A genetic screen for dihydropyridine (DHP)-resistant worms reveals new residues required for DHP-blockage of mammalian calcium channels.
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.
Kwok
Trevor C Y
TC
Department of Molecular Genetics, The Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
Hui
Kwokyin
K
Kostelecki
Wojciech
W
Ricker
Nicole
N
Selman
Guillermo
G
Feng
Zhong-Ping
ZP
Roy
Peter John
PJ
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017124:Conserved Sequence; D004095:Dihydropyridines / Q000378:metabolism / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D004351:Drug Resistance; D004594:Electrophysiology; D023421:Models, Animal; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011110:Polymorphism, Genetic; D051381:Rats; D016415:Sequence Alignment
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; C038806:1,4-dihydropyridine