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|Title:||STRUCTURE-FUNCTION ANALYSES OF DRUG INTERACTIONS WITH L-TYPE CALCIUM CHANNELS|
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|Authors/Affiliations:||1 Phuc Pham*; 1 Taylor Dawson; 1 Stanley Lam; 1 Patrick McCamphill; 1 J. David Spafford; |
1 University of Waterloo, ON, Canada
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|Content:||Introduction: Calcium channel antagonists have a significant clinical role in the treatment of coronary artery disease or hypertension. The current world market for L-type calcium channel blockers is ~6 billion dollars annually, dominated by derivatives of 1,4 dihydroypyridine (DHP). Despite the importance of L-type channel blockers for treatment of heart disease, the structures responsible for dihydropyridine block of L-type channels are not completely known. One unique tool for exploring the residues responsible for dihydropyridine (DHP) block is a molluscan L-type calcium channel homolog, LCav1 isolated from the pond snail, Lymnaea stagnalis. The invertebrate Cav1 channel is nearly indistinguishable to the mammalian heart L-type channel in biophysical features and yet is almost insensitive to DHP (> 140 fold less sensitive), despite bearing all residues previously reported for high affinity blockade.|
Objectives: Our strategy has been to strategically swap residues from the mammalian Cav1.2 channel onto the equivalent invertebrate surrogate channel (LCav1) until we create a mammalian type high affinity binding site with the minimum number of residue changes.
Materials and Methods: Current models establish by photoaffinity labeling and mutagenesis studies delimit the DHP binding region to less than 7.5% (~160 / 2138 amino acids) of the total Cav1.2 Ca2+ channel. These identified DHP binding regions residues included Domain III, segment 5, S5-S6 cytoplasmic linker of Domain III and segments 6 of Domains III and IV (IIIS6, IVS6). Our approach has been to replace the complete DHP binding region into LCav1. And second, our lab has been working closely with structural modeller of L-type channels, Boris Zhorov (Department of Biochemistry and Biomedical Sciences, McMaster University), to define potentially critical residues responsible for the differences in DHP binding between LCav1 and Cav1.2.
Results: We have subcloned a PstI enzyme fragment from the expressible LCav1 channel as a template for mutagenesis, which can be reinsert back into LCav1 channel for functional analyses. We have swapped region containing Domain III, segment 5, S5-S6 cytoplasmic linker using unique, and opportunistic restriction sites (Bsu36I and BsaBI) flanking the insert. DIVS6 was mutated using Quikchange (Stratagene) mutagenesis. We have identified three strategically placed residues that are likely playing a critical role in the difference in DHP binding. These include: Domain III, segment 5 residues: Y1090F, Q1093N, and Domain IV, segment 6 residue: M1506I. We will be characterizing the degree of drug block using patch clamp electrophysiology of transfected wild type and chimeric channels in HEK293T human cell lines. We will pair the electrophysiology work with biochemical work. In particular, we will measure the affinity of radiolabelled drug to purified membranes containing
Conclusion: These studies will be important for correctly addressing which are the critical binding residues that are responsible for L-type channel block. This research will be critical for designing an L-type channel pharmacophore, and a new generation of L-type channel blockers with clinical applicability to a variety of cardiovascular conditions.
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