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Magi-1 scaffolds Na_V1.8 and Slack K_Na channels in dorsal root ganglion neurons regulating excitability and pain.

ABSTRACT: Voltage-dependent sodium (Na_V) 1.8 channels regulate action potential generation in nociceptive neurons, identifying them as putative analgesic targets. Here, we show that Na_V1.8 channel plasma membrane localization, retention, and stability occur through a direct interaction with the postsynaptic density-95/discs large/zonula occludens-1-and WW domain-containing scaffold protein called membrane-associated guanylate kinase with inverted orientation (Magi)-1. The neurophysiological roles of Magi-1 are largely unknown, but we found that dorsal root ganglion (DRG)-specific knockdown of Magi-1 attenuated thermal nociception and acute inflammatory pain and produced deficits in Na_V1.8 protein expression. A competing cell-penetrating peptide mimetic derived from the Na_V1.8 WW binding motif decreased sodium currents, reduced Na_V1.8 protein expression, and produced hypoexcitability. Remarkably, a phosphorylated variant of the very same peptide caused an opposing increase in Na_V1.8 surface expression and repetitive firing. Likewise, in vivo, the peptides produced diverging effects on nocifensive behavior. Additionally, we found that Magi-1 bound to sequence like a calcium-activated potassium channel sodium-activated (Slack) potassium channels, demonstrating macrocomplexing with Na_V1.8 channels. Taken together, these findings emphasize Magi-1 as an essential scaffold for ion transport in DRG neurons and a central player in pain.-Pryce, K. D., Powell, R., Agwa, D., Evely, K. M., Sheehan, G. D., Nip, A., Tomasello, D. L., Gururaj, S., Bhattacharjee, A. Magi-1 scaffolds Na_V1.8 and Slack K_Na channels in dorsal root ganglion neurons regulating excitability and pain.

SUBMITTER: Pryce KD

PROVIDER: S-EPMC6529352 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Magi-1 scaffolds NaV1.8 and Slack KNa channels in dorsal root ganglion neurons regulating excitability and pain.

Pryce Kerri D KD Powell Rasheen R Agwa Dalia D Evely Katherine M KM Sheehan Garrett D GD Nip Allan A Tomasello Danielle L DL Gururaj Sushmitha S Bhattacharjee Arin A

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20190312 6

Voltage-dependent sodium (NaV) 1.8 channels regulate action potential generation in nociceptive neurons, identifying them as putative analgesic targets. Here, we show that NaV1.8 channel plasma membrane localization, retention, and stability occur through a direct interaction with the postsynaptic density-95/discs large/zonula occludens-1-and WW domain-containing scaffold protein called membrane-associated guanylate kinase with inverted orientation (Magi)-1. The neurophysio ...[more]

PMID: 30860870

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Project description:BackgroundDorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.Methodology/principal findingsWe show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na(+) currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I(Na) were preferentially expressed in relatively large neurons, while slowly-inactivating I(Na) was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I(Na) components. Voltage-gated Ca(2+) currents (I(Ca)) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I(Ca) in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.Conclusions/significanceOur present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.

Project description:RationaleIn cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes.ObjectiveThe objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains.Methods and resultsIn patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation.ConclusionsThe NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

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Magi-1 scaffolds Na_V1.8 and Slack K_Na channels in dorsal root ganglion neurons regulating excitability and pain.

Publications

Magi-1 scaffolds Na<sub>V</sub>1.8 and Slack K<sub>Na</sub> channels in dorsal root ganglion neurons regulating excitability and pain.

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