Abstract
Dihydropyridines (DHPs) are L-type calcium channel (Cav1) blockers prescribed to treat several diseases including hypertension. Cav1 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 Cav1 channel crystal structure is lacking, the current model of DHP action has largely been achieved by investigating the role of candidate Cav1 residues in mediating DHP-sensitivity. To better understand DHP-block and identify additional Cav1 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 Cav1 pore-forming (α1) 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 α1C 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 Cav1 channels.
Highlights
Calcium influx into the cell can make both an immediate and a long-term impact on many cellular processes, including membrane excitability, neurotransmitter release, muscle contraction, transcription, and proliferation [1]
Our analyses revealed eight new L-type calcium channel residues that are important for DHPsensitivity and highlight the utility of using a simple animal model system for understanding how drugs interact with their targets
Through a variety of genetic approaches, we demonstrated that the major target of nemadipine is EGL-19 [27], which is the sole Cav1 channel a1 subunit encoded by the C. elegans genome [29]
Summary
Calcium influx into the cell can make both an immediate and a long-term impact on many cellular processes, including membrane excitability, neurotransmitter release, muscle contraction, transcription, and proliferation [1]. Calcium channels of the Cav family, for example, regulate current through voltage dependent mechanisms and exist in three major biophysical states: a resting (non-conducting) state, an activated (conducting) state that is triggered by a rapid change of voltage, and an inactivated (non-conducting) state that follows the conducting state but is refractory to activation. Cav channels are composed of multiple subunits that regulate calcium current through the pore-forming and voltage-sensing a1 subunit, which has four repeating domains (called I–IV), each with six transmembrane segments designated S1–S6 [2,3] (Figure 1). Alterations in Cav abundance or function result in a variety of diseases, including hypertension [4], Timothy Syndrome [5], and congenital stationary night blindness [6]. A better understanding of Cav function and how this function can be modulated by drug-like molecules may lead to better treatments of Cav1-related diseases
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