The volume-regulated anion channel (VRAC) is a crucial part of the regulatory volume decrease in vertebrate cells with various other proposed roles as, for example, in apoptosis, differentiation, and auto/paracrine signaling. Their modular architecture with differing biophysics and potential functions, as well as the lack of specific inhibitors, led to VRAC’s activation mechanism being elusive until today. Furthermore, current methods to study VRACs fail to account for the diversity of these channels. In this thesis, the movement of fluorophores at the C-termini of the VRAC forming protein family LRRC8 upon activation was proven by Förster-resonance energy transfer (FRET) microscopy. Patch-clamp fluorometry validated that FRET variations reflect channel gating. FRET microscopy of fluorescently labeled LRRC8 proteins was, therefore, exploited as a new optical sensor for VRAC activity. The optical sensor revealed isotonic activation of VRAC by the diacylglycerol analog phorbol-12-myristate-13-acetate (PMA), an effect abolished by the whole-cell configuration. VRACs trapped on endomembranes by the reverse dimerization system cannot be activated by decreased intracellular ionic strength. Furthermore, are VRACs rendered active under isotonic conditions by inhibition of the diacylglycerol kinase (DAGK) by dioctanoylglycol. Activation of VRAC by hypotonicity was impaired by the PKD inhibitor CRT 0066101. Concludingly, ionic strength is not the critical regulator of VRAC activity in living cells. Instead, they are activated by diacylglycerol mediated PKD recruitment. Electrophysiological approaches might obscure the effects of drugs and protein manipulations, as seen for PMA. The here established FRET-based optical sensor opens new possibilities to detect activation of VRACs in living cells in a non-invasive, subunit-specific manner.
