Interaction of Diltiazem with an Intracellularly Accessible Binding Site on Ca V1.2

Abstract

Diltiazem inhibits CaV1.2 channels and is widely used in clinical practiceto treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α1 subunit of CaV1.2. Here we elucidate the access path of Diltiazem to its binding site making use of membrane impermeable quaternary derivative qDil and mutant α1 subunits. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion). qDil inhibits CaV1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59±4% at 300 µM) and induced only a low level of tonic (non use-dependent) block (16±2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 reduced sensitivity to intracellularly applied qDil.Our study demonstrates intracellular access of quaternary diltiazem and its interaction with previously identified determinants of BTZ binding pocket. Recovery from block by diltiazem was found to be voltage independent, which is in contrast with the pronounced voltage dependent recovery from block by phenylalkylamines.