RESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos/farmacologia , Hipocampo/efeitos dos fármacos , Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Animais , Fatores de Crescimento de Fibroblastos/genética , Células HEK293 , Humanos , Camundongos Knockout , Peptidomiméticos/farmacologiaRESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteínas/metabolismo , Fatores de Crescimento de Fibroblastos/química , Humanos , Proteínas Luminescentes/química , Microscopia de Fluorescência , Canal de Sódio Disparado por Voltagem NAV1.5/química , Proteínas/químicaRESUMO
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.
Assuntos
Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Neurônios/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/farmacologia , Membrana Celular , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Células HEK293 , Humanos , Imunoprecipitação , Anotação de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.2/química , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Plasticidade Neuronal , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ligação Proteica , Proteoma/genética , Proteoma/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.