Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, 37232, USA.
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37232, USA.
J Physiol. 2021 Jun;599(12):3013-3036. doi: 10.1113/JP281577. Epub 2021 May 26.
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 O 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.
Tumour necrosis factor-α (TNFα) activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs), producing superoxide (O ) 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 O 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 含有阴离子通道与 NADPH 氧化酶 1（Nox1）相关联，并调节超氧化物的产生和肿瘤坏死因子-α（TNFα）信号转导。在这里，我们表明 LRRC8C 和 8D 也与血管平滑肌细胞中的 Nox1 共免疫沉淀。LRRC8C 敲低抑制了 TNFα 诱导的 O 产生、受体内吞、核因子-κB（NF-κB）激活和增殖，而 LRRC8D 敲低增强了 NF-κB 激活。人类动脉粥样硬化和银屑病中 LRRC8 同工型表达的显著变化表明炎症增加的补偿。氧化剂氯胺-T（ChlorT，1 mM）对 LRRC8C 电流的抑制作用较弱（约 25%），但对 LRRC8D 电流的抑制作用较强（约 80%）。8D 的细胞外环（EL1、EL2）结构域的替换赋予 8C 明显更强的 ChlorT 依赖性抑制作用（69%）。ChlorT 暴露损害了随后由 DCPIB 引起的电流阻断，这是通过与 EL1 相互作用发生的，进一步表明存在外部氧化部位。LRRC8A/C 通道在质膜上最有效地维持 Nox1 活性。这可能是由于它们能够在氧化的微环境中保持活性。
肿瘤坏死因子-α（TNFα）激活血管平滑肌细胞（VSMCs）中的 NADPH 氧化酶 1（Nox1），产生超氧化物（O ），这是随后信号转导所必需的。LRRC8 家族蛋白 A-E 构成体积调节阴离子通道（VRAC）。必需的亚基 LRRC8A 与 Nox1 物理结合，并且 VRAC 活性是 Nox 活性和对 TNFα 的炎症反应所必需的。VRAC 电流受氧化剂调节，这表明通道氧化剂敏感性和与 Nox1 的接近度可能在生理上具有相关作用。在 VSMCs 中，LRRC8C 敲低（siRNA）重现了 siLRRC8A 的效果，抑制了 TNFα 诱导的细胞外和内体 O 产生、受体内吞、核因子-κB（NF-κB）激活和增殖。相比之下，siLRRC8D 增强了 NF-κB 激活。Nox1 与 8C 和 8D 共免疫沉淀，并且与 8D 在质膜和囊泡中共定位。我们比较了在 HEK293 细胞中表达的同型和异型 LRRC8C 和 LRRC8D 通道介导的 VRAC 电流。氧化剂氯胺 T（ChlorT，1 mM）对 8C 的抑制作用较弱，但对 8D 电流的抑制作用较强。ChlorT 暴露还损害了随后由 VRAC 阻断剂 DCPIB 引起的电流阻断，这表明存在外部氧化部位。8D 的细胞外环结构域（EL1、EL2）的替换赋予 8C 明显更强的 ChlorT 介导的抑制作用。我们的结果表明，LRRC8A/C 通道活性可以在预期由质膜上的 Nox1 激活引起的氧化微环境中有效地维持。8D：8C 表达比例的增加可能潜在地抑制 TNFα 诱导的炎症反应。LRRC8A/C 通道下调代表了一种减少 TNFα 诱导的炎症反应的新策略。
