Genetic ablation of the fibroblast growth factor (Fgf) 14 gene in mice or a missense mutation in Fgf14 in humans causes ataxia and cognitive deficits. These phenotypes suggest that the neuronally expressed Fgf14 gene is essential for regulating normal neuronal activity. Here, we demonstrate that FGF14 interacts directly with multiple voltage-gated Na + (Nav) channel α subunits heterologously expressed in non-neuronal cells or natively expressed in a murine neuroblastoma cell line. Functional studies reveal that these interactions result in the potent inhibition of Nav channel currents (I Na ) and in changes in the voltage dependence of channel activation and inactivation. Deletion of the unique amino terminus of the splice variant of Fgf14, Fgf14-1b, or expression of the splice variant Fgf14-1a modifies the modulatory effects on I Na , suggesting an important role for the amino terminus domain of FGF14 in the regulation of Na v channels. To investigate the function of FGF14 in neurones, we directly expressed Fgf14 in freshly isolated primary rat hippocampal neurones. In these cells, the addition of FGF14-1a-GFP or FGF14-1b-GFP increased I Na density and shifted the voltage dependence of channel activation and inactivation. In fully differentiated neurones, FGF14-1a-GFP or FGF14-1b-GFP preferentially colocalized with endogenous Nav channels at the axonal initial segment, a critical region for action potential generation. Together, these findings implicate FGF14 as a unique modulator of Nav channel activity in the CNS and provide a possible mechanism to explain the neurological phenotypes observed in mice and humans with mutations in Fgf14.

Fibroblast growth factor 14 (FGF14) belongs to the intracellular FGF homologous factor subfamily of FGF proteins (iFGFs) that are not secreted and do not activate tyrosine kinase receptors. The iFGFs, however, have been shown to interact with the pore-forming (␣) subunits of voltage-gated Na ϩ (Na v ) channels. The neurological phenotypes seen in Fgf14 Ϫ/Ϫ mice and the identification of an FGF14 missense mutation (FGF14 F145S) in a Dutch family presenting with cognitive impairment and spinocerebellar ataxia suggest links between FGF14 and neuronal functioning. Here, we demonstrate that the expression of FGF14 F145S reduces Na v ␣ subunit expression at the axon initial segment, attenuates Na v channel currents, and reduces the excitability of hippocampal neurons. In addition, and in contrast with wild-type FGF14, FGF14 F145S does not interact directly with Na v channel ␣ subunits. Rather, FGF14 F145S associates with wild-type FGF14 and disrupts the interaction between wild-type FGF14 and Na v ␣ subunits, suggesting that the mutant FGF14 F145S protein acts as a dominant negative, interfering with the interaction between wild-type FGF14 and Na v channel ␣ subunits and altering neuronal excitability.

Voltage-gated sodium channels (Na v ) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Na v ␣ subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Na v s at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Na v s.Voltage-gated sodium channels (Na v ) 3 produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. These channels are heteromeric membrane proteins composed of an ␣ subunit, which is sufficient for channel gating, and one or more auxiliary ␤ subunits, which tune voltage dependence and kinetics of channel gating (for review, see Ref.

The Intracellular Fibroblast Growth Factor (iFGF) subfamily includes four members of the structurally related FGF superfamily. Previous studies showed that the iFGFs interact directly with the pore-forming (α) subunits of voltage-gated sodium (Nav) channels and regulate the functional properties of sodium channel currents. Sequence heterogeneity among the iFGFs is thought to confer specificity to this regulation. Here, we demonstrate that the two N-terminal alternatively spliced FGF14 variants, FGF14-1a and FGF14-1b, differentially regulate currents produced by Nav1.2-and Nav1.6 channels. FGF14-1b, but not FGF14-1a, attenuates both Nav1.2 and Nav1.6 current densities. In contrast, co-expression of an FGF14 mutant, lacking the N-terminus, increased Nav1.6 current densities. In neurons, both FGF14-1a and FGF14-1b localized at the axonal initial segment, and deletion of the N-terminus abolished this localization. Thus, the FGF14 Nterminus is required for targeting and functional regulation of Nav channels, suggesting an important function for FGF14 alternative splicing in regulating neuronal excitability.

Background: Fibroblast growth factor 14 (FGF14) binds to and regulates the voltage-gated Na ϩ (Nav) channel.

