Cells need to regulate their volume when exposed to osmotic stress and during processes like cell division, growth, and apoptosis. A key player in regulatory volume decrease is the ubiquitously expressed volume-activated anion channel VRAC which has been studied extensively over the past three decades. However, attempts to identify the underlying molecules have failed repeatedly. We now performed an unbiased genome-wide RNA interference screen using a functional read-out and identified the membrane protein LRRC8A as essential subunit of VRAC. However, LRRC8A needs heteromerization with other members of this gene family to reconstitute VRAC currents in cells in which LRRC8A,B,C,D,E have all been deleted using CRISPR-Cas9. Combinations of different LRRC8 isoforms yield currents with different inactivation kinetics, explaining the differences in VRAC properties observed in vivo. Furthermore, we showed that VRAC conducts also organic osmolytes like taurine. Our work finally provides the basis for investigating the structure-function relationship of this important channel, to elucidate the mechanism by which cell swelling leads to VRAC opening, and to examine the role of VRAC in various pathologies. Our initial identification and characterization of VRAC has been published in Science (2014).
More recently, we showed that the substrate specificity of VRAC depends on its subunit composition, demonstrating that LRRC8 heteromers form its pore. We extended the latter finding by structure-function analysis. LRRC8D increased the selectivity for the organic osmolyte taurine and for the important cancer drug cisplatin. We showed that VRAC plays a dual role in cisplatin sensitivity by mediating its uptake and by promoting apoptosis. Downregulation of LRRC8D may be clinically important in tumor drug resistance.
In addition to inorganic anions, drugs, and taurine, VRAC also transports many neurotransmitters and second messengers such as cGAMP. This suggests a role in extracellular signal transduction, in particular in the nervous system. To explore the physiological functions of VRAC, we have generated several KO mice. In addition, we are generating KI mice in which the addition of epitopes to individual LRRC8 subunits allows their detection by immunohistochemistry in mouse tissues. We showed that insulin-secreting pancreatic β-cells markedly express LRRC8D. Using mice in which we disrupted the essential VRAC subunit Lrrc8a specifically in β-cells, we demonstrated that VRAC modulates insulin secretion. Glucose uptake by β-cells increases cytoplasmic osmolarity. The ensuing cell swelling opens VRAC, leading to depolarization, opening of voltage-dependent Ca-channels, and increased insulin secretion. Hence VRAC also plays a role in intracellular signaling.
Most recently, in a collaboration with Prof. Xiao, we found that VRAC also transports the important messenger molecule cGAMP (cyclicGAMP-AMP). cGAMP is produced by an enzyme called cGAS that is activated by double stranded DNA in the cytoplasm. DNA is present in the cytoplasm e.g. after infection with DNA viruses, or, due to genomic instability, in cancer. cGAMP, after binding to an intracellular receptor called STING, activates the transcription of interferon and thereby stimulates the innate immune response. We showed that cGAMP is transported by VRACs across plasma membranes and thereby can reach non-infected bystander cells in the vicinity, where it enhances the production of interferon. Indeed, KO mice for a VRAC subunit that is particularly important for cGAMP transport showed less interferon response and higher viral titers than WT mice. Hence, VRAC importantly boosts the immune response to DNA viruses.
Neurotransmitter transport by VRAC: Lutter et al., JCS 2017.
VRAC in insulin secretion: Stuhlmann et al., Nature comm. 2018.
VRAC in cGAMP transport and immune response: Zhu et al., Immunity 2020. Research Highlight
Our current research on VRACs focuses on structure-function, its regulation, and in particular on its roles in physiology and pathology. It is now one of our main area of research.