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

Stark, Ryan J.; Nguyen, Hong‐Ngan; Bacon, Matthew; Rohrbough, Jeffrey; Choi, Hyehun; Lamb, Fred S.

doi:10.1007/s00232-022-00271-9

Cited by 4 publications

(5 citation statements)

References 56 publications

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“…5C). As previously demonstrated, LRRC8A localizes to the plasma membrane and to prominent intracellular vesicles 27 . MPRIP co-localized with LRRC8A at the membrane, particularly in areas where membrane extensions were forming, but was completely absent from intracellular vesicles.…”

Section: Identification Of Lrrc8a-associated Proteins Lrrc8 Proteins ...supporting

confidence: 60%

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

Choi

Miller²,

Nguyen³

et al. 2023

Preprint

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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 (O2-·). 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.

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Section: Identification Of Lrrc8a-associated Proteins Lrrc8 Proteins ...supporting

confidence: 60%

“…Cytoplasmic dihydroethidium oxidation by extracellular O 2 -• was measured and inhibited by anion channel blockade or knockdown of Chloride Channel three (ClC-3) expression 53 . We recently demonstrated that ClC-3 influences membrane trafficking of LRRC8A, raising the possibility that this effect of ClC-3 was indirect 27 .…”

Section: Discussionmentioning

confidence: 99%

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

Choi

Miller²,

Nguyen³

et al. 2023

Preprint

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“…Cytoplasmic dihydroethidium oxidation by extracellular O 2 −• was measured and inhibited by anion channel blockade or knockdown of chloride channel three (ClC‐3) expression 57 . We recently demonstrated that ClC‐3 influences membrane trafficking of LRRC8A, raising the possibility that this effect of ClC‐3 was indirect 26 …”

Section: Discussionmentioning

confidence: 99%

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

et al. 2023

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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

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“…MPRIP is oxidized by TNF-α in an LRRC8A-dependent manner 118 and complex assembly may, therefore, be redox regulated. This may be important because association of MPRIP with MYPT1 enhances LRRC8A localizes to the plasma membrane and the surface of intracellular vesicles (see Reference [124] for details). LRRC8A-mCherry and LifeACT-Hyper7 (H 2 O 2 sensor) were coexpressed in cultured LRRC8A knockout aortic VSMCs.…”

Section: Figure 1 Loss Of Ecsod Promotes Apoptosis Signaling-related ...mentioning

confidence: 99%

“…Figure 2. Colocalization of LRRC8A (leucine-rich repeat containing 8A) with H2O2 in a single vascular smooth muscle cell (VSMC).LRRC8A localizes to the plasma membrane and the surface of intracellular vesicles (see Reference[124] for details). LRRC8A-mCherry and LifeACT-Hyper7 (H 2 O 2 sensor) were coexpressed in cultured LRRC8A knockout aortic VSMCs.…”

mentioning

confidence: 99%

Vascular Inflammation and Smooth Muscle Contractility: The Role of Nox1-Derived Superoxide and LRRC8 Anion Channels

Lamb,

Choi,

Miller

et al. 2024

Hypertension

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Vascular inflammation underlies the development of hypertension, and the mechanisms by which it increases blood pressure remain the topic of intense investigation. Proinflammatory factors including glucose, salt, vasoconstrictors, cytokines, wall stress, and growth factors enhance contractility and impair relaxation of vascular smooth muscle cells. These pathways share a dependence upon redox signaling, and excessive activation promotes oxidative stress that promotes vascular aging. vascular smooth muscle cell phenotypic switching and migration into the intima contribute to atherosclerosis, while hypercontractility increases systemic vascular resistance and vasospasm that can trigger ischemia. Here, we briefly review factors that drive the initiation and progression of this vasculopathy in vascular smooth muscle cells. Emphasis is placed on the contribution of reactive oxygen species generated by the Nox1 NADPH oxidase which produces extracellular superoxide (O 2 •− ). The mechanisms of O 2 •− signaling remain poorly defined, but recent evidence demonstrates physical association of Nox1 with leucine-rich repeat containing 8 family volume-sensitive anion channels . These may provide a pathway for influx of O 2 •− to the cytoplasm, creating an oxidized cytoplasmic nanodomain where redox-based signals can affect both cytoskeletal structure and vasomotor function. Understanding the mechanistic links between inflammation, O 2 •− and vascular smooth muscle cell contractility may facilitate targeting of anti-inflammatory therapy in hypertension.

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

Cited by 4 publications

References 56 publications

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

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

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

Vascular Inflammation and Smooth Muscle Contractility: The Role of Nox1-Derived Superoxide and LRRC8 Anion Channels

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