JAK2 regulates Nav1.6 channel function via FGF14Y158 phosphorylation

Wadsworth, Paul A.; Singh, A.K.; Nguyen, Nghi; Dvorak, Nolan M.; Tapia, Cynthia M.; Russell, William K.; Stephan, Clifford; Laezza, Fernanda

doi:10.1016/j.bbamcr.2020.118786

Cited by 17 publications

(53 citation statements)

References 70 publications

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“…The CLuc-FGF14, CD4-Nav1.6-NLuc constructs and the pcDNA3.1 vector (Invitrogen, Carlsbad, CA) were engineered and characterized as previously described (Goetz et al, 2009;Shavkunov et al, 2012;Shavkunov et al, 2013;Shavkunov et al, 2015;Ali et al, 2016Ali et al, , 2018Wadsworth et al, 2019Wadsworth et al, , 2020Singh et al, 2020). The plasmid pGL3 expressing fulllength Firefly (Photinus pyralis) luciferase was a gift from P. Sarkar (Department of Neurology, UTMB).…”

Section: Plasmid Constructsmentioning

confidence: 99%

“…For transient transfections, the Lipofectamine 2000 protocol was followed (Invitrogen, Waltham, MA, United States), and the amount of cDNA used was 1 µg for each. For whole-cell, patch-clamp recordings HEK293-Nav1.6 cells were washed and replated at very low density prior to incubating the cells with peptides for recordings (Ali et al, 2016(Ali et al, , 2018Wadsworth et al, 2019Wadsworth et al, , 2020Singh et al, 2020).…”

Section: Cell Culturementioning

confidence: 99%

“…LCA readings were performed at 2 min intervals for 20-30 min, integration time 0.5 s, while cells were maintained at 37°C throughout the measurements. Detailed LCA method can be found in previous studies (Shavkunov et al, 2012;Ali et al, 2014Ali et al, , 2016Ali et al, , 2018Hsu et al, 2015;Wadsworth et al, 2019Wadsworth et al, , 2020Singh et al, 2020).…”

Section: In Cell Split Luciferase Assaymentioning

confidence: 99%

“…To evaluate the hypothesis that PLEV and/or EYYV could modulate FGF14:Nav1.6 complex assembly, an in-cell assay was used with a HEK293 cell line (hereafter coded as "Clone V" cells) stably expressing both CLuc-FGF14 and CD4-Nav1.6-NLuc (Shavkunov et al, 2012;Shavkunov et al, 2013;Shavkunov et al, 2015;Ali et al, 2016Ali et al, , 2018Wadsworth et al, 2019Wadsworth et al, , 2020Dvorak et al, 2020Dvorak et al, , 2021Singh et al, 2020;Wang et al, 2020). In this assay, upon binding of FGF14 to the Nav1.6 C-terminal tail, there is reconstitution of the NLuc and CLuc halves of the luciferase enzyme, which produces luminescence in the presence of substrate D-luciferin (Figures 2A,D).…”

Section: In-cell Testing Of Plev and Eyyv Using The Split Luciferase Complementation Assaymentioning

confidence: 99%

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Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

Singh

Dvorak

Tapia

et al. 2021

Front. Mol. Biosci.

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The voltage-gated Na+ (Nav) channel is a primary molecular determinant of the initiation and propagation of the action potential. Despite the central role of the pore-forming α subunit in conferring this functionality, protein:protein interactions (PPI) between the α subunit and auxiliary proteins are necessary for the full physiological activity of Nav channels. In the central nervous system (CNS), one such PPI occurs between the C-terminal domain of the Nav1.6 channel and fibroblast growth factor 14 (FGF14). Given the primacy of this PPI in regulating the excitability of neurons in clinically relevant brain regions, peptides targeting the FGF14:Nav1.6 PPI interface could be of pre-clinical value. In this work, we pharmacologically evaluated peptides derived from FGF14 that correspond to residues that are at FGF14’s PPI interface with the CTD of Nav1.6. These peptides, Pro-Leu-Glu-Val (PLEV) and Glu-Tyr-Tyr-Val (EYYV), which correspond to residues of the β12 sheet and β8-β9 loop of FGF14, respectively, were shown to inhibit FGF14:Nav1.6 complex assembly. In functional studies using whole-cell patch-clamp electrophysiology, PLEV and EYYV were shown to confer differential modulation of Nav1.6-mediated currents through mechanisms dependent upon the presence of FGF14. Crucially, these FGF14-dependent effects of PLEV and EYYV on Nav1.6-mediated currents were further shown to be dependent on the N-terminal domain of FGF14. Overall, these data suggest that the PLEV and EYYV peptides represent scaffolds to interrogate the Nav1.6 channel macromolecular complex in an effort to develop targeted pharmacological modulators.

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Section: Plasmid Constructsmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: In Cell Split Luciferase Assaymentioning

confidence: 99%

Section: In-cell Testing Of Plev and Eyyv Using The Split Luciferase Complementation Assaymentioning

confidence: 99%

See 2 more Smart Citations

Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

Singh

Dvorak

Tapia

et al. 2021

Front. Mol. Biosci.

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“…We complemented our imaging studies with electrophysiology and found that inhibition of AKT in primary hippocampal neuronal cultures exerts the strongest effect on neuronal excitability as indicated by increased firing frequency and decreased action potential current threshold ( Figures 5B–D ). The lack of changes in passive properties and input resistance ( Table 2 ) point toward increasing in Nav channel availability ( Ali et al, 2018 ; Scala et al, 2018 ; Wadsworth et al, 2020 ) though this does not rule out alterations in other ion channels, which will need further investigation. Understanding the concomitance of the two phenotypes produced upon inhibition of AKT, namely sensitivity of βIV spectrin and increased neuronal excitability, might provide new hypotheses toward understanding AIS plasticity and related neuropsychiatric and neurodegenerative disorders.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of AKT Signaling Alters βIV Spectrin Distribution at the AIS and Increases Neuronal Excitability

Hsu

Kayasandık

et al. 2021

Front. Mol. Neurosci.

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The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, βIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing βIV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on βIV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in βIV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders.

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Nav1.6 promotes the progression of human follicular thyroid carcinoma cells via JAK-STAT signaling pathway

Liu

Fan

et al. 2022

Pathology - Research and Practice

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JAK2 regulates Nav1.6 channel function via FGF14Y158 phosphorylation

Cited by 17 publications

References 70 publications

Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

Inhibition of AKT Signaling Alters βIV Spectrin Distribution at the AIS and Increases Neuronal Excitability

Nav1.6 promotes the progression of human follicular thyroid carcinoma cells via JAK-STAT signaling pathway

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