Finding modulation sites of the Volume regulating anion channel through a synthetic ligand, a potential supplementary drug for the chemotherapeutics cisplatin and carboplatin

The Volume Regulating Anion Channel (VRAC) is a ubiquitous transmembrane channel involved in regulatory volume decrease to counter osmotic swelling. It is regulated by a protein binding the intracellular LRRD. Interestingly, it has been discovered that the VRAC has been involved in the uptake of anticancer chemotherapeutics cisplatin and carboplatin. An activating synthetic ligand binding the regulating LRRD was developed in 2021. This work set out to find the amino acids on the synthetic ligand building salt bridges with the LRRD. As salt bridges are very strong interactions, altering the amino acids at these sites could alter binding strength. Thus, we call them modulation sites. To use the synthetic ligand to increase the specificity of chemotherapeutics like cisplatin and carboplatin modulation of binding strength is necessary. From the crystal structure of the synthetic ligand binding the LRRD, residues were identified, which possibly engaged in a salt bridge. To test whether they could be used for binding strength modulation, double mutants were engineered, where two of those residues were replaced by an alanine each, to see if binding would be disrupted when these salt bridges would be abolished. Size-exclusion chromatography and SDS-Page confirmed disruption of binding and these amino acids as modulation sites.