Leucine-rich Repeat-containing 8 Research Articles

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.

Lysosomal LRRC8 complex regulates lysosomal pH, morphology and systemic glucose metabolism.

The lysosome integrates anabolic signalling and nutrient-sensing to regulate intracellular growth pathways. The leucine-rich repeat containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signalling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show LRRC8A regulates leucine-stimulated mTOR, lysosome size, number, pH, and expression of lysosomal proteins LAMP2, P62, LC3B, suggesting impaired autophagic flux. Mutating a LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signalling and altered lysosomal morphology and pH observed in LRRC8A KO cells. In vivo , LRRC8A-L706A;L707A KI mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance characterized by reduced skeletal muscle glucose-uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8 mediated metabolic signalling function that regulates lysosomal activity, systemic glucose homeostasis and insulin-sensitivity.

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.

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.

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.

Abstract P189: Hypercontractility Of Pudendal Artery And Corpus Cavernosum Is Associated With LRRC8A-VRAC And Superoxide Anion Production In Diabetic Vasculogenic Erectile Dysfunction

Erectile dysfunction (ED) may be an early marker for cardiovascular diseases and it isoften associated with an hypercontractility of the corpus cavernosum (CC) and pudendalartery (PA) in consequence of increased reactive oxygen species (ROS), which favorsthe flaccid state of the penis. Leucine Rich Repeat Containing 8 (LRRC8) VolumeRegulated Anion Channels (VRACs) regulates Nox1 and modulates the extracellularproduction of superoxide anion (O2 •- ). Downregulation of LRRC8 is associated withdecreased production of O2 •- and we have demonstrated improved relaxation andreduced contraction of the pudendal artery in LRRC8A knockout mice. In this study, wehypothesize that inhibition of Nox or LRRC8A decreases phenylephrine (PE)-inducedcontraction in the CC and PA of diabetic mice. To test this hypothesis, the CC and PA ofdiabetic (dbdb-/-) mice were mounted in myographs, and the contraction elicited by PEwas performed in the presence of apocynin (non-selective NOX inhibitor), GKT137831(NOX1/4 inhibitor) and montelukast (LRRC8A inhibitor). In the PA, the potency of PE wasincreased in diabetic mice, and apocynin (1 mM) restored the contraction of PE to thenormal levels. In the CC, the maximal contraction of PE was increased in diabetic animals,and in the presence of 3 mM of apocynin, the contractile response was markedlydiminished in both control and diabetic mice. The LRRC8A inhibitor, montelukast,significantly reduced PE-induced contraction of the CC reaching maximal contraction of69 + 7% and 65 + 10% in the control and diabetic group, respectively, compared to thecontraction 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 notsignificant (82 + 14% of the PE-maximal contraction). In the CC and PA, LRRC8Amodulates the contractile response 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 arteriesand CC in vasculogenic ED.

Abstract P308: Leucine Rich Repeat Containing 8A Anion Channels Modulate Vasodilation In Angiotensin II-induced Hypertensive Mice

Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) comprise Volume-regulated anion channels (VRACs) which are activated by tumor necrosis factor alpha (TNFα) and angiotensin II (AngII). We have previously investigated vascular reactivity in mesenteric arteries from male wild type (WT) and smooth muscle-specific LRRC8A knockout (KO) mice. KO vessels displayed enhanced responsiveness to both endothelium-dependent (acetylcholine, ACh), and independent (sodium nitroprusside, SNP) vasodilators. We hypothesized that LRRC8A KO mice would be protected from AngII-induced hypertension. Blood pressure (BP) was measured by radiotelemetry. Resting BP was unaltered compared to WT and AngII infusion by osmotic minipump (alzet, 90 ng/min for 14 days) induced a similar degree of hypertension in awake mice (nighttime) but the difference between day and night pressures was significantly larger in KO mice. Mesenteric arteries were isolated from WT and KO mice after 14 days and vascular reactivity was quantified by wire myograph. Relaxation responses to both ACh and SNP relaxation were impaired in AngII hypertensive compared to normotensive WT mice. However, KO vessels relaxed better to both vasodilators in rings from both vehicle and AngII-treated mice (ACh LogEC 50 , WT+AngII -5.3 ± 0.27 vs. KO+AngII -6.5 ± 0.19, p < 0.05, n=4-6; SNP LogEC 50 , WT+AngII -7.4 ± 0.10 vs. KO+AngII -8.1 ± 0.10, p < 0.05, n=3-6). These results demonstrate a dissociation between elevated blood pressure and impaired vasodilator function. The data suggest that LRRC8A anion channels play a complex role in the development of AngII-induced hypertension.

