Abstract
BACKGROUND AND PURPOSE: Diltiazem inhibits Ca(V)1.2 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 Ca(V)1.2. 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 APPROACH: Ca(V)1.2 composed of α1, α2-δ and β2a subunits were expressed in tsA-201 cells and barium currents through Ca(V)1.2 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 RESULTS: Quaternary derivative d-cis-diltiazem inhibited Ca(V)1.2 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 Ca(V)1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil. CONCLUSION AND IMPLICATIONS: The data show that use-dependent block of in Ca(V)1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.