Functional Modulation of Voltage-Gated Sodium Channels by a FGF14-Based Peptidomimetic.

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.

Parallel fiber to Purkinje cell synaptic impairment in a mouse model of spinocerebellar ataxia type 27.

Genetically inherited mutations in the fibroblast growth factor 14 (FGF14) gene lead to spinocerebellar ataxia type 27 (SCA27), an autosomal dominant disorder characterized by heterogeneous motor and cognitive impairments. Consistently, genetic deletion of Fgf14 in Fgf14 (-/-) mice recapitulates salient features of the SCA27 human disease. In vitro molecular studies in cultured neurons indicate that the FGF14 (F145S) SCA27 allele acts as a dominant negative mutant suppressing the FGF14 wild type function and resulting in inhibition of voltage-gated Na(+) and Ca(2+) channels. To gain insights in the cerebellar deficits in the animal model of the human disease, we applied whole-cell voltage-clamp in the acute cerebellar slice preparation to examine the properties of parallel fibers (PF) to Purkinje neuron synapses in Fgf14 (-/-) mice and wild type littermates. We found that the AMPA receptor-mediated excitatory postsynaptic currents evoked by PF stimulation (PF-EPSCs) were significantly reduced in Fgf14 (-/-) animals, while short-term plasticity, measured as paired-pulse facilitation (PPF), was enhanced. Measuring Sr(2+)-induced release of quanta from stimulated synapses, we found that the size of the PF-EPSCs was unchanged, ruling out a postsynaptic deficit. This phenotype was corroborated by decreased expression of VGLUT1, a specific presynaptic marker at PF-Purkinje neuron synapses. We next examined the mGluR1 receptor-induced response (mGluR1-EPSC) that under normal conditions requires a gradual build-up of glutamate concentration in the synaptic cleft, and found no changes in these responses in Fgf14 (-/-) mice. These results provide evidence of a critical role of FGF14 in maintaining presynaptic function at PF-Purkinje neuron synapses highlighting critical target mechanisms to recapitulate the complexity of the SCA27 disease.

Split-luciferase complementation assay to detect channel-protein interactions in live cells.

The understanding of ion channel function continues to be a significant driver in molecular pharmacology. In this field of study, protein-protein interactions are emerging as fundamental molecular determinants of ion channel function and as such are becoming an attractive source of highly specific targets for drug development. The investigation of ion channel macromolecular complexes, however, still relies on conventional methods that are usually technically challenging and time-consuming, significantly hampering our ability to identify, characterize and modify ion channel function through targeted molecular approaches. As a response to the urgent need of developing rapid and albeit accurate technologies to survey ion channel molecular complexes, we describe a new application of the split-luciferase complementation assay to study the interaction of the voltage-gated Na + channel with the intracellular fibroblast growth factor 14 and its dynamic regulation in live cells. We envision that the flexibility and accessibility of this assay will have a broad impact in the ion channel field complementing structural and functional studies, enabling the interrogation of protein-channel dynamic interactions in complex cellular contexts and laying the basis for new frameworks in drug discovery campaigns.

Quantitative proteomics reveals protein-protein interactions with fibroblast growth factor 12 as a component of the voltage-gated sodium channel 1.2 (nav1.2) macromolecular complex in Mammalian brain.

Voltage-gated sodium channels (Nav1.1-Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein-protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.

The fibroblast growth factor 14·voltage-gated sodium channel complex is a new target of glycogen synthase kinase 3 (GSK3).

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na(+) (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na(+) currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.