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*

Mark R Emmett,Alexander S Shavkunov,Fernanda Laezza,Miroslav N Nenov,Wei-Chun J Hsu,Richard D Leduc,Neli I Panova-Elektronova,Syed R Ali,Norelle C Wildburger,Carol L Nilsson,Cheryl F Lichti,Ekaterina Mostovenko

doi:10.1074/mcp.m114.040055

Abstract

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.

Highlights

From the ‡Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555-0617; §Neuroscience Graduate Program, Graduate School of Biomedical Sciences, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-0617; ¶UTMB Cancer Center, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-1074; ʈDepartment of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-0617; **National Center for Genome Analysis Support, Indiana University, 107 S Indiana Ave., Bloomington, Indiana, 47408
Using a target-directed affinity purification (AP) approach combined with quantitative mass spectrometry (MS), we identified proteins constituting the putative interactome of Nav1.2, one of three dominant Nav channel isoforms in the mammalian brain, from native tissue [1, 2, 4, 8]
Monoclonal antiNav1.2 antibody K69/3 targeted against the C-terminal domain (1885–2005) of Nav1.2, which has been independently tested and validated in previous proteomic studies [28], was used for affinity purification of the channel complex

Summary

Introduction

From the ‡Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555-0617; §Neuroscience Graduate Program, Graduate School of Biomedical Sciences, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-0617; ¶UTMB Cancer Center, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-1074; ʈDepartment of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-0617; **National Center for Genome Analysis Support, Indiana University, 107 S Indiana Ave., Bloomington, Indiana, 47408. The Nav channel and the protein constituents that comprise the protein–protein interaction network are all part of a macromolecular complex that modulates the spatiotemporal dynamics of neuronal input and output playing a critical role in synaptic transmission, signal integration, and neuronal plasticity. Using a target-directed AP approach combined with quantitative MS, we identified proteins constituting the putative interactome of Nav1.2, one of three dominant Nav channel isoforms in the mammalian brain, from native tissue [1, 2, 4, 8] Among these putative interactors, the fibroblast growth factor 12 (FGF12), a member of the intracellular FGF family [5, 13, 14], stood out as one of the most abundant coprecipitating proteins with ϳ30fold enrichment over other interactors. The identified channel/ protein interaction between FGF12 and Nav1.2 provides new insights for structural and functional interpretation of neuronal excitability, synaptic transmission, and plasticity in the normal and diseased brain

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