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

Richard L Kraus,Steffen Hering,Manfred Grabner,Dominique Ostler,Jörg Striessnig

doi:10.1074/jbc.273.42.27205

Abstract

Benzothiazepine Ca2+ antagonists (such as (+)-cis-diltiazem) interact with transmembrane segments IIIS6 and IVS6 in the alpha1 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 alpha1 subunit chimera (ALDIL) expressed in Xenopus laevis oocytes. The most dramatic decrease of block by 100 microM 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.

Highlights

The most dramatic decrease of block by 100 ␮M 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%)
Alanine Scanning Mutagenesis of Segment IIIS6 —We have previously shown that the photoreactive diltiazem analogue [3H]benziazem photoaffinity labels transmembrane segments IVS6 and IIIS6, suggesting that both segments are in close contact with the bound drug molecule and contain high affinity BTZ-binding determinants [8]
Mutagenesis was carried out in a diltiazem and PAA-sensitive ␣1 subunit chimera ALDIL in which the segments corresponding to the photolabeled regions and the respective S5-S6 linkers consisted of L-type sequence, whereas the remaining sequence consisted of ␣1A sequence (Fig. 1, see “Experimental Procedures”)

Summary

Introduction

The most dramatic decrease of block by 100 ␮M 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%). Taken together our data reveal that mutation of two conserved residues, Phe1164 and Val1165, on the cytoplasmic end of transmembrane segment IIIS6, change activation and inactivation gating and are major determinants for use-dependent diltiazem block.

Results

Conclusion