Characterization of molecular mechanisms underlying VRAC activation

Multicellular organisms evolved mechanisms to accurately regulate cellular volume to counteract swelling or shrinkage which can in turn affect cell integrity. The Volume Regulated Anion Channel (VRAC) is ubiquitously found in all vertebrate cells and turned out to be a key player in the cell-intrinsic regulatory processes which tend to restore the original volume upon osmotic challenges. In combination with potassium export mechanisms, this is achieved by the swelling-induced increased VRAC activity allowing release of halide ions and organic osmolytes, that subsequently drive water efflux through the membrane, in the so-called regulatory volume decrease process. Difficulties encountered in the molecular identification of the channel greatly undermined biophysical characterization of VRAC and the mechanism underlying channel activation is not resolved. Several biochemical and mechanical events have been implicated in the capability of VRAC to respond to a hypotonic challenge. The most relevant among these include: a foreground role for reactive oxygen species (ROS) and channel oxidation; the involvement of phosphorylation with several kinases and signalling pathways leading to a differential modulation of the channel; and finally the sensing of low intracellular ionic strength. Molecular identification of genes encoding VRAC and recently solved cryo-EM structures of LRRC8A homomers revealed that the channel is a hexamer formed by members of the leucine rich repeat containing protein 8 (LRRC8) family, with the obligatory LRRC8A subunit, essential for channel functioning, and at least one of the other closely related members, which in total comprise five paralogues (LRRC8A-E). Little is known about the relevant stoichiometry, even if the high variability of the expression patterns depending on the cell type suggests that subunit composition is modified to meet physiological demand. In this project, I followed parallel strategies to obtain a deeper insight the relevant trigger events which underlie channel activation. By means of site-directed mutagenesis and electrophysiological analysis I elucidated mechanism of oxidation dependent VRAC modulation with the goal to understand which structural motifs are involved. Using oocytes as a heterologous expression system, I confirmed that oxidation sensitivity is subunit specific, a finding that was previously supported by the work of (Gradogna et al, 2017): LRRC8A-LRRC8E (8A-8E) heteromers are activated by oxidation, whereas 8A-8C heteromers are inhibited. A chimeric approach allowed to identify regions responsible of the divergent effect mediated by the application of oxidant component. Swapping the C-terminus leucine-rich repeat domains (LRRD) between 8E and 8C subunits resulted in a corresponding interchange of the respective oxidation sensitivity. Further specific targeted site-directed mutagenesis led to the identification of cysteines C424 and C448 unique to the 8E subunit, as those responsible of the dramatic increase of the current upon oxidative stimulation, with both 8A/8EC424F and 8A/8EC448S heteromers losing their capability to activate upon oxidant application. In a parallel set of experiments I investigated the role played by phosphorylation in swelling dependent VRAC activation. On this purpose I mutated several residues with a high prediction score to be target of kinases from the first intracellular loop (IL1) of LRRC8A. By means of a chimeric strategy I finally disclosed that mutating residue T169 to alanine (A) or serine (S), gave rise to channels that completely lost their ability to undergo activation upon hypotonic stimulation. Surprisingly, mutation T169S retained activation by low intracellular ionic strength. Overall, the results obtained in this thesis provide strong evidence in support of a role of oxidation in the subunit dependent VRAC modulation. In parallel, the identification of T169 as a determinant residue for channel functioning lays the groundwork to further investigation to disclose whether its importance might rely on phosphorylation or other unknown mechanisms.

Multicellular organisms evolved mechanisms to accurately regulate cellular volume to counteract swelling or shrinkage which can in turn affect cell integrity. The Volume Regulated Anion Channel (VRAC) is ubiquitously found in all vertebrate cells and turned out to be a key player in the cell-intrinsic regulatory processes which tend to restore the original volume upon osmotic challenges. In combination with potassium export mechanisms, this is achieved by the swelling-induced increased VRAC activity allowing release of halide ions and organic osmolytes, that subsequently drive water efflux through the membrane, in the so-called regulatory volume decrease process. Difficulties encountered in the molecular identification of the channel greatly undermined biophysical characterization of VRAC and the mechanism underlying channel activation is not resolved. Several biochemical and mechanical events have been implicated in the capability of VRAC to respond to a hypotonic challenge. The most relevant among these include: a foreground role for reactive oxygen species (ROS) and channel oxidation; the involvement of phosphorylation with several kinases and signalling pathways leading to a differential modulation of the channel; and finally the sensing of low intracellular ionic strength. Molecular identification of genes encoding VRAC and recently solved cryo-EM structures of LRRC8A homomers revealed that the channel is a hexamer formed by members of the leucine rich repeat containing protein 8 (LRRC8) family, with the obligatory LRRC8A subunit, essential for channel functioning, and at least one of the other closely related members, which in total comprise five paralogues (LRRC8A-E). Little is known about the relevant stoichiometry, even if the high variability of the expression patterns depending on the cell type suggests that subunit composition is modified to meet physiological demand. In this project, I followed parallel strategies to obtain a deeper insight the relevant trigger events which underlie channel activation. By means of site-directed mutagenesis and electrophysiological analysis I elucidated mechanism of oxidation dependent VRAC modulation with the goal to understand which structural motifs are involved. Using oocytes as a heterologous expression system, I confirmed that oxidation sensitivity is subunit specific, a finding that was previously supported by the work of (Gradogna et al, 2017): LRRC8A-LRRC8E (8A-8E) heteromers are activated by oxidation, whereas 8A-8C heteromers are inhibited. A chimeric approach allowed to identify regions responsible of the divergent effect mediated by the application of oxidant component. Swapping the C-terminus leucine-rich repeat domains (LRRD) between 8E and 8C subunits resulted in a corresponding interchange of the respective oxidation sensitivity. Further specific targeted site-directed mutagenesis led to the identification of cysteines C424 and C448 unique to the 8E subunit, as those responsible of the dramatic increase of the current upon oxidative stimulation, with both 8A/8EC424F and 8A/8EC448S heteromers losing their capability to activate upon oxidant application. In a parallel set of experiments I investigated the role played by phosphorylation in swelling dependent VRAC activation. On this purpose I mutated several residues with a high prediction score to be target of kinases from the first intracellular loop (IL1) of LRRC8A. By means of a chimeric strategy I finally disclosed that mutating residue T169 to alanine (A) or serine (S), gave rise to channels that completely lost their ability to undergo activation upon hypotonic stimulation. Surprisingly, mutation T169S retained activation by low intracellular ionic strength. Overall, the results obtained in this thesis provide strong evidence in support of a role of oxidation in the subunit dependent VRAC modulation. In parallel, the identification of T169 as a determinant residue for channel functioning lays the groundwork to further investigation to disclose whether its importance might rely on phosphorylation or other unknown mechanisms.