(G-255) The Peripheral Interactions of the Cardiac Sodium Channel: Binding Interactions of FGF12A and CaM to the Nav 1.5 Channel

Introduction::

The cardiac sodium current carried by the voltage-gated Na⁺ channel, Nav1.5, initiates action potentials in cardiac myocytes to stimulate muscle contractions. Inherited mutations to these channels can lead to arrhythmias that include the Long QT Syndrome (LQT3) or Brugada Syndrome (BrS)^1,3. Of the known peripheral proteins to the Nav1.5 channel, Fibroblast Growth Factor (FGF12A or FHF1A) and Calmodulin (CaM) regulate steady-state inactivation and mutations of their interaction sites are linked to LQT3 and BrS². These proteins are known to interact with the C-terminal domain of Nav1.5 (Nav CTD); however, the binding mechanisms of FGF12A and CaM remain unclear. In particular, the interdependence of FGF12A and CaM for Nav CTD binding and the calcium (Ca²⁺) dependence of these interactions remains unknown. It is predicted that internal Ca²⁺ concentration may alter the relative amount of CaM bound to FGF12A and the Nav CTD. Here we observed the binding interactions between the Nav CTD, FGF12A, and CaM using Fluorescence Resonance Energy Transfer (FRET) microscopy to determine the stoichiometric relationship among these proteins. Understanding the auxiliary interactions on current inactivation will shed further light on the Nav CTD contribution to proper channel function, its regulation, and how mutations to interaction sites can lead to arrhythmias and cardiomyopathies.

Materials and Methods::

Mammalian HEK293 cells were transfected with DNA constructs tagged with either Cyan Fluorescent Protein (CFP) FRET Donor fluorophores or Yellow Fluorescent Protein (YFP) FRET Acceptor fluorophores. The relevant DNA constructs for this experiment were Nav CTD-YFP, and Nav CTD-YFP with IQ/AA mutation at the CaM binding domain (CaMBD), CaM-CFP, FGF12A-YFP, FGF12A-CFP, and FGF12A-YFP with a deleted CaM binding site, and Venus-YPF (for spurious binding fluorescence). Cells were then imaged in 1X HBSS buffer with FRET microscopy to observe the binding interactions of these three proteins as Donor/Acceptor pairs for experimental conditions (Figure 1A). FRET Donor-only conditions contain only CFP-tagged constructs, while FRET Acceptor-only conditions contain only YFP-tagged constructs. Spurious FRET conditions contain a CFP-tagged construct and Venus-YFP to test for background FRET fluorescence. The Ca²⁺ dependence of interactions is tested using serial dilutions of Ca²⁺ ionomycin solution at 0 mM, 1 mM, 5 mM, 10 mM, and 20 mM for changes in FRET fluorescence.

Results, Conclusions, and Discussions::

The experimental pairing of FGF12A-CFP and CaM-YFP showed a Ca²⁺-dependent interaction (Figure 1B); increasing extracellular Ca²⁺ concentration resulted in a higher FRET signal from FGF12A, indicative of an increase in binding of CaM and FGF12A. Therefore, the FGF12A-CaM interaction was observed to be Ca²⁺-dependent and would be expected to change depending on physiological Ca²⁺ levels. When comparing Nav CTD to mutated Nav CTD IQ/AA FRET, altered CaMBD (IQ/AA) binding sites showed a significant difference in FGF12A binding. Furthermore, CaM binding site deletion on FGF12A also showed a significant difference in Nav CTD binding. Nav CTD and FGF12A binding interaction is CaMBD/CaM binding site dependent. When testing Nav CTD and CaM interactions in a high Ca²⁺ condition (10 mM), there was no significant increase in apparent FRET. Changes in extracellular Ca²⁺ via an ionomycin solution do not significantly interfere with Nav CTD and CaM binding in vitro conditions. Elucidating these binding interactions will help us to understand better the effects of varying physiological Ca²⁺ concentrations on the inactivation of Nav1.5 current and direct us in further exploration of CaM and FGF12A peripheral binding mechanisms in Nav1.5 channel malfunction in arrhythmias and cardiomyopathies.

Acknowledgements (Optional): : AHA (Grant # GR0023814), WashU CardS Fellowship Program

References (Optional): :

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² Abriel H. (2010). Cardiac sodium channel Na(v)1.5 and interacting proteins: Physiology and pathophysiology. Journal of molecular and cellular cardiology, 48(1), 2–11. https://doi.org/10.1016/j.yjmcc.2009.08.025

³ Garcia-Elias, A., & Benito, B. (2018). Ion Channel Disorders and Sudden Cardiac Death. International journal of molecular sciences, 19(3), 692. https://doi.org/10.3390/ijms19030692