2.1 Determine if sodium channel polymorphisms can modify the effects of LQT3 and BrS mutations.
First we will determine if the H558R polymorphism can restore the function of other LQT3 and BrS mutations. Different mutations that have been identified in patients with LQTS and BrS will be reproduced on the cardiac sodium channel by using site-directed mutagenesis. The functional expression experiments will be done by co-expressing these mutations with the H558R polymorphism in HEK-293 cells. Patch-clamping will be used to characterize the biophysical properties of the sodium channel. We will study if the H558R polymorphism can restore the function of different type of mutations (trafficking, folding or functional mutations). Then we will investigate if other sodium channel polymorphisms can also rescue defective sodium channels. We will co-express these polymorphisms with different LQTS and BrS mutations to determine if they can also restore their function and therefore test our hypothesis that polymorphisms are important to explain incomplete penetrance in both LQTS and BrS.
2.2 Investigate the mechanisms by which sodium channel polymorphisms modulate the functional defects of mutated sodium channel.
We have previously demonstrated that the P2006A mutation alters the stability of the inactivation phase producing a persistent non-inactivating sodium current. Interestingly, in presence of the H558R polymorphism, the mutation produced sodium currents that behaved like wild type (WT). Additionally, we have previously demonstrated that the H558R polymorphism can restore trafficking of the R282H BrS mutation. Therefore, using Fluorescence Resonance Energy Transfer (FRET) combined with patch clamping, and pull down assays we will investigate the mechanisms by which the H558R polymorphism restores the functional defects of the mutated sodium channel.
2.3 Develop gene therapy approaches, using genetic polymorphisms, to rescue dysfunctional channels.
We will create peptides containing the polymorphisms that were shown to restore the function of defective sodium channels in vitro and use them to develop a gene therapy approach to rescue these dysfunctional ion channels.
