BACKGROUND Neutrophils form the first line of innate host defense against invading microorganisms. We previously showed that F0F1 ATP synthase (F-ATPase), which is widely known as mitochondrial respiratory chain complex V, is expressed in the plasma membrane of human neutrophils and is involved in regulating cell migration. Whether F-ATPase performs cellular functions through other pathways remains unknown. METHODS Blue native polyacrylamide gel electrophoresis followed by nano-ESI-LC MS/MS identification and bioinformatic analysis were used to identify protein complexes containing F-ATPase. Then, the identified protein complexes containing F-ATPase were verified by immunoblotting, immunofluorescence colocalization, immunoprecipitation, real-time RT-PCR and agarose gel electrophoresis. Immunoblotting, flow cytometry and a LPS-induced mouse lung injury model were used to assess the effects of the F-ATPase-containing protein complex in vitro and in vivo. RESULTS We found that the voltage-gated calcium channel (VGCC) α2δ-1 subunit is a binding partner of cell surface F-ATPase in human neutrophils. Further investigation found that the physical connection between the two proteins may exist between the F1 part (α and β subunits) of F-ATPase and the α2 part of VGCC α2δ-1. Real-time RT-PCR and PCR analyses showed that Cav2.3 (R-type) is the primary type of VGCC expressed in human neutrophils. Research on the F-ATPase/Cav2.3 functional complex indicated that it can regulate extracellular Ca2+ influx, thereby modulating ERK1/2 phosphorylation and reactive oxygen species production, which are typical features of neutrophil activation. In addition, the inhibition of F-ATPase can reduce neutrophil accumulation in the lungs of mice that were intratracheally instilled with lipopolysaccharide, suggesting that the inhibition of F-ATPase may prevent neutrophilic inflammation-induced tissue damage. CONCLUSIONS In this study, we identified a mechanism by which neutrophil activity is modulated, with simultaneous regulation of neutrophil-mediated pulmonary damage. These results show that surface F-ATPase of neutrophils is a potential innate immune therapeutic target.

中文翻译：

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F0F1 ATP合酶通过与Cav2.3相互作用来调节人中性粒细胞的细胞外钙内流，并调节中性粒细胞在脂多糖挑战的肺中的积累。

背景技术中性粒细胞形成先天宿主防御入侵微生物的第一线。我们以前显示F0F1 ATP合酶（F-ATPase），广泛称为线粒体呼吸链复合体V，在人嗜中性粒细胞的质膜中表达，并参与调节细胞迁移。F-ATPase是否通过其他途径执行细胞功能尚不清楚。方法采用蓝色天然聚丙烯酰胺凝胶电泳，然后通过纳米ESI-LC MS / MS鉴定和生物信息学分析，鉴定含有F-ATPase的蛋白质复合物。然后，通过免疫印迹，免疫荧光共定位，免疫沉淀，实时RT-PCR和琼脂糖凝胶电泳对鉴定出的含有F-ATPase的蛋白质复合物进行验证。免疫印迹 流式细胞仪和LPS诱导的小鼠肺损伤模型用于评估体内和体外含F-ATPase的蛋白质复合物的作用。结果我们发现电压门控钙通道（VGCC）α2δ-1亚基是人类嗜中性粒细胞中细胞表面F-ATPase的结合伴侣。进一步的研究发现，两种蛋白质之间的物理联系可能存在于F-ATPase的F1部分（α和β亚基）和VGCCα2δ-1的α2部分之间。实时RT-PCR和PCR分析表明，Cav2.3（R型）是人类嗜中性粒细胞表达的VGCC的主要类型。对F-ATPase / Cav2.3功能复合物的研究表明，它可以调节细胞外Ca2 +内流，从而调节ERK1 / 2磷酸化和活性氧的产生，这是嗜中性粒细胞活化的典型特征。此外，F-ATPase的抑制作用可以减少经气管内滴注脂多糖的小鼠肺中的中性粒细胞积累，这表明F-ATPase的抑制作用可以预防中性粒细胞炎症引起的组织损伤。结论在这项研究中，我们确定了一种调节嗜中性粒细胞活性并同时调节嗜中性粒细胞介导的肺损伤的机制。这些结果表明嗜中性粒细胞的表面F-ATP酶是潜在的先天免疫治疗靶标。同时调节中性粒细胞介导的肺损伤。这些结果表明嗜中性粒细胞的表面F-ATP酶是潜在的先天免疫治疗靶标。同时调节中性粒细胞介导的肺损伤。这些结果表明嗜中性粒细胞的表面F-ATP酶是潜在的先天免疫治疗靶标。