Indiana University-Purdue University Indianapolis (IUPUI) / The cardiac voltage-gated sodium channels (Nav1.5) play a specific and critical role in regulating cardiac electrical activity by initiating and propagating action potentials in the heart. The association between Nav1.5 dysfunctions and generation of various types of cardiac arrhythmia disease, including long-QT3 and Brugada syndrome, is well established. Many types of post-translational modifications have been shown to regulate Nav1.5 biophysical properties, including phosphorylation, glycosylation and ubiquitination. However, our understanding about how post-translational lipid modification affects sodium channel function and cellular excitability, is still lacking. The goal of this dissertation is to characterize Nav1.5 palmitoylation, one of the most common post-translational lipid modification and its role in regulating Nav1.5 function and cardiac excitability. In our studies, three lines of biochemistry evidence were shown to confirm Nav1.5 palmitoylation in both native expression background and heterologous expression system. Moreover, palmitoylation of Nav1.5 can be bidirectionally regulated using 2-Br-palmitate and palmitic acid. Our results also demonstrated that enhanced palmitoylation in both cardiomyocytes and HEK293 cells increases sodium channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation by 2-Br-palmitiate increases closed-state channel inactivation and reduces myocyte excitability. Our computer simulation results confirmed that the observed modification in Nav1.5 gating properties by protein palmitoylation are adequate for the alterations in cardiac excitability. Mutations of potential palmitoylation sites predicted by CSS-Palm bioinformatics tool were introduced into wild-type Nav1.5 constructs using site-directed mutagenesis. Further studies revealed four cysteines (C981, C1176, C1178, C1179) as possible Nav1.5 palmitoylation sites. In particular, a mutation of one of these sites(C981) is associated with cardiac arrhythmia disease. Cysteine to phenylalanine mutation at this site largely enhances of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Therefore, C981 might be the most important site that regulates Nav1.5 palmitoylation. In summary, this dissertation research identified novel post-translational modification on Nav1.5 and revealed important details behind this process. Our data provides new insights on how post-translational lipid modification alters cardiomyocyte excitability and its potential role in arrhythmogenesis.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/12840 |
| Date | 09 December 2016 |
| Creators | Pei, Zifan |
| Contributors | Cummins, Theodore R., Oxford, Gerry S., Hudmon, Andy, Rubart-von der Lohe, Michael, Sheets, Patrick L. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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