(085) Montelukast, a LRRC8A Inhibitor Decreases the Contractile Response to Phenylephrine in the Corpus Cavernosum of Diabetic Mice

Abstract Introduction Increased reactive oxygen species are often correlated with erectile dysfunction (ED) by enhancing contractility and favoring the flaccid state of the penis (which is mainly mediated by α-adrenergic innervation) and decreasing the relaxation of the corpus cavernosum (CC) associated with decreased nitric oxide (NO) bioavailability. Leucine Rich Repeat Containing 8 (LRRC8) Volume Regulated Anion Channels (VRACs) regulates Nox1 and modulates the extracellular production of superoxide anion (O2•-). Downregulation of LRRC8 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 In this study, we aim to test the hypothesis that inhibition of Nox or LRRC8A decreases phenylephrine (PE)-induced contraction in the CC of diabetic mice. Methods To test this hypothesis, the CC of diabetic (dbdb-/-, 16-week-old) mice were mounted in myographs, and the contraction elicited by PE was performed in the absence and repeated in the presence of the LRRC8 inhibitor, montelukast or Nox1/Nox4 inhibitor, GKT137831. The maximal contraction in the absence of the inhibitor was taken as 100%, and the contraction in the presence of the inhibitor was calculated as a percentage of the maximal contraction attained in the absence of the inhibitor. Results Contraction induced by phenylephrine in the CC of diabetic mice (1.27 + 0.2 mN) was similar to that observed in the control mice (1.26 + 0.4 mN). In the presence of the LRRC8 inhibitor, montelukast, PE-induced contraction of the CC was significantly reduced, reaching maximal contraction of 69 + 7% and 65 + 10% in the control and diabetic group, respectively. 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 maximal contraction). Conclusions This is the first study demonstrating that, in the CC, LRRC8 modulates the contractile response induced by α-adrenergic stimulation and this may be associated with O2•- production, which plays a role in the development of ED. Since a great number of diabetic patients are not benefited by phosphodiesterase 5 inhibitors therapy, LRRC8 might be a potential therapeutic target for the development of new drugs to improve sexual function in diabetes. Disclosure No

LRRC8A Anion Channel Associates with Myosin Phosphatase Rho Interacting Protein (MPRIP) to Regulate Vascular Function

Volume-regulated anion channels (VRACs) are activated by tumor necrosis factor alpha (TNFα) and angiotensin II. VRACs are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) and LRRC8A is required for all VRACs. LRRC8A associates with NADPH oxidase 1 (Nox1) and modulates production of extracellular superoxide in vascular smooth muscle cells (VSMCs). We investigated vascular reactivity in mesenteric arteries from wild type (WT) and smooth muscle-specific LRRC8A knockout (KO) mice. KO vessels displayed enhanced responsiveness to multiple vasodilator agents; acetylcholine, sodium nitroprusside, PKA (forskolin) or PKG (BAY60-2770) activators and a Rho kinase inhibitor (Y-27632), compared to WT. This was associated with decreased RhoA activity and reduced ROCK-dependent MYPT1 phosphorylation at T853. To explore the mechanism of enhanced relaxation we immunoprecipitated LRRC8A-associated proteins from WT and KO VSMCs and analyzed these by mass spectroscopy. Myosin Phosphatase Rho-Interacting protein (MPRIP) was identified only in WT lysates. MPRIP is a scaffolding protein that binds to RhoA, MYPT1, and actin and regulates activity of the myosin light chain phosphatase complex. Association of LRRC8A and MPRIP was confirmed using IP/western blot, proximity ligation assay (PLA) and immunohistochemistry. LRRC8A binds to the second plextrin homology domain of MPRIP. Knockdown of MPRIP (siRNA) increased protein expression of LRRC8A in cultured VSMCs. To explore the relationship between Nox1/LRRC8A, superoxide and intracellular redox signaling events sulfenylation of MPRIP cysteine residues (-SOH) was quantified using DCP-Bio1 (a biotin-labeled dimedone derivative) pulldown with streptavidin beads and western blotting. Sulfenylation was significantly increased 3 min following TNFα stimulation (2.3 fold, n = 3, p < 0.05). It remains to be determined how this impacts association of MPRIP with its four binding partners. Disruption of interaction between Nox1/LRRC8A and MPRIP/RhoA/MYPT1/actin may underly enhanced vasodilation in VSMC-specific LRRC8A null blood vessels. This multi-protein complex provides a potential mechanism by which oxidants produced by Nox1 impact vasomotor function, linking inflammation to increased vascular reactivity and hypertension. NIGMS (5R35GM138191-02), NIDDK (1R01DK132948-01), NHLBI (1R01HL160975-01A1) This is the full abstract presented at the American Physiology Summit 2023 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.

