F0F1 ATP synthase regulates extracellular calcium influx in human neutrophils by interacting with Cav2.3 and modulates neutrophil accumulation in the lipopolysaccharide-challenged lung

Baoyi Zhu,Yan Guo,Tian Zhang,Dai Wang,Dongxing Zhu,Ai Mai,Zhanfang Kang,Zhengfu Feng,Jun Gao,Ting Weijen,Dazhong Yin

doi:10.1186/s12964-020-0515-3

Abstract

BackgroundNeutrophils 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.MethodsBlue 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.ResultsWe 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.ConclusionsIn 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.Graphical abstract

Highlights

Neutrophils form the first line of innate host defense against invading microorganisms
The mitochondrial membrane potential will increase in the presence of oligomycin because oligomycin can hyperpolarize mitochondria by blocking proton re-entry from the intermembrane space to the matrix via its effects on F0-ATP synthase [9]; Fossati et al found that oligomycin had no effect on the mitochondrial membrane potential in neutrophils [1]
The voltage-gated calcium channel (VGCC) α2δ-1 subunit was identified as a binding partner of cell surface F-ATPase After the purification and viability determination of neutrophils (Additional file 2), plasma membranes were purified and lysed in 2% digitonin to obtain protein complexes, and blue native polyacrylamide gel electrophoresis (BN-PAGE) was used to identify proteins that could interact with F-ATPase

Summary

Introduction

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. Studies on mitochondrial function in neutrophils commonly assumed that this mitochondrial system does not participate in oxidative phosphorylation. F-ATPase is commonly assumed to be respiratory chain complex V in the inner membrane of mitochondria; this complex consists of a transmembrane proton transport domain, F0, and an ATP synthesis catalytic domain, F1 [7]. The mitochondrial membrane potential will increase in the presence of oligomycin because oligomycin can hyperpolarize mitochondria by blocking proton re-entry from the intermembrane space to the matrix via its effects on F0-ATP synthase [9]; Fossati et al found that oligomycin had no effect on the mitochondrial membrane potential in neutrophils [1]. Strictly speaking, F-ATPase rather than mitochondria participates in the regulatory process described above

Methods

Results

Discussion

Conclusion