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

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

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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 patchclamp 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.

Original languageEnglish (US)
Pages (from-to)1288-1300
Number of pages13
JournalMolecular and Cellular Proteomics
Volume14
Issue number5
DOIs
StatePublished - May 1 2015

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NAV1.2 Voltage-Gated Sodium Channel
Voltage-Gated Sodium Channels
Macromolecular Substances
Fibroblast Growth Factors
Proteomics
Brain
Proteins
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Neuronal Plasticity
NAV1.9 Voltage-Gated Sodium Channel
Proteome
NAV1.1 Voltage-Gated Sodium Channel
Action Potentials
Neurons
Purification
Plasticity
Electrophysiology
Multiprotein Complexes
Confocal microscopy
Brain Diseases

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

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

In: Molecular and Cellular Proteomics, Vol. 14, No. 5, 01.05.2015, p. 1288-1300.

Research output: Contribution to journalArticle

Wildburger, Norelle C. ; Ali, Syed R. ; Hsu, Wei Chun J ; Shavkunov, Alexander S. ; Nenov, Miroslav N. ; Lichti, Cheryl F. ; LeDuc, Richard D. ; Mostovenko, Ekaterina ; Panova-Elektronova, Neli I. ; Emmett, Mark ; Nilsson, Carol L. ; Laezza, Fernanda. / 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. In: Molecular and Cellular Proteomics. 2015 ; Vol. 14, No. 5. pp. 1288-1300.
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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 patchclamp 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.",
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AU - Ali, Syed R.

AU - Hsu, Wei Chun J

AU - Shavkunov, Alexander S.

AU - Nenov, Miroslav N.

AU - Lichti, Cheryl F.

AU - LeDuc, Richard D.

AU - Mostovenko, Ekaterina

AU - Panova-Elektronova, Neli I.

AU - Emmett, Mark

AU - Nilsson, Carol L.

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