FGF12A confers calcium sensitivity to Nav1.5 inactivation via dynamic calmodulin recruitment

Voltage-gated Na⁺ (Nav) channels, including Nav1.5, initiate cardiac and neuronal action potentials. Regulation of Nav1.5 inactivation is linked to multiple accessory proteins that bind its C-terminal domain (CTD), including calmodulin (CaM) and intracellular fibroblast growth factors (iFGFs). Previous results demonstrate that Ca²⁺-saturated CaM binds the FGF12A N-terminus. The role of intracellular Ca²⁺ ([Ca²⁺]_i) in regulating Nav1.5 gating, either directly or via auxiliary proteins such as CaM, is controversial. We hypothesize that CaM binding to the Nav1.5 CTD and to FGF12A synergistically alters channel inactivation in a previously unobserved calcium-dependent manner. We performed fluorescence resonance energy transfer (FRET) imaging in live cells to observe the interaction between the YFP (acceptor)-tagged FGF12A and Nav1.5 α subunit to the CFP (donor)-tagged CaM. At resting [Ca²⁺]_i a twofold difference between acceptor and donor FRET efficiencies was observed, signifying that a single CaM protein is present on the Nav1.5 CTD even in presence of FGF12A. After [Ca²⁺]_i was increased the donor and acceptor FRET efficiencies equalized, indicating that a second CaM is recruited to the Nav1.5:FGF12A complex. We then compared the voltage-dependent gating kinetics of Nav1.5 with FGF12A across calcium conditions. At low [Ca²⁺]_i steady-state inactivation of Nav1.5 with FGF12A was significantly shifted towards hyperpolarized potentials. At resting and elevated [Ca²⁺]_i inactivation was dramatically altered in the depolarizing direction. These results demonstrate that the FGF12A:CaM complex enables Ca²⁺-dependent modulation of Nav1.5's voltage-dependent gating kinetics. KEY POINTS: The dependence of Nav1.5 gating on intracellular Ca²⁺ concentration remains controversial, and the underlying mechanisms of regulation are unclear. Fluorescence resonance energy transfer (FRET)-based imaging is capable of detecting changes in subunit stoichiometry in live cells. Increased intracellular Ca²⁺ leads to additional calmodulin (CaM) binding within the Nav1.5 complex as measured using FRET. Ca²⁺-dependent binding of CaM to FGF12A on the Nav1.5 complex alters inactivation gating.