Abstract
BACKGROUND AND PURPOSE: The synthetic compound efsevin was recently identified to suppress arrhythmogenesis in models of cardiac arrhythmia, making it a promising candidate for antiarrhythmic therapy. Its activity was shown to be dependent on the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. Here, we investigated the molecular mechanism of the efsevin-VDAC2 interaction. EXPERIMENTAL APPROACH: To evaluate the functional interaction of efsevin and VDAC2, we measured currents through recombinant VDAC2 in planar lipid bilayers. Using molecular ligand-protein docking and mutational analysis, we identified the efsevin binding site on VDAC2. Finally, physiological consequences of the efsevin-induced modulation of VDAC2 were analysed in HL-1 cardiomyocytes. KEY RESULTS: In lipid bilayers, efsevin reduced VDAC2 conductance and shifted the channel's open probability towards less anion-selective closed states. Efsevin binds to a binding pocket formed by the inner channel wall and the pore-lining N-terminal α-helix. Exchange of amino acids N207, K236 and N238 within this pocket for alanines abolished the channel's efsevin-responsiveness. Upon heterologous expression in HL-1 cardiomyocytes, both channels, wild-type VDAC2 and the efsevin-insensitive VDAC2(AAA) restored mitochondrial Ca(2+) uptake, but only wild-type VDAC2 was sensitive to efsevin. CONCLUSION AND IMPLICATIONS: In summary, our data indicate a direct interaction of efsevin with VDAC2 inside the channel pore that leads to modified gating and results in enhanced SR-mitochondria Ca(2+) transfer. This study sheds new light on the function of VDAC2 and provides a basis for structure-aided chemical optimization of efsevin.