Heteromeric LRRC8 channel assemblies and their impact on channel activity.

Volume regulated anion channels (VRAC) are activated in response to the osmotic swelling of vertebrate cells to catalyze the efflux of anions and small osmolytes. VRACs are multimeric channels that are composed of leucine-rich repeat-containing 8 (LRRC8) proteins, a protein family encompassing five paralogues in mammals (LRRC8A-E). Endogenous VRACs are known to form heteromeric assemblies of LRRC8A with at least one further LRRC8 isoform. Until recently, only structures of homomeric LRRC8 channels were described, while the structures of heteromeric channels remained elusive. Combining results from targeted quantification mass spectrometry and cryoEM with proteins obtained from heterologous overexpression, we investigated the subunit stoichiometry and structure of heteromeric LRRC8A/C channels. For these proteins, we observed a ratio of LRRC8A:C subunits of 2:1, which was confirmed in cryo-EM structures where LRRC8A and LRRC8C subunits cluster as pairs in a hexameric protein assembly. This subunit arrangement increases the pore size in LRRC8A/C channels compared to homomeric LRRC8A. Moreover, we found a tight interaction between LRR domains of LRRC8A subunit pairs of heteromers, reflecting a similar arrangement observed in homomeric LRRC8A. Conversely, the same parts of LRRC8C subunits were not resolved, indicating increased flexibility of their cytosolic regions. The structure thus suggests that the LRRC8C subunits perturb the tight packing of the LRRC8A subunits in heteromeric channels. Similar destabilizing effects were previously observed in LRRC8A homomers binding synthetic nanobodies with activating phenotype. There the binders induced a destabilization of the LRR domain leading to an increase of the open probability of homomeric channels. Collectively, our results suggest that the incorporation of LRRC8C in heteromeric LRRC8A/C channels destabilizes the tight packing of LRRC8A subunits to improve the activation properties of the channels.

Contribution of large-pore channels to inflammation induced by microorganisms.

Plasma membrane ionic channels selectively permeate potassium, sodium, calcium, and chloride ions. However, large-pore channels are permeable to ions and small molecules such as ATP and glutamate, among others. Large-pore channels are structures formed by several protein families with little or no evolutionary linkages including connexins (Cxs), pannexins (Panxs), innexin (Inxs), unnexins (Unxs), calcium homeostasis modulator (CALHMs), and Leucine-rich repeat-containing 8 (LRRC8) proteins. Large-pore channels are key players in inflammatory cell response, guiding the activation of inflammasomes, the release of pro-inflammatory cytokines such as interleukin-1 beta (IL-1ß), and the release of adenosine-5'-triphosphate (ATP), which is considered a danger signal. This review summarizes our current understanding of large-pore channels and their contribution to inflammation induced by microorganisms, virulence factors or their toxins.

Chloride Channel-3 (ClC-3) Modifies the Trafficking of Leucine-Rich Repeat-Containing 8A (LRRC8A) Anion Channels.

