title: Bioluminescence methodology for the detection of protein-protein interactions within the voltage-gated sodium channel macromolecular complex.
abstract: Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Shavkunov
authors.forename: Neli
authors.initials: N
authors.lastname: Panova
authors.forename: Anesh
authors.initials: A
authors.lastname: Prasai
authors.forename: Ron
authors.initials: R
authors.lastname: Veselenak
authors.forename: Nigel
authors.initials: N
authors.lastname: Bourne
authors.forename: Svetla
authors.initials: S
authors.lastname: Stoilova-McPhie
authors.forename: Fernanda
authors.initials: F
authors.lastname: Laezza
mesh_terms: D000465:Algorithms; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival; D004247:DNA / Q000235:genetics; D005346:Fibroblast Growth Factors / Q000235:genetics; D048888:Fireflies / Q000737:chemistry; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents; D015640:Ion Channel Gating / Q000502:physiology*; D008156:Luciferases / Q000737:chemistry; D049449:Luminescence; D008164:Luminescent Proteins / Q000032:analysis*; D008958:Models, Molecular; D062557:NAV1.6 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D015222:Sodium Channels / Q000737:chemistry / Q000502:physiology*
chemical_list: D000596:Amino Acids; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D062557:NAV1.6 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; C100681:SCN8A protein, human; D015222:Sodium Channels; C112971:fibroblast growth factor 14; D005346:Fibroblast Growth Factors; D004247:DNA; D008156:Luciferases