Periodic Reporting for period 5 - VOLSIGNAL (Volume regulation and extracellular signalling by anion channels)
Okres sprawozdawczy: 2023-10-01 do 2024-09-30
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 aimed 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 strived at molecularly identifying other anion channels as the essential first step for determining their biological roles, and identified the molecular composition of the acid-activated anion channel ASOR. Reaching these objectives were the basis to fundamentally new insights into molecular, cellular and organismal processes highly relevant for health and disease.
In VOLSIGNAL projects, we thoroughly characterized the properties and functions of the volume-regulated anion channel VRAC (molecularly identified (Voss et al., Science 2014) in my first ERC grant) and the acid-activated anion channel ASOR, the molecular identification of which was a major aim of the concluded VOLSIGNAL grant. This aim was achieved in 2019 (4). Their characterization included structure-function analysis with mutagenesis and electrophysiology (1, 4), as well as Cryo-EM (Nakamura et al., Commun. Biol. 2018; 8 & 9), characterization of their cellular roles (5, 7) and organismal roles, mostly using mouse models (3, 6, 10).
VRAC is a heterohexamer of up to 5 different LRRC8 subunits. The specific composition determines VRAC properties such as regulation and permeation. VRACs not only conduct inorganic anions, but also organic molecules including neurotransmitters and drugs. In structure/function studies, we identified in the framework of VOLSIGNAL residues of swelling-activated LRRC8/VRAC channels that line its pore by classical mutagenesis/biophysical analysis methods (1). We had suggested that LRRC8 N-termini fold back into the pore and form a selectivity filter. This was confirmed in our cryo-EM study together with Prof. Liao, which showed that LRRC8 channels display two selectivity filters in series (8). Molecular dynamics suggested that VRAC activation by low ionic strength involves partial unfolding of pore-inserted N-termini. This also changes ion selectivity as confirmed by electrophysiology. In another cryo-EM collaboration, with Osamu Nureki, Tokyo, we found that homomultimers of LRRC8D have wider pores (Nakamura et al., Commun. Biol. 2018), providing an explanation for the fact that incorporation of this subunit increases the permeability for organic substrates.
A major focus was on the biological roles of VRACs in the cell and organism. We generated KO mice – in part conditional – for all 5 LRRC8 subunits, as well as KI mice expressing epitope-tagged subunits under the control of the respective promoters (2, 3, 6, 10). These were used to investigate roles of VRAC in various tissues. We disrupted the essential VRAC subunit Lrrc8a specifically in insulin-secreting pancreatic β-cells and demonstrated that the channel plays a modulatory role in glucose-induced insulin secretion (3). Opening of VRAC by glucose-induced β-cell swelling depolarizes the cell, opens voltage-dependent Ca2+ channels leading to exocytosis of insulin granules. Germ cell-specific, but not Sertoli cell-specific Lrrc8a disruption led to male infertility, probably by affecting sperm cell volume regulation (2). We also determined the expression pattern of all 5 LRRC8 subunits in the kidney using our KI mice, and investigated effects of disrupting each individual subunit on kidney function (6). We concluded 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). We also investigated the expression patterns of all Lrrc8 subunits in the inner ear and determined the effect of disrupting individual Lrrc8 subunits (10). Inner-ear specific disruption of Lrrc8a, or the double-KO of Lrrc8d and Lrrc8e, led to deafness in mice. This is not (only) due to a degeneration of the organ of Corti, which occurs over many weeks, but most likely to the (measured) decrease of the endocochlear potential that is necessary for current generation through mechanosensitive channels of sensory hair cells. We could attribute this effect to a secondary loss of the K+ channel Kir4.1 in the stria vascularis. We hypothesized that this downregulation is owed to oxidative stress in the inner ear caused by a lack of glutathione transport through VRAC (10). Together with Hui Xiao, Shanghai, we discovered that VRAC can also conduct cGAMP, an important immunomodulator. cGAMP transport from virally infected to non-infected cells serves to convey a danger signal to neighboring cells, augmenting the immune response in viral infection (5). cGAMP transport through VRAC likely has roles in other settings such as cancer immunity.
In a major breakthrough, we identified, in 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. In structure/function studies, we identified pore residues and showed that it plays a role in acid-induced cell death. Together with Steve Long, New York, we obtained the Cryo-EM structure of the open channel, which displays a highly unusual metamorphosis of the transmembrane domains upon gating, and clarified the activation by protons (9). We also identified a crucial cell biological role of ASOR: It provides the Cl- conductance that is needed for the shrinkage of macropinosomes (7). This shrinkage is crucial for downstream trafficking steps. RAS-mutant tumor cells grew better when TMEM206 was disrupted because they could use albumin, taken up by macropinocytosis, more efficiently (8). Given the nearly ubiquitous expression pattern, ASOR/TMEM206 likely plays a role in endocytosis in many, if not most, tissues.