Chloride channel-3 (ClC-3) Cl-/H+ antiporters and leucine-rich repeat-containing 8 (LRRC8) family anion channels have both been associated with volume-regulated anion currents (VRACs). VRACs are often altered in ClC-3 null cells but are absent in LRRC8A null cells. To explore the relationship between ClC-3, LRRC8A, and VRAC we localized tagged proteins in human epithelial kidney (HEK293) cells using multimodal microscopy. Expression of ClC-3-GFP induced large multivesicular bodies (MVBs) with ClC-3 in the delimiting membrane. LRRC8A-RFP localized to the plasma membrane and to small cytoplasmic vesicles. Co-expression demonstrated co-localization in small, highly mobile cytoplasmic vesicles that associated with the early endosomal marker Rab5A. However, most of the small LRRC8A-positive vesicles were constrained within large MVBs with abundant ClC-3 in the delimiting membrane. Dominant negative (S34A) Rab5A prevented ClC-3 overexpression from creating enlarged MVBs, while constitutively active (Q79L) Rab5A enhanced this phenotype. Thus, ClC-3 and LRRC8A are endocytosed together but independently sorted in Rab5A MVBs. Subsequently, LRRC8A-labeled vesicles were sorted to MVBs labeled by Rab27A and B exosomal compartment markers, but not to Rab11 recycling endosomes. VRAC currents were significantly larger in ClC-3 null HEK293 cells. This work demonstrates dependence of LRRC8A trafficking on ClC-3 which may explain the association between ClC-3 and VRACs.

Recent Advances in the Structural Biology of the Volume-Regulated Anion Channel LRRC8.

Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC), which is activated by cell swelling and releases chloride ions (Cl−) or other osmolytes to counteract cell swelling. Although the LRRC8 protein family was identified as the molecular entity of VRAC only in 2014, due to recent advances in cryo-electron microscopy (cryo-EM), various LRRC8 structures, including homo-hexameric LRRC8A and LRRC8D structures, as well as inhibitor-bound and synthetic single-domain antibody-bound homo-hexameric LRRC8A structures, have been reported, thus extending our understanding of the molecular mechanisms of this protein family. In this review, we describe the important features of LRRC8 provided by these structures, particularly the overall architectures, and the suggested mechanisms underlying pore inhibition and allosteric modulation by targeting the intracellular leucine-rich repeat (LRR) domain.

Leucine rich repeat containing 8A anion channels modulate vascular reactivity in the pudendal artery

ABSTRACT Introduction Erectile dysfunction (ED) is characterized by an inability to develop or maintain penile erection during sexual activity. The internal pudendal artery (IPA) is the primary supplier of genital blood flow. Reactive oxygen species (ROS) generation is necessary for intracellular signaling, but excessive ROS production can promote ED. NADPH oxidase (Nox) produces extracellular superoxide (O2 •-) in the vasculature and we have demonstrated that Leucine Rich Repeat Containing 8 (LRRC8) Volume Regulated Anion Channels (VRACs) physically associate with and regulate Nox1. LRRC8A downregulation or channel inhibition decreases extracellular O2 •- production by Nox1 and dramatically reduces the inflammatory response to tumor necrosis factor alpha. Objective We hypothesize that smooth muscle-specific LRRC8A knockout (KO) increases vascular relaxation in IPA. Methods The IPAs from male wildtype (WT) and LRRC8A KO (smooth muscle-specific) mice were used in wire myograph studies. Results Following phenylephrine (PE, 2x10-6 M)-induced contraction, cumulative relaxation-response curves to acetylcholine (ACh, 10-9 to 3x10-6 M), sodium nitroprusside (SNP, 10-9 to 10-5 M), and a soluble guanylyl cyclase activator (BAY60-2770, 10-9 to 10-6 M) were measured. Endothelium-dependent relaxation to ACh was increased in LRRC8A KO compared to WT (WT LogEC50 -7.3 ± 0.08; LRRC8A KO LogEC50 -7.7 ± 0.12; *p<0.05, n=5). Relaxation to SNP or BAY60-2770 showed no difference between WT and KO. The LRRC8A-targeted VRAC inhibitor, montelukast (10 μM) reduced PE (10-6 M)-induced contractile responses in the IPA (56 % decrease). Conclusions These data demonstrate that LRRC8A plays an important role in controlling vascular reactivity that may be associated with ED. We speculate that LRRC8 channels are novel pharmacologic targets. Inhibition of the channels may reduce oxidative stress and improve sexual function. Disclosure Work supported by industry: no.

