Interaction of diltiazem with an intracellularly accessible binding site on CaV1.2

Abstract

BACKGROUND AND PURPOSEDiltiazem inhibits CaV1.2 channels and is widely used in clinical practice to 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. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α1 subunits.EXPERIMENTAL APPROACHCaV1.2 composed of α1, α2-δ and β2a subunits were expressed in tsA-201 cells and barium currents through CaV1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion).KEY RESULTSQuaternary derivative d-cis-diltiazem inhibited 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 that have previously been shown to reduce the sensitivity of CaV1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil.CONCLUSION AND IMPLICATIONSThe data show that use-dependent block of in CaV1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.