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.
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; ncwildbu@utmb.edu felaezza@utmb.edu.
Norelle C
NC
Wildburger
From the ‡Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555-0617;
Syed R
SR
Ali
‖Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-0617;
Wei-Chun J
WC
Hsu
From the ‡Department of Pharmacology and Toxicology, 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;
Alexander S
AS
Shavkunov
From the ‡Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555-0617;
Miroslav N
MN
Nenov
From the ‡Department of Pharmacology and Toxicology, 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;
Cheryl F
CF
Lichti
**National Center for Genome Analysis Support, Indiana University, 107 S Indiana Ave., Bloomington, Indiana, 47408.
Richard D
RD
LeDuc
From the ‡Department of Pharmacology and Toxicology, 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;
Ekaterina
E
Mostovenko
From the ‡Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555-0617;
Neli I
NI
Panova-Elektronova
¶UTMB Cancer Center, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-1074; ‖Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas, 77555-0617;
Mark R
MR
Emmett
From the ‡Department of Pharmacology and Toxicology, 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;
Carol L
CL
Nilsson
From the ‡Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555-0617; ncwildbu@utmb.edu felaezza@utmb.edu.
Fernanda
F
Laezza
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane; D005346:Fibroblast Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D058977:Molecular Sequence Annotation; D062551:NAV1.2 Voltage-Gated Sodium Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009473:Neuronal Plasticity; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D062551:NAV1.2 Voltage-Gated Sodium Channel; D020543:Proteome; D011994:Recombinant Proteins; C000604599:fibroblast growth factor 12, rat; D005346:Fibroblast Growth Factors; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2