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.
