MOLECULAR MECHANISM OF DILTIAZEM INTERACTION WITH L-TYPE CA2+ CHANNELS

Benzothiazepine Ca2+ antagonists (such as (+)-cis-diltiazem) interact with transmembrane segments IIIS6 and TVS6 in the alpha(1) subunit of L-type Ca2+ channels, We investigated the contribution of individual I IIS6 amino acid residues for diltiazem sensitivity by employing alanin e scanning mutagenesis in a benzothiazepine-sensitive alpha(1) subunit chimera (AL(DIL)) expressed in Xenopus laevis oocytes, The most drama tic decrease of block by 100 mu M diltiazem (AL(DIL) 45 +/- 4.8% inhib ition) during trains of 100-ms pulses (0.1 Hz, -80 mV holding potentia l) was found after mutation of adjacent IIIS6 residues Phe(1164)(21 +/ - 3%) and Val(1165) (8.5 +/- 1.4%). Diltiazem delayed current recovery by promoting a slowly recovering current component. This effect was s imilar in AL(DIL) and F1164A but largely prevented in V1165A. Both mut ations slowed inactivation kinetics during a pulse. The reduced diltia zem block can therefore be explained by slowing of inactivation kineti cs (F1164A and V1165A) and accelerated recovery from drug block (V1165 A), The bulkier diltiazem derivative benziazem still efficiently block ed V1165A. From these functional and from additional radioligand bindi ng studies with the dihydropyridine (+)-[H-3]isradipine we propose a m odel in which Val(1165) controls dissociation of the bound diltiazem m olecule, and where bulky substituents on the basic nitrogen of diltiaz em protrude toward the adjacent dihydropyridine binding domain.