Abstract
Verapamil is a prototypical phenylalkylamine (PAA), and it was the first calcium channel blocker to be used clinically. It tonically blocks L-type channels in the inner pore with micromolar affinity, and its affinity increases at depolarized membrane potentials. In T-type calcium channels, verapamil blocks with micromolar affinity and has modestly increased affinity at depolarized potentials. We found that a related PAA, 4-desmethoxyverapamil (D888), is comparable with verapamil both in affinity and in state-dependence. Permanently charged verapamil was more effective intracellularly than neutral verapamil. Charged PAAs were able to access their binding site from both inside and outside the cell. Furthermore, membrane-impermeant [2-(trimethylammonium)ethyl]methanethiosulfonate was able to access the inner pore from outside of the cell. We examined a homology model of the T-type calcium channel to look for possible routes of drug entry. Mutation of L1825W produced a channel that was blocked significantly more slowly by charged verapamil from the outside, with an increase in apparent affinity when the drug was applied from the inside. Data suggest that T-type channels have a back pathway through which charged drugs can access the inner pore of the channel without passing through the plasma membrane.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01-HL65680, T32-HL007381].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.069492.
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ABBREVIATIONS:
- CaV3.1
- the T-type voltage-gated calcium channel isoform
- PAA
- phenylalkylamine
- MTSET
- [2-(trimethylammonium)ethyl] methanethiosulfonate
- WT
- wild type
- HEK
- human embryonic kidney
- D888
- 4-desmethoxyverapamil
- MTS
- methanethiosulfonate
- QX314
- 2-((2,6-dimethylphenyl)amino)-N,N,N-triethyl-2-oxoethanaminium
- QX222
- 2-((2,6-dimethylphenyl)amino)-N,N,N-trimethyl-2-oxoethanaminium.
- Received October 19, 2010.
- Accepted December 13, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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