelling and Glutamate Toxicity.

Cytotoxic neuronal swelling and glutamate excitotoxicity are two hallmarks of ischemic stroke. However, the underlying molecular mechanisms are not well understood. Here, it is reported that SWELL1, the essential subunit of the volume-regulated anion channel (VRAC), plays a dual role in ischemic injury by promoting neuronal swelling and glutamate excitotoxicity. SWELL1 expression is upregulated in neurons and astrocytes after experimental stroke in mice. The neuronal SWELL1 channel is activated by intracellular hypertonicity, leading to Cl- influx-dependent cytotoxic neuronal swelling and subsequent cell death. Additionally, the SWELL1 channel in astrocytes mediates pathological glutamate release, indicated by increases in neuronal slow inward current frequency and tonic NMDAR current. Pharmacologically, targeting VRAC with a new inhibitor, an FDA-approved drug Dicumarol, attenuated cytotoxic neuronal swelling and cell death, reduced astrocytic glutamate release, and provided significant neuroprotection in mice when administered either before or after ischemia. Therefore, these findings uncover the pleiotropic effects of the SWELL1 channel in neurons and astrocytes in the pathogenesis of ischemic stroke and provide proof of concept for therapeutically targeting it in this disease.

Interactomic exploration of LRRC8A in volume-regulated anion channels

Ion channels are critical in enabling ion movement into and within cells and are important targets for pharmacological interventions in different human diseases. In addition to their ion transport abilities, ion channels interact with signalling and scaffolding proteins, which affects their function, cellular positioning, and links to intracellular signalling pathways. The study of “channelosomes” within cells has the potential to uncover their involvement in human diseases, although this field of research is still emerging. LRRC8A is the gene that encodes a crucial protein involved in the formation of volume-regulated anion channels (VRACs). Some studies suggest that LRRC8A could be a valuable prognostic tool in different types of cancer, serving as a biomarker for predicting patients’ outcomes. LRRC8A expression levels might be linked to tumour progression, metastasis, and treatment response, although its implications in different cancer types can be varied. Here, publicly accessible databases of cancer patients were systematically analysed to determine if a correlation between VRAC channel expression and survival rate exists across distinct cancer types. Moreover, we re-evaluated the impact of LRRC8A on cellular proliferation and migration in colon cancer via HCT116 LRRC8A-KO cells, which is a current topic of debate in the literature. In addition, to investigate the role of LRRC8A in cellular signalling, we conducted biotin proximity-dependent identification (BioID) analysis, revealing a correlation between VRAC channels and cell-cell junctions, mechanisms that govern cellular calcium homeostasis, kinases, and GTPase signalling. Overall, this dataset improves our understanding of LRRC8A/VRAC and explores new research avenues while identifying promising therapeutic targets and promoting inventive methods for disease treatment.

ATP-elicited Cation Fluxes Promote Volume-regulated Anion Channel LRRC8/VRAC Transport cGAMP for Antitumor Immunity.

The cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway is instrumental to antitumor immunity, yet the underlying molecular and cellular mechanisms are complex and still unfolding. A new paradigm suggests that cancer cells' cGAS-synthesized cGAMP can be transferred to tumor-infiltrating immune cells, eliciting STING-dependent IFN-β response for antitumor immunity. Nevertheless, how the tumor microenvironment may shape this process remains unclear. In this study, we found that extracellular ATP, an immune regulatory molecule widely present in the tumor microenvironment, can potentiate cGAMP transfer, thereby boosting the STING signaling and IFN-β response in murine macrophages and fibroblasts. Notably, genetic ablation or chemical inhibition of murine volume-regulation anion channel LRRC8/volume-regulated anion channel (VRAC), a recently identified cGAMP transporter, abolished ATP-potentiated cGAMP transfer and STING-dependent IFN-β response, revealing a crucial role of LRRC8/VRAC in the cross-talk of extracellular ATP and cGAMP. Mechanistically, ATP activation of the P2X family receptors triggered Ca2+ influx and K+ efflux, promoting reactive oxygen species production. Moreover, ATP-evoked K+ efflux alleviated the phosphorylation of VRAC's obligate subunit LRRC8A/SWELL1 on S174. Mutagenesis studies indicated that the phosphorylation of S174 on LRRC8A could act as a checkpoint for VRAC in the steady state and a rheostat of ATP responsiveness. In an MC38-transplanted tumor model, systemically blocking CD39 and ENPP1, hydroxylases of extracellular ATP and cGAMP, respectively, elevated antitumor NK, NKT, and CD8+ T cell responses and restrained tumor growth in mice. Altogether, this study establishes a crucial role of ATP in facilitating LRRC8/VRAC transport cGAMP in the tumor microenvironment and provides new insight into harnessing cGAMP transfer for antitumor immunity.

