期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：33
DOI：10.1073/pnas.2025320118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
The cardiac sodium channel (Na
v1.5) is crucial for generating a regular heartbeat. It is thus not surprising that Na
v1.5 mutations have been linked to life-threatening arrhythmias. Interestingly, Na
v1.5 activity can also be altered by posttranslational modifications, such as tyrosine phosphorylation. Our combination of protein engineering and molecular modeling has revealed that the detrimental effect of a long QT3 patient mutation is only exposed when a proximal tyrosine is phosphorylated. This suggests a dynamic cross-talk between the genetic mutation and a neighboring phosphorylation, a phenomenon that could be important in other classes of proteins. Additionally, we show that phosphorylation can affect the channel’s sensitivity toward clinically relevant drugs, a finding that may prove important when devising patient-specific treatment plans.
The voltage-gated sodium channel Na
v1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Na
v1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Na
v1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Na
v1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Na
v1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Na
v1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens.
关键词：enprotein engineering;cardiac arrhythmia;pharmacology;sodium channel inactivation;personalized medicine