Smooth Muscle LRRC8A Anion Channel Knockout Promotes Vasodilation and Protects Against TNFα‐induced Vascular Dysfunction

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine, is associated with cardiovascular risk. Volume‐regulated anion channels (VRACs) are activated by TNFα and are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E). LRRC8A is required for all VRACs. We previously determined that VRAC inhibitors or LRRC8A knockdown suppress the inflammatory response to TNFα in cultured vascular smooth muscle cells, however, the role of these channels in vasomotor function has not been studied. We hypothesized that loss of LRRC8A would prevent the impairment of vascular function by TNFα. We created vascular smooth muscle‐specific (Sm22alpha‐Cre) LRRC8A knockout (KO) mice and isolated mesenteric arteries from male mice for wire myograph recording. Vessel segments were incubated with TNFα (10 ng/mL) or vehicle for 2 days in tissue culture. Contractile responses to norepinephrine (NE) were unaltered in LRRC8A KO mice but enhanced by TNFα in wild type (WT) than in KO rings (WT+TNFα 284 ± 18.9 %, KO+TNFα 222 ± 7.1 % of KCl contraction, *p<0.05, n = 5‐6). In the vehicle‐treated group both endothelium‐dependent (acetylcholine, ACh) and ‐independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings contracted with phenylephrine (PE). Exposure to TNFα impaired relaxation to both ACh and SNP in WT vessels but had no effect on relaxation of LRRC8A KO vessels (black *p<0.05 compared to WT control, red *p<0.05 compared to TNFα‐treated, n = 6‐8). The enhanced relaxation responses of KO vessels were recapitulated in WT rings by 20 min of exposure to a VRAC inhibitor, carbenoxolone (CBX; 100 μM). Thus, in addition to its established role in TNFα signaling, LRRC8A anion channel activity modulates vascular reactivity with loss of function enhancing vasodilator function. VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of inflammation‐induced vascular dysfunction.

Functional Interaction Between Caveolin 1 and LRRC8-Mediated Volume-Regulated Anion Channel.

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5′-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl– currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism.

Leucine-rich repeat-containing 8 (LRRC8) volume-regulated anion channels (VRACs) play important physiological roles in diverse cell types and may represent therapeutic targets for various diseases. To date, however, the pharmacological tools for evaluating the druggability of VRACs have been limited to inhibitors, as no activators of the channel have been reported. We therefore performed a fluorescence-based high-throughput screening (HTS) of 1,184 Food and Drug Administration-approved drugs for compounds that increase VRAC activity. The most potent VRAC potentiator identified was zinc pyrithione (ZPT), which is used commercially as an antifouling agent and for treating dandruff and other skin disorders. In intracellular Yellow Fluorescent Protein YFP(F46L/H148Q/I152L)-quenching assays, ZPT potentiates the rate and extent of swelling-induced iodide influx dose dependently with a half-maximal effective concentration (EC50) of 5.7 µM. Whole cell voltage-clamp experiments revealed that coapplication of hypotonic solution and 30 µM ZPT to human embryonic kidney 293 or human colorectal carcinoma 116 cells increases the rate of swelling-induced VRAC activation by approximately 10-fold. ZPT potentiates swelling-induced VRAC currents after currents have reached a steady state and activates currents in the absence of cell swelling. Neither ZnCl2 nor free pyrithione activated VRAC; however, treating cells with a mixture of ZnCl2 and pyrithione led to robust channel activation. Finally, the effects of ZPT on VRAC were inhibited by reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and NAD(P)H oxidase inhibitor diphenyleneiodonium chloride, suggesting the mechanism of action involves ROS generation. The discovery of ZPT as a potentiator/activator of VRAC demonstrates the utility of HTS for identifying small-molecule modulators of VRAC and adds to a growing repertoire of pharmacological tool compounds for probing the molecular physiology and regulation of this important channel.

Cryo-EM structure of the volume-regulated anion channel LRRC8D isoform identifies features important for substrate permeation

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). LRRC8A and at least one of the other LRRC8 isoforms assemble into heteromers to generate VRAC transport activities. Despite the availability of the LRRC8A structures, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. Here, we present the structure of the human LRRC8D isoform, which enables the permeation of organic substrates through VRAC. The LRRC8D homo-hexamer structure displays a two-fold symmetric arrangement, and together with a structure-based electrophysiological analysis, revealed two key features. The pore constriction on the extracellular side is wider than that in the LRRC8A structures, which may explain the increased permeability of organic substrates. Furthermore, an N-terminal helix protrudes into the pore from the intracellular side and may be critical for gating.

CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor.

Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 (LRRC8) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate Gq-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- Gq-calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC50 of ~17 µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.