LRRC8/VRAC volume-regulated anion channels are crucial for hearing

Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClC-K/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K+ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.

DCPIB Attenuates Ischemia–Reperfusion Injury by Regulating Microglial M1/M2 Polarization and Oxidative Stress

The inflammatory response plays an indispensable role in ischemia–reperfusion injury, the most significant of which is the inflammatory response caused by microglial polarization. Anti-inflammatory therapy is also an important remedial measure after failed vascular reconstruction. Maintaining the internal homeostasis of the brain is a crucial measure for suppressing the inflammatory response. The mechanism underlying the relationship between DCPIB, a selective blocker of volume-regulated anion channels (VRAC), and inflammation induced by cerebral ischemia–reperfusion injury is currently unclear. The purpose of this study was to investigate the relationship between DCPIB and microglial M1/M2 polarization-mediated inflammation after cerebral ischemia–reperfusion injury. C57BL/6 mice were subjected to transient middle cerebral artery occlusion (tMCAO). DCPIB was administered by a lateral ventricular injection within 5 min after reperfusion. Behavioral assessments were conducted at 1, 3, and 7 days after tMCAO/R. Pathological injuries were evaluated using TTC assay, HE and Nissl staining, brain water content measurement, and immunofluorescence staining. The levels of inflammatory cytokines were analyzed using qPCR and ELISA. Additionally, the phenotypic variations of microglia were examined using immunofluorescence staining. In mouse tMCAO/R model, DCPIB administration markably reduced mortality, improved behavioral performance, and alleviated pathological injury. DCPIB treatment significantly inhibited the inflammatory response, promoted the conversion of M1 microglia to M2 microglia via the MAPK signaling pathway, and ultimately protected neurons from the microglia-mediated inflammatory response. In addition, DCPIB inhibited oxidative stress induced by cerebral ischemia–reperfusion injury. In conclusion, DCPIB attenuates cerebral ischemia–reperfusion injury by regulating microglial M1/M2 polarization and oxidative stress.

LRRC8A-VRAC modulates the contractile response by managing the superoxide anion influx in diabetic vasculogenic erectile dysfunction

Introduction: Erectile dysfunction (ED) may be an early marker for cardiovascular diseases, and it is often associated with an hypercontractility of the corpus cavernosum (CC) and pudendal artery (PA) because of increased reactive oxygen species (ROS). Leucine Rich Repeat Containing 8 (LRRC8) Volume Regulated Anion Channels (VRACs) regulates Nox1 and modulates the influx of extracellular superoxide anion (O2•-), contributing to the creation of an oxidized domain, enhancing calcium sensitivity of the smooth muscle which promotes contraction. Objective: We hypothesize that downregulation of NOX1 or LRRC8A would decrease the contractile responses to phenylephrine (PE) and electrical field stimulation (EFS) in the CC, PA and mesenteric resistance artery (MRA) of diabetic mice (dbdb−/−). Methods: The CC and PA of diabetic (dbdb−/−) mice were mounted in myographs. The contraction elicited by PE or EFS was performed in the presence of apocynin (non-selective NOX inhibitor) and GKT137831 (NOX1/4 inhibitor). In LRRC8A KO dbdb−/− mice, contractions to PE and EFS were obtained in the absence or in the presence of GKT137831.Results: In the CC, the maximal contraction to PE was increased in dbdb−/− animals, and in the presence of 3 mM of apocynin, the contractile response was markedly diminished in both control and dbdb−/−. Incubation with GKT137831 reduced the contraction elicited by PE only in the dbdb−/− group (68 ± 9% of the maximal contraction), whereas in the control group this reduction was not significant (82 ± 14% of the PE-maximal contraction). In the PA, the potency of PE was increased in the dbdb−/− group, and apocynin 1mM restored the contraction to normal levels. In the LRRC8A KO dbdb−/− mice, the contraction to PE in the MRA and PA was reduced by 30% compared to dbdb−/−. The response to EFS in CC was similar to dbdb−/−, however, GKT137831 reduced the contraction to EFS with lesser magnitude of inhibition in the LRRC8A KO dbdb−/− mice (35% of inhibition) compared to dbdb−/− mice (50% of inhibition), suggesting that the participation of O2•- in the contractile response was reduced by the lack of functional LRRC8A. Conclusion: LRRC8A modulates the contractile response by managing the influx of reactive oxygen species (ROS) in smooth muscle cells. Inhibition of LRRC8A may be a therapeutic target to prevent hypercontractility of arteries and CC in conditions where there are marked increases in ROS production, such as diabetes. NIH – R01 132948; Institute on Cardiovascular Disease Research, University of South Carolina. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Smooth Muscle-Specific Knockout of LRRC8A Anion Channels Improves Vasodilation in Renal Arteries from Diabetic Mice

Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) comprise volume-regulated anion channels (VRACs). We previously have shown that knockout (KO) or block of LRRC8A anion channel attenuates reactive oxygen species (ROS) production by Nox1, impairs TNFα-induced inflammation, and increases vascular relaxation in mesenteric arteries. These changes are associated with reduced RhoA activation. Diabetic (db/db−/−) mice have previously been shown to develop vascular dysfunction that is characterized by increased ROS production and enhanced RhoA activation. Diabetic nephropathy causes chronic renal disease and can lead to end-stage renal failure. Oxidative stress plays a role in both the initiation and progression of this process. We hypothesized that LRRC8A KO would protect vascular function in db/db−/− mice and promote vascular relaxation in renal arteries. Smooth-muscle specific LRRC8A KO mice were created in a db/db−/− background. Renal arteries were isolated from db/db−/− WT and db/db−/− LRRC8A KO mice vascular contraction and relaxation were measured. Contractile responses to phenylephrine (PE) were unaltered in LRRC8A KO compared to WT vessels. However, both endothelium-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings that were contracted with PE (Emax ACh: WT 76.8 ± 5.5 vs. KO 92.2 ± 3.3 %, LogEC50 SNP: WT -7.6 ± 0.14 vs. KO -8.4 ± 0.14, n = 4-5, * p<0.05). These data demonstrate preserved vasodilation in smooth muscle-specific LRRC8A KO blood vessels. This may lead to increased renal blood flow in the setting of db/db−/−-related diabetes. Ongoing work will determine if improved vasodilation is associated with preserved renal function in these animals. If so, VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of diabetes-induced renal dysfunction. This work was supported by DK132948 and HL160975. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB256: New mechanistic insights into cisplatin resistance in muscle-invasive bladder cancer

Abstract The standard of care for eligible patients with muscle-invasive bladder cancer (MIBC) is cisplatin-based neoadjuvant chemotherapy followed by radical cystectomy. However, platinum-based treatments leave up to 70% of MIBC patients with residual disease, and the 5-year survival rate for these individuals is under 30%. Understanding cisplatin resistance mechanisms will improve MIBC treatment by developing more effective therapeutics and precision medicine strategies. We previously identified NPEPPS, an M1 aminopeptidase, as a novel and potentially targetable mediator of platinum drug response. Recently, we have established that NPEPPS controls platinum drug import through its interactions with volume regulated anion channels (VRACs), which have been shown to import cisplatin to cells directly. We have shown that NPEPPS interacts with key VRAC subunit SWELL1 at the cell membrane and that intact SWELL1 is required for NPEPPS-mediated platinum resistance. We further discovered that NPEPPS cannot bind SWELL1 when its enzymatic aminopeptidase function is disrupted genetically or pharmacologically. In vivo studies showed that catalytic-dead NPEPPS-mutant MIBC xenografts were sensitive to cisplatin treatment compared to NPEPPS-wildtype xenografts. Patient cohort data analysis supported the model that the expression of NPEPPS and its relative expression with VRAC proteins are associated with overall survival in treatment-resistant MIBC patients. These findings shed light on how bladder cancer cells resist chemotherapy by disrupting the transport of drugs into cells and suggest new ways to target NPEPPS to enhance the effectiveness of platinum-based therapeutics in treating bladder cancer. Citation Format: Lily Elizabeth R. Feldman, Saswat Mohapatra, Robert Jones, Charlene Tilton, Tahlita Zuiverloon, James Costello, Dan Theodorescu. New mechanistic insights into cisplatin resistance in muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB256.

