Periodic Reporting for period 4 - VOLSIGNAL (Volume regulation and extracellular signalling by anion channels)
Período documentado: 2022-04-01 hasta 2023-09-30
Anion channels perform a plethora of crucial functions in cells and the organism, but have been studied much less than cation channels and the molecular identity of many anion channels has remained enigmatic. VolSignal aims at clarifying the properties of volume-regulated VRAC anion channels we had only recently identified as LRRC8 heteromers and to investigate their role in signal transduction and pathologies. Further, we aim at molecularly identifying other anion channels as the essential first step for determining their biological roles. Reaching these objectives will lead to fundamentally new insights into cellular and organismal processes highly relevant for health and disease.
Following our identification of residues of swelling-activated LRRC8/VRAC channels that line its ion-selective pore by classical mutagenesis/biophysical analysis methods (1), we more recently collaborated with the group of Prof. Juan Liao (Shanghai) to extend these results with cryo-EM studies (8). These studies confirmed our previous results, and led to deep insights into the pore structure of LRRC8 channels – importantly, they display two selectivity filters in series. Furthermore, molecular dynamics simulations suggested that activation of VRAC by low ionic strength involves conformational changes of pore-inserted N-termini, significantly advancing our understanding of VRAC gating and regulation. Our main focus, however, was on the biological roles of VRACs in the cell and organism. Using a mouse model in which we disrupted the essential VRAC subunit Lrrc8a specifically in insulin-secreting β-cells of the pancreas we demonstrated that the channel plays an important modulatory role in glucose-induced insulin secretion (3). Opening of VRAC by glucose-induced β-cell swelling reduces the voltage across the outer cell membrane, opens voltage-dependent Ca2+ channels and thereby leads to the release of insulin granules. Recently we discovered another, unexpected role of VRAC in extracellular signal transduction. We found that LRRC8/VRAC channels transport the important biological messenger molecule cGAMP (cyclic GMP-AMP) that is produced in cells in response to the presence of double-stranded DNA in the cell interior (cytoplasm), where it does not belong. However, DNA is present in the cytoplasm upon certain viral infections and in cancer cells. cGAMP, which is a danger signal, leads to the production of protective interferons. Our study (5) now shows that cGAMP can be transferred to non-affected bystander cells and thereby boost the innate immune response, as shown directly with mouse models in which we had disrupted a Lrrc8 subunit important for cGAMP transport. This work identified an unsuspected role of LRRC8/VRAC in innate immunity response against DNA viruses and probably also cancer. Using cell-type specific KO mice, we investigated its role in male fertility. Disruption of VRAC in male germ cells severely disturbed, probably because of impaired cell volume regulation, the development of sperm cells (2). In another project, we determined the expression pattern of all five LRRC8 subunits in the kidney using KI mice expressing epitope-tagged subunits and investigated the effect of the KO of each individual LRRC8 subunit on kidney function. This led to the conclusion that LRRC8A/D may form basolateral exit sites for metabolites in the proximal tubule, a nephron segment which degenerates upon disruption of either Lrrc8a or Lrrc8d (6).
In another major breakthrough, we identified, using a sophisticated genome-wide siRNA screen, the protein constituting the acid-activated anion channel ASOR (4), closing another gap in our knowledge of anion channels. ASOR is a multimer of TMEM206 proteins. We already identified pore residues and showed that it plays a role in acid-induced cell death (4). In collaboration with Steve Long, we obtained for the first time the structure of the open channel, which involves a highly unusual metamorphosis of the transmembrane domains, and clarified the activation by extracellular/luminal protons (9). Further, we identified a crucial cell biological role of ASOR: It provides the Cl- conductance that is needed in parallel to a TPC channel-mediated Na+ conductance for the shrinkage of macropinosomes (7). This shrinkage is crucial for downstream trafficking steps. As a consequence, RAS-mutant tumor cells grew better when TMEM206 was disrupted because they could use albumin, that is taken up by macropinocytosis, more efficiently (7).
In another major breakthrough, we identified, using a sophisticated genome-wide siRNA screen, the protein constituting the acid-activated anion channel ASOR (4), closing another gap in our knowledge of anion channels. ASOR is a multimer of TMEM206 proteins. We already identified pore residues and showed that it plays a role in acid-induced cell death (4). In collaboration with Steve Long, we obtained for the first time the structure of the open channel, which involves a highly unusual metamorphosis of the transmembrane domains, and clarified the activation by extracellular/luminal protons (9). Further, we identified a crucial cell biological role of ASOR: It provides the Cl- conductance that is needed in parallel to a TPC channel-mediated Na+ conductance for the shrinkage of macropinosomes (7). This shrinkage is crucial for downstream trafficking steps. As a consequence, RAS-mutant tumor cells grew better when TMEM206 was disrupted because they could use albumin, that is taken up by macropinocytosis, more efficiently (7).
Our work on the characterization of LRRC8/VRAC channels is clearly beyond the state of the art. Our work on VRAC and insulin secretion is medically important, and the recent, unexpected discovery that VRAC transports cGAMP, plays a role in innate immunity against DNA viruses and will likely be of relevance for other important pathologies like cancer. We expect many more novel insights on the role of VRAC in signal transduction and pathologies until completion of this project.
The discovery of TMEM206 as constituting the ASOR channel, which has been known physiologically for about 10 years, but whose molecular identity has remained obscure, is a major breakthrough that now opens the door to analyze its structure-function relationship and, more importantly, its role in physiology and pathology. Indeed, in the few years after its discovery we already identified in cryo-EM studies the novel mechanism by which acidification causes a dramatic change in transmembrane toplogy that leads to channel opening. We also identified a crucial role in macropinocytosis that may be relevant for cancer. Using our mouse model, we pursue studies on the role of ASOR in endocytic processes at the organismal level in various tissues.
The discovery of TMEM206 as constituting the ASOR channel, which has been known physiologically for about 10 years, but whose molecular identity has remained obscure, is a major breakthrough that now opens the door to analyze its structure-function relationship and, more importantly, its role in physiology and pathology. Indeed, in the few years after its discovery we already identified in cryo-EM studies the novel mechanism by which acidification causes a dramatic change in transmembrane toplogy that leads to channel opening. We also identified a crucial role in macropinocytosis that may be relevant for cancer. Using our mouse model, we pursue studies on the role of ASOR in endocytic processes at the organismal level in various tissues.