Molecular Mechanism of Diltiazem Interaction with L-type Ca2+ Channels

Abstract Benzothiazepine Ca2+antagonists (such as (+)-cis-diltiazem) interact with transmembrane segments IIIS6 and IVS6 in the α1 subunit of L-type Ca2+ channels. We investigated the contribution of individual IIIS6 amino acid residues for diltiazem sensitivity by employing alanine scanning mutagenesis in a benzothiazepine-sensitive α1 subunit chimera (ALDIL) expressed inXenopus laevis oocytes. The most dramatic decrease of block by 100 μm diltiazem (ALDIL 45 ± 4.8% inhibition) during trains of 100-ms pulses (0.1 Hz, −80 mV holding potential) was found after mutation of adjacent IIIS6 residues Phe1164(21 ± 3%) and Val1165 (8.5 ± 1.4%). Diltiazem delayed current recovery by promoting a slowly recovering current component. This effect was similar in ALDIL and F1164A but largely prevented in V1165A. Both mutations slowed inactivation kinetics during a pulse. The reduced diltiazem block can therefore be explained by slowing of inactivation kinetics (F1164A and V1165A) and accelerated recovery from drug block (V1165A). The bulkier diltiazem derivative benziazem still efficiently blocked V1165A. From these functional and from additional radioligand binding studies with the dihydropyridine (+)-[3H]isradipine we propose a model in which Val1165 controls dissociation of the bound diltiazem molecule, and where bulky substituents on the basic nitrogen of diltiazem protrude toward the adjacent dihydropyridine binding domain.