Voltage-gated sodium channels (Na_vs) are tightly regulated by multiple conserved auxiliary proteins, including the four fibroblast growth factor homologous factors (FGFs), which bind the Na_v EF-hand like domain (EFL), and calmodulin (CaM), a multifunctional messenger protein that binds the Na_V IQ motif. The EFL domain and IQ motif are contiguous regions of Na_V cytosolic C-terminal domains (CTD), placing CaM and FGF in close proximity. However, whether the FGFs and CaM act independently, directly associate, or operate through allosteric interactions to regulate channel function is unknown. Titrations monitored by steady-state fluorescence spectroscopy, structural studies with solution NMR, and computational modeling demonstrated for the first time that both domains of (Ca²⁺)₄-CaM (but not apo CaM) directly bind two sites in the N-terminal domain (NTD) of A-type FGF splice variants (FGF11A, FGF12A, FGF13A, and FGF14A) with high affinity. The weaker of the (Ca²⁺)₄-CaM-binding sites was known via electrophysiology to have a role in long-term inactivation of the channel but not known to bind CaM. FGF12A binding to a complex of CaM associated with a fragment of the Na_V1.2 CTD increased the Ca²⁺-binding affinity of both CaM domains, consistent with (Ca²⁺)₄-CaM interacting preferentially with its higher-affinity site in the FGF12A NTD. Thus, A-type FGFs can compete with Na_V IQ motifs for (Ca²⁺)₄-CaM. During spikes in the cytosolic Ca²⁺ concentration that accompany an action potential, CaM may translocate from the Na_V IQ motif to the FGF NTD, or the A-type FGF NTD may recruit a second molecule of CaM to the channel.

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