Periodic Reporting for period 1 - MOVINGHEART (How pathological mutations and drugs alter the intracellular movements of the cardiac sodium channel (Nav1.5).)
Período documentado: 2023-09-06 hasta 2025-09-05
The dysfunction of the cardiac sodium channel NaV1.5 which plays a key role in initiating the heart’s electrical activity, is responsible for inherited cardiac diseases and life-threatening arrythmias. This dysfunction is frequently the results of an impaired inactivation process of NaV1.5. Thus, this research project MovingHeart aims at a better understanding of the molecular mechanisms governing activation and inactivation of NaV1.5 to in the long-term improve their targeting. For that, several objectives have been settled. The first objective was to develop fluorescently engineered-NaV1.5 by using the cutting-edge technology of tandem protein trans-splicing (tPTS), enabling visualization of conformational changes during NaV1.5 gating. Then, the fluorescently engineered-NaV1.5 had to be precisely characterized by electrophysiological techniques. Finally, these characterized fluorescent sensors have been used to investigate the molecular motions of NaV1.5 during activation and inactivation and upon the application of clinically relevant drugs. Moreover, the same workflow has been used to engineer phosphomimic-containing NaV1.5 enabling us to study the impact of phosphorylation on pathogenic and splice variants and on the regulation by cytosolic protein partners.
During this project we have expressed fluorescently engineered-NaV1.5 by incorporating rhodamine-based fluorophores into its intracellular interdomain linkers using tandem protein trans-splicing (tPTS). This has enabled the development of two fluorescent NaV1.5 with fluorescent dyes incorporated into the DI-DII and DIII-DIV linkers. These constructs were successfully expressed in living oocytes, their expression was optimized, and we have characterized their biophysical properties by using two-electrode voltage clamp (TEVC). Then, the DIII-DIV fluorescent sensors were used to study the molecular motions happening during activation and inactivation by voltage-clamp fluorometry (VCF), allowing simultaneous measurement of ionic currents and conformational changes. VCF enabled us to obtain the first real-time visualization of conformational changes in the DIII-DIV linker of NaV1.5 in living cells. We precisely studied the voltage dependence, kinetic parameters, and the effects of clinically relevant drugs, cytosolic proteins, and toxins, providing unprecedented insight into NaV1.5 behavior. We then compared the behavior of pathological NaV1.5 variants with the wild-type channel using the engineered sensors. Here, the project pivoted, following the same experimental workflow, to explore the crosstalk between the phosphorylation state of a DIII-DIV linker located site with pathogenic variants and with cytosolic auxiliary proteins. This has revealed an unexpected regulatory mechanism involving phosphorylation and cytosolic protein-protein association, exceeding the original scope and offering new therapeutic perspectives.
Together, this project improves the understanding of the molecular understanding of NaV1.5 function and regulation, paving the way for opportunities of improved diagnostics and targeted treatments for cardiac arrhythmias.
Together, this project improves the understanding of the molecular understanding of NaV1.5 function and regulation, paving the way for opportunities of improved diagnostics and targeted treatments for cardiac arrhythmias.
This project has delivered results that improve our understanding of the molecular functioning of NaV1.5. By successfully engineering fluorescent NaV1.5 channels using tandem protein trans-splicing (tPTS), it has enabled real-time visualization of conformational changes in the DIII-DIV linker, which is a critical region for NaV1.5 inactivation. This represents a technological and methodological improvement, as it was the first time that tPTS was applied to incorporate fluorescent probes into the intracellular domains of an ion channel and the first time that VCF revealed intracellular molecular motions for NaV1.5 expressed in living cells. The workflow used in this project, on the tPTS-based engineering of ion channels, could be extended to other membrane proteins on further research projects, offering a versatile platform for studying dynamic molecular processes in real time.The more precise understanding of the molecular functioning and regulation of NaV1.5 will also be useful to develop better targeted therapies for cardiac diseases.