Regulation of voltage-gated ion channels in excitable cells by the ubiquitin ligases Nedd4 and Nedd4-2

Bongiorno, Daria; Schuetz, Friderike; Poronnik, Philip; Adams, David J.

doi:10.4161/chan.5.1.13967

Cited by 37 publications

(34 citation statements)

References 112 publications

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“…To test this hypothesis, we expressed chimeric constructs carrying the single transmembrane domain protein CD4, depleted of its intracellular C-terminal tail (CD4-ΔC-tail), fused in frame with either the I–II loop (CD4-Na v 1.2-I-II loop), the II–III loop (CD4-Na v 1.2-II-III loop), or the C-terminal tail of the Na v 1.2 (CD4-Na v 1.2-C-tail), intracellular domains of the Na v 1.2 channel rich in trafficking motifs and phosphorylation sites [36–38]. These constructs were transiently expressed in COS-7 cells, and the CD4 surface pool was labeled using an Alexa 488-conjugated antibody against the extracellular domain of CD4 applied on live cells, followed by fixation and staining with HCS Cell Mask ™ Deep Red Plasma membrane stain to label the cell contour (Fig.…”

Section: 1 Resultsmentioning

confidence: 99%

“…The E3 ubiquitin ligase family neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) has been shown to be involved in Na v 1.2 internalization [38] and might be part of the GSK3-dependent mechanism described in this study. Co-expression of NEDD4 or NEDD4-2 significantly impairs Na v 1.2 function, leading to suppression of Na + currents.…”

Section: 1 Discussion and Conclusionmentioning

confidence: 99%

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The Nav1.2 channel is regulated by GSK3

James

Nenov²,

Wildburger

et al. 2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Phosphorylation plays an essential role in regulating the voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. Herein, we posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Nav channels. Methods We used patch-clamp electrophysiology to record sodium currents from Nav1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. Results We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Nav1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Nav1.2-encoded currents. Neither mRNA nor total protein expression were changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Nav1.2 channel indicates that cell surface expression of CD4-Nav1.2-Ctail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T1966 at the C-terminal tail of Nav1.2. Conclusion These findings provide evidence for a new mechanism by which GSK3 modulate Nav channel function via its C-terminal tail. General Significance These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.

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Section: 1 Resultsmentioning

confidence: 99%

Section: 1 Discussion and Conclusionmentioning

confidence: 99%

The Nav1.2 channel is regulated by GSK3

James

Nenov²,

Wildburger

et al. 2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…It is unlikely that the effects on mechanical allodynia are due to peripheral changes in the activation threshold of low-threshold mechanical afferent fibers, since basal mechanical sensitivity was minimally or not at all affected by rAAV-NEDD4-2 (nor was it after NEDD4-2 knockout). The unexpected mechanical hypoalgesic effect of rAAV2/6-NEDD4-2 might be due to the ability of ubiquitin ligase to regulate other ion channels, such as voltage-gated potassium or chloride channels (45), which could inversely affect cellular excitability depending on the targeted neuronal subpopulation. Despite its effect on the I Na of fast DRG neurons, other effects of the viral vector rAAV2/6 on cells or fibers in the peripheral nerve cannot be ruled out.…”

Section: Discussionmentioning

confidence: 99%

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Laedermann

Cachemaille²,

Kirschmann³

et al. 2013

J. Clin. Invest.

119

160

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Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Na v s) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na v s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Na v s, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na v 1.7-specific inhibitor, ProTxII, allowed the isolation of Na v 1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na v 1.7 and Na v 1.8 currents. The redistribution of Na v 1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L -/-). SNS-Nedd4L -/-mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na v 1.7 and Na v 1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na v s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

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“…In addition to Na v , Voltage-gated K+ channels play major roles in modulating electrical excitability in neurons. For instance, KCNQ2/3/5contain a PY motif and is subjected to Nedd4-2 dependent downregulation in a Xenopus oocyte expression system (Ekberg et al, 2007;Pongs, 2008;Bongiorno and Poronnik, 2011). This regulation and modulation of K+ channels might also be a key point in the excitability generated after a peripheral nerve injury.…”

Section: Discussionmentioning

confidence: 97%