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

doi:10.1074/jbc.273.42.27205

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

From the Institut für Biochemische Pharmakologie, A-6020 Innsbruck, Austria

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 (AL_DIL) expressed inXenopus laevis oocytes.

The most dramatic decrease of block by 100 μm diltiazem (AL_DIL 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 AL_DIL 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.

Footnotes

↵* This work was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung (P12641-MED to J. S., P12649-MED to S. H.), the Österreichische Nationalbank (to J. S.), and the Austrian Academy of Sciences (to R. L. K.). BZDC-NHS and benzazepines were generously provided by Dr. D. Marecak (supported by National Institutes of Health Grant NS 29632 to G. D. Prestwich) and S. D. Kimball (Bristol-Myers Squibb), respectively.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Institut für Biochemische Pharmakologie, Peter-Mayrstraße 1, A-6020 Innsbruck, Austria. Tel.: 43-512-507-3164; Fax: 43-512-588627; E-mail:joerg.striessnig{at}uibk.ac.at.
Abbreviations:

diltiazem

(+)-cis-diltiazem

Bz-BAZ

(3R,4S)-cis-1-[2-[3-(benzoylamino)propyl]amino-ethyl]-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromethyl)-2H-1-benzazepine-2-one

BTZ

benzothiazepine

DHP

dihydropyridine

DMBODIPY-BAZ

(3R,4S)-cis-1-[2-[[3-[[3-[4,4-difluoro-3a,4-dihydro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]proprionyl]amino]propyl]amino]ethyl]-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromethyl)-2H-1-benzazepine-2-one

I_Ba

barium inward current

PAA

phenylalkylamine.
- Received June 16, 1998.