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PLoS Genet. 2008 May 9;4(5):e1000067. doi: 10.1371/journal.pgen.1000067.

A genetic screen for dihydropyridine (DHP)-resistant worms reveals new residues required for DHP-blockage of mammalian calcium channels.

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1
Department of Molecular Genetics, The Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.

Erratum in

  • PLoS Genet. 2009 Feb;5(2). doi: 10.1371/annotation/c38f7645-c9d8-4371-b363-5a9099c75e83.

Abstract

Dihydropyridines (DHPs) are L-type calcium channel (Ca(v)1) blockers prescribed to treat several diseases including hypertension. Ca(v)1 channels normally exist in three states: a resting closed state, an open state that is triggered by membrane depolarization, followed by a non-conducting inactivated state that is triggered by the influx of calcium ions, and a rapid change in voltage. DHP binding is thought to alter the conformation of the channel, possibly by engaging a mechanism similar to voltage dependent inactivation, and locking a calcium ion in the pore, thereby blocking channel conductance. As a Ca(v)1 channel crystal structure is lacking, the current model of DHP action has largely been achieved by investigating the role of candidate Ca(v)1 residues in mediating DHP-sensitivity. To better understand DHP-block and identify additional Ca(v)1 residues important for DHP-sensitivity, we screened 440,000 randomly mutated Caenorhabditis elegans genomes for worms resistant to DHP-induced growth defects. We identified 30 missense mutations in the worm Ca(v)1 pore-forming (alpha(1)) subunit, including eleven in conserved residues known to be necessary for DHP-binding. The remaining polymorphisms are in eight conserved residues not previously associated with DHP-sensitivity. Intriguingly, all of the worm mutants that we analyzed phenotypically exhibited increased channel activity. We also created orthologous mutations in the rat alpha(1C) subunit and examined the DHP-block of current through the mutant channels in culture. Six of the seven mutant channels examined either decreased the DHP-sensitivity of the channel and/or exhibited significant residual current at DHP concentrations sufficient to block wild-type channels. Our results further support the idea that DHP-block is intimately associated with voltage dependent inactivation and underscores the utility of C. elegans as a screening tool to identify residues important for DHP interaction with mammalian Ca(v)1 channels.

PMID:
18464914
PMCID:
PMC2362100
DOI:
10.1371/journal.pgen.1000067
[Indexed for MEDLINE]
Free PMC Article
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