Fibroblast growth factor homologous factors serve as a molecular rheostat in tuning arrhythmogenic cardiac late sodium current.

Voltage-gated sodium (Nav1.5) channels support the genesis and brisk spatial propagation of action potentials in the heart. Disruption of NaV1.5 inactivation results in a small persistent Na influx known as late Na current (I Na,L), which has emerged as a common pathogenic mechanism in both congenital and acquired cardiac arrhythmogenic syndromes. Here, using low-noise multi-channel recordings in heterologous systems, LQTS3 patient-derived iPSCs cardiomyocytes, and mouse ventricular myocytes, we demonstrate that the intracellular fibroblast growth factor homologous factors (FHF1-4) tune pathogenic I Na,L in an isoform-specific manner. This scheme suggests a complex orchestration of I Na,L in cardiomyocytes that may contribute to variable disease expressivity of NaV1.5 channelopathies. We further leverage these observations to engineer a peptide-inhibitor of I Na,L with a higher efficacy as compared to a well-established small-molecule inhibitor. Overall, these findings lend insights into molecular mechanisms underlying FHF regulation of I Na,L in pathophysiology and outline potential therapeutic avenues.

Probing ion channel macromolecular interactions using fluorescence resonance energy transfer.

Ion channels are macromolecular complexes whose functions are exquisitely tuned by interacting proteins. Fluorescence resonance energy transfer (FRET) is a powerful methodology that is adept at quantifying ion channel protein-protein interactions in living cells. For FRET experiments, the interacting partners are tagged with appropriate donor and acceptor fluorescent proteins. If the fluorescently-labeled molecules are in close proximity, then photoexcitation of the donor results in non-radiative energy transfer to the acceptor, and subsequent fluorescence emission of the acceptor. The stoichiometry of ion channel interactions and their relative binding affinities can be deduced by quantifying both the FRET efficiency and the total number of donors and acceptors in a given cell. In this chapter, we discuss general considerations for FRET analysis of biological interactions, various strategies for estimating FRET efficiencies, and detailed protocols for construction of binding curves and determination of stoichiometry. We focus on implementation of FRET assays using a flow cytometer given its amenability for high-throughput data acquisition, enhanced accessibility, and robust analysis. This versatile methodology permits mechanistic dissection of dynamic changes in ion channel interactions.