(055) LRRC8A-VRAC Controls Superoxide Anion Flux in Diabetic Vasculogenic Erectile Dysfunction

Abstract Introduction Erectile dysfunction (ED) may be an early marker for cardiovascular diseases, and it is often associated with an hypercontractility of the corpus cavernosum (CC) as a consequence of increased reactive oxygen species (ROS), which favors the flaccid state of the penis. Leucine Rich Repeat Containing 8 (LRRC8A) Volume Regulated Anion Channels (VRACs) regulates Nox1 and modulates the extracellular production of superoxide anion (O2•-). Downregulation of LRRC8A is associated with decreased production of O2•- and we have demonstrated improved relaxation and reduced contraction of the pudendal artery in LRRC8A knockout mice. Objective We hypothesized that the role of LRRC8A in contractile responses to phenylephrine (PE) and electrical field stimulation (EFS) in the CC of diabetic mice would be exaggerated compared to that in normal mice. Methods To test this hypothesis, the CC of diabetic (dbdb-/-) mice was mounted in a myograph, and contraction elicited by PE or EFS was performed in the presence of apocynin (non-selective NOX inhibitor), GKT137831 (NOX1/4 inhibitor) and montelukast (LRRC8A inhibitor). In LRRC8A KO dbdb-/- mice, contractions to PE and EFS were obtained in the absence or in the presence of GKT137831. Results In the CC, the maximal contraction to PE was increased in diabetic animals, and in the presence of 3 mM of apocynin, the contractile response was markedly diminished in both control and diabetic mice. The LRRC8A inhibitor, montelukast, significantly reduced PE-induced contraction of the CC reaching maximal contraction of 69 + 7% and 65 + 10% in the control and diabetic group, respectively, compared to the contraction in the absence of the inhibitor. In the presence of the Nox1/Nox4 inhibitor, GKT137831, the contraction elicited by PE was reduced only in the diabetic group (68 + 9% of the maximal contraction), whereas in the control group this reduction was not significant (82 + 14% of the PE-maximal contraction). In the LRRC8A KO dbdb-/- mice, the contraction to EFS was similar to dbdb-/-, however, GKT137831 reduced the contraction to EFS with lesser magnitude of inhibition in the LRRC8A KO dbdb-/- mice (35% of inhibition) compared to dbdb-/- mice (50% of inhibition), suggesting that the participation of O2•- in the contractile response was reduced by the lack of functional LRRC8A. Conclusions LRRC8A modulates the contractile response of the CC induced by α-adrenergic stimulation and in diabetes, this may be associated with O2•- production, which plays a role in the development of ED. Inhibition of LRRC8A may be a therapeutic target to prevent hypercontractility of arteries and CC in vasculogenic ED. Disclosure No.

Sphingosine-1-phosphate activates volume-regulated anion channels through Gβγ dimers

Sphingosine-1-phosphate activates volume-regulated anion channels through Gβγ dimers

Comprehensive review on the novel immunotherapy target: Leucine-rich repeat-containing 8A/volume-regulated anion channel.

Leucine-rich repeat-containing 8A (LRRC8A) is a key component of the volume-regulated anion channel (VRAC) that influences essential homeostatic processes in various immune cells. These processes include the regulation of cell volume and membrane potential and the facilitation of the transport of organic agents used as anticancer drugs and immune-stimulating factors. Therefore, understanding the structure-function relationship of LRRC8A, exploring its physiological role in immunity, assessing its efficacy in treating diseases, and advancing the development of compounds that regulate its activity are important research frontiers. This review emphasized the emerging field of LRRC8A, outlined its structure and function, and summarized its role in immune cell development and immune cell-mediated antiviral and antitumor effects. Additionally, it explored the potential of LRRC8A as an immunotherapeutic target, offering insights into resolving persistent challenges and future research directions.

Hypoxia, Ion Channels and Glioblastoma Malignancy.

The malignancy of glioblastoma (GBM), the most aggressive type of human brain tumor, strongly correlates with the presence of hypoxic areas within the tumor mass. Oxygen levels have been shown to control several critical aspects of tumor aggressiveness, such as migration/invasion and cell death resistance, but the underlying mechanisms are still unclear. GBM cells express abundant K+ and Cl- channels, whose activity supports cell volume and membrane potential changes, critical for cell proliferation, migration and death. Volume-regulated anion channels (VRAC), which mediate the swelling-activated Cl- current, and the large-conductance Ca2+-activated K+ channels (BK) are both functionally upregulated in GBM cells, where they control different aspects underlying GBM malignancy/aggressiveness. The functional expression/activity of both VRAC and BK channels are under the control of the oxygen levels, and these regulations are involved in the hypoxia-induced GBM cell aggressiveness. The present review will provide a comprehensive overview of the literature supporting the role of these two channels in the hypoxia-mediated GBM malignancy, suggesting them as potential therapeutic targets in the treatment of GBM.