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Functional crosstalk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5

View ORCID ProfileIacopo Galleano, Hendrik Harms, Koushik Choudhury, View ORCID ProfileKeith Khoo, View ORCID ProfileLucie Delemotte, View ORCID ProfileStephan Alexander Pless
doi: https://doi.org/10.1101/2020.12.11.417683
Iacopo Galleano
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, 2100 Denmark
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Hendrik Harms
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, 2100 Denmark
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Koushik Choudhury
2Science for Life Laboratory, Department of Applied Physics, KTH Royal Institute of Technology, Solna, SE-171 65 Sweden
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Keith Khoo
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, 2100 Denmark
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Lucie Delemotte
2Science for Life Laboratory, Department of Applied Physics, KTH Royal Institute of Technology, Solna, SE-171 65 Sweden
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Stephan Alexander Pless
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, 2100 Denmark
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  • For correspondence: stephan.pless@sund.ku.dk
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Abstract

The voltage-gated sodium channel Nav1.5 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, Nav1.5 undergoes extensive post-translational 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 post-translational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semi-synthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both 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 Nav1.5. 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 Nav1.5 sensitivity towards the clinically used anti-arrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 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.

Significance statement The cardiac sodium channel (Nav1.5) is crucial for generating a regular heartbeat. It is thus not surprising that mutations in its sequence have been linked to life-threatening arrhythmias. Interestingly, Nav1.5 activity can also be altered by posttranslational modifications, such as tyrosine phosphorylation. Our combination of protein engineering and molecular modeling studies 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 crosstalk 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 towards clinically-relevant drugs, a finding that may prove important when devising patient-specific treatment plans.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Additional data and revised discussion

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 20, 2021.
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Functional crosstalk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5
Iacopo Galleano, Hendrik Harms, Koushik Choudhury, Keith Khoo, Lucie Delemotte, Stephan Alexander Pless
bioRxiv 2020.12.11.417683; doi: https://doi.org/10.1101/2020.12.11.417683
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Functional crosstalk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5
Iacopo Galleano, Hendrik Harms, Koushik Choudhury, Keith Khoo, Lucie Delemotte, Stephan Alexander Pless
bioRxiv 2020.12.11.417683; doi: https://doi.org/10.1101/2020.12.11.417683

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