Interaction of diltiazem with an intracellularly accessible binding site on Ca(V)1.2.
OBJECTIVE
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.


METHODS
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).


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.


CONCLUSIONS
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.
Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse, Vienna, Austria.
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Shabbir
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Beyl
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Timin
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Schellmann
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Erker
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Hohaus
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Hockerman
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Hering
D000595:Amino Acid Sequence; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000378:metabolism* / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability; D004110:Diltiazem / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D050505:Mutant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D004110:Diltiazem