Interaction of Diltiazem with an Intracellularly Accessible Binding Site on Ca(V)1.2

Overview

Journal Br J Pharmacol

Publisher Wiley

Specialty Pharmacology

Date 2010 Oct 27

PMID 20973779

Citations 6

Authors

W Shabbir

S Beyl

E N Timin

D Schellmann

T Erker

A Hohaus

G H Hockerman

S Hering

Affiliations

Soon will be listed here.

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.

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References

Hering S, Savchenko A, Strubing C, Lakitsch M, Striessnig J . Extracellular localization of the benzothiazepine binding domain of L-type Ca2+ channels. Mol Pharmacol. 1993; 43(5):820-6. View

Hescheler J, Pelzer D, Trube G, TRAUTWEIN W . Does the organic calcium channel blocker D600 act from inside or outside on the cardiac cell membrane?. Pflugers Arch. 1982; 393(4):287-91. DOI: 10.1007/BF00581411. View

Lee K, Tsien R . Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells. Nature. 1983; 302(5911):790-4. DOI: 10.1038/302790a0. View

Zhang H, Liao P, Wang J, Yu D, Soong T . Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Ca(v)1.2 calcium channels. Br J Pharmacol. 2010; 160(7):1631-40. PMC: 2936836. DOI: 10.1111/j.1476-5381.2010.00798.x. View

Mathias R, Cohen I, Oliva C . Limitations of the whole cell patch clamp technique in the control of intracellular concentrations. Biophys J. 1990; 58(3):759-70. PMC: 1281016. DOI: 10.1016/S0006-3495(90)82418-8. View

Hohaus A, Beyl S, Kudrnac M, Berjukow S, Timin E, Marksteiner R . Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder. J Biol Chem. 2005; 280(46):38471-7. PMC: 3189691. DOI: 10.1074/jbc.M507013200. View

Dilmac N, Hilliard N, Hockerman G . Molecular determinants of Ca2+ potentiation of diltiazem block and Ca2+-dependent inactivation in the pore region of cav1.2. Mol Pharmacol. 2003; 64(2):491-501. DOI: 10.1124/mol.64.2.491. View

Kass R, Arena J, Chin S . Block of L-type calcium channels by charged dihydropyridines. Sensitivity to side of application and calcium. J Gen Physiol. 1991; 98(1):63-75. PMC: 2229044. DOI: 10.1085/jgp.98.1.63. View

Hamill O, Marty A, Neher E, Sakmann B, Sigworth F . Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981; 391(2):85-100. DOI: 10.1007/BF00656997. View

10.

Hockerman G, Dilmac N, Scheuer T, Catterall W . Molecular determinants of diltiazem block in domains IIIS6 and IVS6 of L-type Ca(2+) channels. Mol Pharmacol. 2000; 58(6):1264-70. DOI: 10.1124/mol.58.6.1264. View

11.

FLECKENSTEIN A . Cardiovascular protection by Ca antagonists. Eur Heart J. 1980; 1(Suppl B):15-21. DOI: 10.1093/eurheartj/1.suppl_2.15. View

12.

Smirnov S, Aaronson P . pH-dependent block of the L-type Ca2+ channel current by diltiazem in human mesenteric arterial myocytes. Eur J Pharmacol. 1998; 360(1):81-90. DOI: 10.1016/s0014-2999(98)00656-6. View

13.

Triggle D . Calcium channel antagonists: clinical uses--past, present and future. Biochem Pharmacol. 2007; 74(1):1-9. DOI: 10.1016/j.bcp.2007.01.016. View

14.

Striessnig J, Murphy B, Catterall W . Dihydropyridine receptor of L-type Ca2+ channels: identification of binding domains for [3H](+)-PN200-110 and [3H]azidopine within the alpha 1 subunit. Proc Natl Acad Sci U S A. 1991; 88(23):10769-73. PMC: 53012. DOI: 10.1073/pnas.88.23.10769. View

15.

Catterall W . From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron. 2000; 26(1):13-25. DOI: 10.1016/s0896-6273(00)81133-2. View

16.

Berjukow S, Gapp F, Aczel S, Sinnegger M, Mitterdorfer J, Glossmann H . Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor. J Biol Chem. 1999; 274(10):6154-60. DOI: 10.1074/jbc.274.10.6154. View

17.

Motoike H, Bodi I, Nakayama H, Schwartz A, Varadi G . A region in IVS5 of the human cardiac L-type calcium channel is required for the use-dependent block by phenylalkylamines and benzothiazepines. J Biol Chem. 1999; 274(14):9409-20. DOI: 10.1074/jbc.274.14.9409. View

18.

Tikhonov D, Zhorov B . Molecular modeling of benzothiazepine binding in the L-type calcium channel. J Biol Chem. 2008; 283(25):17594-604. DOI: 10.1074/jbc.M800141200. View

19.

Hering S, Aczel S, Grabner M, Doring F, Berjukow S, Mitterdorfer J . Transfer of high sensitivity for benzothiazepines from L-type to class A (BI) calcium channels. J Biol Chem. 1996; 271(40):24471-5. DOI: 10.1074/jbc.271.40.24471. View

20.

Berjukov S, Aczel S, Beyer B, Kimball S, Dichtl M, Hering S . Extra- and intracellular action of quaternary devapamil on muscle L-type Ca(2+)-channels. Br J Pharmacol. 1996; 119(6):1197-202. PMC: 1915905. DOI: 10.1111/j.1476-5381.1996.tb16022.x. View