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‐l‐and WW domain‐containing scaffold protein called membrane‐associated guanylate kinase with inverted orientation (Magi)‐l. 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 Nav1.8 protein expression. A competing cell‐penetrating peptide mimetic derived from the Nav1.8 WW binding motif decreased sodium currents, reduced Nav1.8 protein expression, and produced hypoexcitability. Remarkably, a phosphorylated variant of the very same peptide caused an opposing increase in Nav1.8 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 Nav1.8 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 Nav1.8 and Slack KNa channels in dorsal root ganglion neurons regulating excitability and pain. FASEB J. 33, 7315–7330 (2019). http://www.fasebj.org

The sodium‐activated potassium subunit Slack (Kcnt1) is highly expressed in sensory neurons and contributes to firing accommodation. Previous studies have shown that protein kinase A‐induced internalization of Slack channels contributes to hyperexcitability in DRG neurons. These results suggest that the molecular modulators of Slack channel trafficking could serve as novel therapeutic targets in the treatment of pain. In this study, we probed the molecular determinants of Slack channel trafficking by focusing on the evolutionary conserved last 10 amino acids sequence of the C‐terminus. In this region there is a post‐synaptic density‐95/discs large/zonulaoccludens‐1 (PDZ) binding domain. Using a synthetic peptide comprised of these 10 amino acids, we performed patch‐clamp recordings on isolated DRG neurons. We found that incubating neurons with the peptide resulted in hyperexcitability whereas, a scrambled peptide did not alter firing. Biotinylation assays confirmed a significant reduction in channel expression at the membrane upon peptide incubation; suggesting that the anchoring of Slack channels to the membrane is likely mediated through the PDZ‐binding motif. Studies have found MAGI‐1, a scaffolding protein, and Slack to both be enriched in the dorsal horn of the spinal cord. Using co‐immunolocalization assays, we demonstrated that MAGI‐1 localized with Slack at the cell membrane. We next confirmed that MAGI‐1 interacts with Slack via the PDZ binding motif by immunoprecipitation assays. Our results suggest that Slack is stabilized at the cell membrane by its ability to bind PDZ containing proteins. Dissociation from the PDZ scaffold leads to hyperexcitability. MAGI‐1 is a potential Slack anchoring protein and strengthening this interaction may be a novel analgesic strategy for pain.