VRAC is a heterohexamer of up to 5 different LRRC8 subunits. The specific composition determines VRAC properties such as regulation and permeation. VRACs not only conduct inorganic anions, but also organic molecules including neurotransmitters and drugs. In structure/function studies, we identified in the framework of VOLSIGNAL residues of swelling-activated LRRC8/VRAC channels that line its pore by classical mutagenesis/biophysical analysis methods (1). We had suggested that LRRC8 N-termini fold back into the pore and form a selectivity filter. This was confirmed in our cryo-EM study together with Prof. Liao, which showed that LRRC8 channels display two selectivity filters in series (8). Molecular dynamics suggested that VRAC activation by low ionic strength involves partial unfolding of pore-inserted N-termini. This also changes ion selectivity as confirmed by electrophysiology. In another cryo-EM collaboration, with Osamu Nureki, Tokyo, we found that homomultimers of LRRC8D have wider pores (Nakamura et al., Commun. Biol. 2018), providing an explanation for the fact that incorporation of this subunit increases the permeability for organic substrates.
A major focus was on the biological roles of VRACs in the cell and organism. We generated KO mice – in part conditional – for all 5 LRRC8 subunits, as well as KI mice expressing epitope-tagged subunits under the control of the respective promoters (2, 3, 6, 10). These were used to investigate roles of VRAC in various tissues. We disrupted the essential VRAC subunit Lrrc8a specifically in insulin-secreting pancreatic β-cells and demonstrated that the channel plays a modulatory role in glucose-induced insulin secretion (3). Opening of VRAC by glucose-induced β-cell swelling depolarizes the cell, opens voltage-dependent Ca2+ channels leading to exocytosis of insulin granules. Germ cell-specific, but not Sertoli cell-specific Lrrc8a disruption led to male infertility, probably by affecting sperm cell volume regulation (2). We also determined the expression pattern of all 5 LRRC8 subunits in the kidney using our KI mice, and investigated effects of disrupting each individual subunit on kidney function (6). We concluded 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). We also investigated the expression patterns of all Lrrc8 subunits in the inner ear and determined the effect of disrupting individual Lrrc8 subunits (10). Inner-ear specific disruption of Lrrc8a, or the double-KO of Lrrc8d and Lrrc8e, led to deafness in mice. This is not (only) due to a degeneration of the organ of Corti, which occurs over many weeks, but most likely to the (measured) decrease of the endocochlear potential that is necessary for current generation through mechanosensitive channels of sensory hair cells. We could attribute this effect to a secondary loss of the K+ channel Kir4.1 in the stria vascularis. We hypothesized that this downregulation is owed to oxidative stress in the inner ear caused by a lack of glutathione transport through VRAC (10). Together with Hui Xiao, Shanghai, we discovered that VRAC can also conduct cGAMP, an important immunomodulator. cGAMP transport from virally infected to non-infected cells serves to convey a danger signal to neighboring cells, augmenting the immune response in viral infection (5). cGAMP transport through VRAC likely has roles in other settings such as cancer immunity.
In a major breakthrough, we identified, in 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. In structure/function studies, we identified pore residues and showed that it plays a role in acid-induced cell death. Together with Steve Long, New York, we obtained the Cryo-EM structure of the open channel, which displays a highly unusual metamorphosis of the transmembrane domains upon gating, and clarified the activation by protons (9). We also identified a crucial cell biological role of ASOR: It provides the Cl- conductance that is needed for the shrinkage of macropinosomes (7). This shrinkage is crucial for downstream trafficking steps. RAS-mutant tumor cells grew better when TMEM206 was disrupted because they could use albumin, taken up by macropinocytosis, more efficiently (8). Given the nearly ubiquitous expression pattern, ASOR/TMEM206 likely plays a role in endocytosis in many, if not most, tissues.
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. Our finding that VRAC is important for hearing and kidney function suggests that many more organismal functions of this channel may be found. Our work suggests that the transport of signaling molecules, in addition to volume regulation, is a biologically crucial function of VRAC.
The discovery of TMEM206 as constituting the ASOR channel, which has been known physiologically for about 10 years, but whose molecular identity had 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 topology 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 had 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 topology 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.