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*

• DOI: 10.1074/mcp.m114.040055
• Publication Date: 2015-02-28T03:47:25Z
• Authors: Norelle C. Wildbu...; Syed R. Ali; Wei-Chun J. Hsu; Alexander S. Shav...; Miroslav N. Nenov; Cheryl F. Lichti; Richard D. LeDuc; Ekaterina Mostovenko; Neli I. Panova-El...; Mark R. Emmett; Carol L. Nilsson; Fernanda Laezza
• Keywords: Proteome; Immunoprecipitation; Nav1.5

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