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.

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)

“…1E, n ϭ 3, p ϭ 0.70 for Cluc-FGF14, p ϭ 0.89 for CD4-Nav1.6-NLuc, one-way ANOVA). These features combined with the assay sensitivity, reliability, and favorable signal-to-noise ratio characterized in previous studies (14) prompted us to conclude that LCA would be suitable for small molecule screenings.…”

“…Complex-In previous studies, we introduced the LCA (26) to detect the assembly of FGF14 with the C-terminal tail of the Nav1.6 channel in live cells (14). In this assay, the C-terminal and the N-terminal fragments of the P. pyralis luciferase are fused, respectively, to FGF14, CLuc-FGF14, and a chimera expressing CD4 fused to the Nav1.6 C-tail, CD4-Nav1.6-NLuc (Fig.…”

“…Toward this end, we have applied the LCA (14) to screen a library of kinase inhibitors against the FGF14⅐Nav1.6 C-tail complex and identified GSK3 inhibitors as hits. Through a combination of LCA validations, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that pharmacological inhibition of GSK3 reduces the assembly of the FGF14⅐Nav channel complex, modifies FGF14-dependent modulation of Na ϩ currents, and induces subcellular redistribution of the native FGF14⅐Nav channel complex in hippocampal neurons.…”

“…Signal intensity for each well was calculated as an average value of peak luminescence derived from three adjacent time points and expressed as percent control. Dose-response curves and IC 50 values were obtained by fitting the data with a nonlinear regression (14).…”

Identification of Amino Acid Residues in Fibroblast Growth Factor 14 (FGF14) Required for Structure-Function Interactions with Voltage-gated Sodium Channel Nav1.6

“…The cells were then transiently transfected or co-transfected with the appropriate plasmids using Lipofectamine 2000 (Invitrogen). For cotransfections the DNA concentration of plasmid pairs was adjusted, based on previous studies, to achieve an equal ratio of protein production (51,57,62).…”