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

The spinal nerve injection protocol was adopted from Chang et al. (37) and optimized for spinal nerve injection in mice. Three days after baseline thermal behavior was established, mice were anesthetized using isoflurane (induction: 4%; maintenance: 2%) and placed in a prone position. A 3-cm posterior longitudinal skin incision was made at the lumbar segment of the spine. The ipsilateral paraspinal muscles were carefully separated, using a pair of sterile toothpicks, from their attachments at the L4 to S1 levels of the vertebral column.A total of 1.5 ml of polyethylenimine–short hairpin RNA (shRNA) plasmid DNA polyplexes at an N/P ratio of 6 was slowly injected directly in the spinal nerve of the right hind paw using a syringe connected to a 26-gauge needle. After injection, the needle was held at the spinal nerve for 1 min to prevent leakage.

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.