LRRC8A anion channels modulate vascular reactivity via association with myosin phosphatase rho interacting protein.

Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 µM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.

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.

Oxidant-resistant LRRC8A/C anion channels support superoxide production by NADPH oxidase 1.

KEY POINTS: LRRC8A-containing anion channels associate with NADPH oxidase 1 (Nox1) and regulate superoxide production and tumour necrosis factor-α (TNFα) signalling. Here we show that LRRC8C and 8D also co-immunoprecipitate with Nox1 in vascular smooth muscle cells. LRRC8C knockdown inhibited TNFα-induced O2•- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation while LRRC8D knockdown enhanced NF-κB activation. Significant changes in LRRC8 isoform expression in human atherosclerosis and psoriasis suggest compensation for increased inflammation. The oxidant chloramine-T (ChlorT, 1 mM) weakly (∼25%) inhibited LRRC8C currents but potently (∼80%) inhibited LRRC8D currents. Substitution of the extracellular loop (EL1, EL2) domains of 8D into 8C conferred significantly stronger (69%) ChlorT-dependent inhibition. ChlorT exposure impaired subsequent current block by DCPIB, which occurs through interaction with EL1, further implicating external oxidation sites. LRRC8A/C channels most effectively sustain Nox1 activity at the plasma membrane. This may result from their ability to remain active in an oxidized microenvironment. ABSTRACT: Tumour necrosis factor-α (TNFα) activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs), producing superoxide (O2•- ) required for subsequent signalling. LRRC8 family proteins A-E comprise volume-regulated anion channels (VRACs). The required subunit LRRC8A physically associates with Nox1, and VRAC activity is required for Nox activity and the inflammatory response to TNFα. VRAC currents are modulated by oxidants, suggesting that channel oxidant sensitivity and proximity to Nox1 may play a physiologically relevant role. In VSMCs, LRRC8C knockdown (siRNA) recapitulated the effects of siLRRC8A, inhibiting TNFα-induced extracellular and endosomal O2•- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation. In contrast, siLRRC8D potentiated NF-κB activation. Nox1 co-immunoprecipitated with 8C and 8D, and colocalized with 8D at the plasma membrane and in vesicles. We compared VRAC currents mediated by homomeric and heteromeric LRRC8C and LRRC8D channels expressed in HEK293 cells. The oxidant chloramine T (ChlorT, 1 mM) weakly inhibited 8C, but potently inhibited 8D currents. ChlorT exposure also impaired subsequent current block by the VRAC blocker DCPIB, implicating external sites of oxidation. Substitution of the 8D extracellular loop domains (EL1, EL2) into 8C conferred significantly stronger ChlorT-mediated inhibition of 8C currents. Our results suggest that LRRC8A/C channel activity can be effectively maintained in the oxidized microenvironment expected to result from Nox1 activation at the plasma membrane. Increased ratios of 8D:8C expression may potentially depress inflammatory responses to TNFα. LRRC8A/C channel downregulation represents a novel strategy to reduce TNFα-induced inflammation.

LRRC8A anion channels modulate vasodilation via association with Myosin Phosphatase Rho Interacting Protein (MPRIP).

Background: In vascular smooth muscle cells (VSMCs), LRRC8A volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A physically associates with NADPH oxidase 1 (Nox1) and supports its production of extracellular superoxide (O 2 -• ). Methods and Results: Mice lacking LRRC8A exclusively in VSMCs (Sm22α-Cre, KO) were used to assess the role of VRACs in TNFα signaling and vasomotor function. KO mesenteric vessels contracted normally to KCl and phenylephrine, but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). 48 hours of ex vivo exposure to TNFα (10ng/ml) markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 µM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 35 proteins that interacted with LRRC8A. Pathway analysis revealed actin cytoskeletal regulation as the most closely associated function of these proteins. Among these proteins, the Myosin Phosphatase Rho-Interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots which revealed LRRC8A binding at the second Pleckstrin Homology domain of MPRIP. siLRRC8A or CBX treatment decreased RhoA activity in cultured VSMCs, and MYPT1 phosphorylation at T853 was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Conclusions: Interaction of Nox1/LRRC8A with MPRIP/RhoA/MYPT1/actin may allow redox regulation of the cytoskeleton and link Nox1 activation to both inflammation and vascular contractility.