Long-term depression (LTD) is a well-known form of synaptic plasticity of principal neurons in the mammalian brain. Whether such changes occur in interneurons is still controversial. CA3 hippocampal interneurons expressing Ca2+-permeable AMPA receptors exhibited LTD after tetanic stimulation of CA3 excitatory inputs. LTD was independent of NMDA receptors and required both Ca2+ influx through postsynaptic AMPA receptors and activation of presynaptic mGluR7-like receptors. These results point to the capability of interneurons to undergo plastic changes of synaptic strength through joint activation of pre- and postsynaptic glutamate receptors.

The voltage-gated Na ؉ (Nav) channel provides the basis for electrical excitability in the brain. This channel is regulated by a number of accessory proteins including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF family. In addition to forming homodimers, FGF14 binds directly to the Nav1.6 channel C-tail, regulating channel gating and expression, properties that are required for intrinsic excitability in neurons. Seeking amino acid residues with unique roles at the protein-protein interaction interface (PPI) of FGF14⅐Nav1.6, we engineered model-guided mutations of FGF14 and validated their impact on the FGF14⅐Nav1.6 complex and the FGF14: FGF14 dimer formation using a luciferase assay. Divergence was found in the ␤-9 sheet of FGF14 where an alanine (Ala) mutation of Val-160 impaired binding to Nav1.6 but had no effect on FGF14:FGF14 dimer formation. . Altogether these studies indicate that the ␤-9 sheet and the N terminus of FGF14 are well positioned targets for drug development of PPI-based allosteric modulators of Nav channels.Voltage-gated sodium (Nav) 2 channels are responsible for the initiation and propagation of the action potential in excitable cells. Nine isoforms of Nav channels (Nav1.1-Nav1.9) have been characterized functionally, and evidence for a tenth one (Na x ) has been provided (1-12). Nav channels are differentially expressed in organs, with Nav1.1, -1.2, -1.3, and -1.6 found primarily in the central and peripheral nervous systems, Nav1.4 in the adult skeletal muscle, Nav1.5 in cardiac muscle, and Nav1.7, -1.8, and -1.9 primarily in the peripheral nervous system (3,4,7,12,13). With such widespread expression, it is not surprising that numerous diseases have been ascribed to mutations of specific Nav channel isoforms (4, 14). These include the Dravet syndrome and other types of epilepsy (15-17); pain-related syndromes, such as congenital insensitivity to pain (18, 19), primary erythromelalgia (20), and paroxysmal extreme pain disorder (21, 22); and cardiac arrhythmias with congenital long QT syndrome (LQTS) type 3 (23, 24); and Brugada syndrome (25). Furthermore, SNPs and/or copy variants within Nav channel genes have been associated recently with autism (Nav1.2) (26). Nav channels blockers are currently used in combined therapy for bipolar disorder (27, 28), depression (29, 30), and schizophrenia (31), extending the role of Nav channels to virtually all brain disorders both neurological and psychiatric (14,26,32). Their centrality in the pathophysiology of so many disruptive diseases has made Nav channels key pharmacological target sites for antiepileptic, analgesic, antiarrhythmic, and psychiatric drugs (11,14,33,34). Unfortunately, current Nav channel blockers lack specificity, as they are directed against molecular domains conserved across all Nav isoforms. As such, therapies based on these medications can result in severe side effects, such as Stevens-Johnson syndrome, blood dyscrasias, and ataxia (35). Although some success has been achieved in developing more ta...

Protein-protein interactions (PPI) offer unexploited opportunities for CNS drug discovery and neurochemical probe development. Here, we present ZL181, a novel peptidomimetic targeting the PPI interface of the voltage-gated Na channel Nav1.6 and its regulatory protein fibroblast growth factor 14 (FGF14). ZL181 binds to FGF14 and inhibits its interaction with the Nav1.6 channel C-tail. In HEK-Nav1.6 expressing cells, ZL181 acts synergistically with FGF14 to suppress Nav1.6 current density and to slow kinetics of fast inactivation, but antagonizes FGF14 modulation of steady-state inactivation that is regulated by the N-terminal tail of the protein. In medium spiny neurons in the nucleus accumbens, ZL181 suppresses excitability by a mechanism that is dependent upon expression of FGF14 and is consistent with a state-dependent inhibition of FGF14. Overall, ZL181 and derivatives could lay the ground for developing allosteric modulators of Nav channels that are of interest for a broad range of CNS disorders.