Molecular Mechanisms of Human ERG1 Channel Blockade by Ceramides

Drug-induced blockade of IKrcardiac current conducted by the hERG1 channel is associated with Long-QT syndrome and the potentially-lethal ventricular tachycardia known as Torsade-des-Pointes. The widely accepted mechanism for drug-induced LQTS considers drug binding to the water-filled internal cavity of the hERG1 pore-domain. However, recently reported modulation and blockade of IKr by lipophilic molecules have challenged this idea. Here we combined molecular dynamics (MD) simulations, mutagenesis, and electrophysiology to provide mechanistic insights into lipophilic hERG1 blockade by ceramides. The sphingolipid probe induced significant left-shift of activation voltage, faster deactivation rates, and a current blockade comparable to commonly reported hERG1 blockers. Microseconds-long MD simulations elucidated ceramide-specific binding locations at the interface between the pore and voltage-sensing domains. Finally, modelling and mutagenesis showed that membrane-localized ceramides bind to several residues known to be critical for high-affinity drug blockade of IKr. This finding highlights the allosteric mechanisms involving lipophilic access in drug-induced LQTS.