Molecular Mechansims And Celluar Functions Of CGAS Regulation Of LRRC8/VRAC Channel In Innate Immunity And Anti–tumor Therapy

The volume-regulated anion channel(VRAC)which regulates hypotonic response and cell volume in response to cell swelling has only been found seven years ago.It is widely expressed in various mammalian tissues and needed to form heteromultimeric channels by LRRC8A and its homologous subunit LRRC8B-E in the plasma membrane.VRAC is rapidly activated under the hypoosmotic pressure in the extracellular environment.In addition to conducting Cl~-efflux to reduce intracellular osmotic pressure and prevent cell expansion to maintain normal physiological functions.VRAC are involved in regulating growth and development or affecting diabetes and stroke by transporting diverse organic molecules,including aspartic acid and glutamate,neurotransmitter GABA and anticancer drug cisplatin.Our Previous data shows that LRRC8A/E VRAC,can transport cGAMP,a natural agonist of STING.However,the molecule mechanisms underlying LRRC8A/E VRAC regulation remain unclear.The ds DNA released from pathogenic microbes and tumor cells recognized by cGAS(CyclicGMP–AMP synthase),which then synthesizes cGAMP by the catalysis of ATP and GTP.cGAMP then activates STING,which dissociates from the ER to recruit TBK1,and further phosphorylates IRF3 to induce the induction of IFN-?and inflammatory cytokines/chemokines to initiate innate immune response.Many researches suggest that in addition to binding with ds DNA to catalyze the synthesis of cGAMP,the intracellular localization and physiological function of cGAS need to be further investigated.Based on our previous results that LRRC8A/E VRAC regulates antiviral immune response by transporting cGAMP across the cell membrane.We sought to further elucidate the mechanisms underlying the activation of VRAC/LRRC8 channels.Combining with electrophysiological patch clamp technology and liquid chromatography-mass spectrometry(LC-MS/MS),we found that LRRC8/VRAC could transport cGAMP produced by virus-infected cells to non-infected cells to activate STNG signaling and type I IFNs.In addition,the activation of the VRAC channel is regulated by inflammatory factors TNF,IL-1 and serum.In addition to synthesizing cGAMP,cGAS also participates in upregulating VRAC channels in transmitting cGAMP or cisplatin upon stimulation with cytokines or serum.Mechanistically,we found that cGAS regulates the transport of cGAMP and cisplatin by VRAC through binding with PI(4,5)P2 localized on the plasma membrane.Finally,we further confirmed that cGAS plays an important role in cisplatin-mediated cancer therapy through a transplanted tumor model in vivo.Overall,our study revealed novel mechanism and function of LRRC8/VRAC in transporting cGAMP and cisplatin.The molecular mechanism of cGAS regulating LRRC8/VRAC channel activity was previously unappreciated,thus would serve as new idea and strategy for developing vaccines and optimizing anti-tumor immunotherapy.