Identifying the function of the NMDA NR1 C2 subunit through its interaction with Magi-2 during inflammatory pain.

Much is understood about the structure and gating properties of NMDA receptors (NMDAR), but the function of the carboxy-terminal splice variant of the NR1 subunit, NR1 C2 has never been identified. By studying the scaffolding protein Magi-2 in animal models of inflammatory pain, we discovered how NR1 C2 protein is specifically regulated. We found that Magi-2 deficiency resulted in decreased pain behavior and a concomitant reduction in NR1 C2 protein. Magi-2 contains WW domains, domains typically found in ubiquitin ligases. We identified an atypical WW-binding domain within NR1 C2 which conferred susceptibility to Nedd4-1 ubiquitin-ligase dependent degradation. We used lipidated peptidomimetics derived from the NR1 C2 sequence and found that NR1 C2 protein levels and pain behavior can be pharmacologically targeted. The function of NR1 C2 is to give lability to a pool of NMDAR, important for pain signaling.

Thermal hyperalgesia and dynamic weight bearing share similar recovery dynamics in a sciatic nerve entrapment injury model.

Chronic constriction injuries (CCI) of the sciatic nerve are widely used nerve entrapment animal models of neuropathic pain. Two common pain behaviors observed following CCI are thermal hyperalgesia and mechanical allodynia, measured by the Hargreaves and von Frey tests, respectively. While thermal hyperalgesia tends to recover by 30 days, mechanical allodynia can persist for many more months thereafter. Consequently, mechanical allodynia has been used extensively as a measure of 'chronic pain' focusing on the circuitry changes that occur within the spinal cord. Here, using the sciatic nerve cuff variant of CCI in mice, we propose that in contrast to these evoked measures of nociceptive hypersensitivity, dynamic weight bearing provides a more clinically relevant behavioral measure for ongoing pain during nerve injury. We found that the effect of sciatic nerve cuff on the ratio of weight bearing by the injured relative to uninjured hindlimbs more closely resembled that of thermal hyperalgesia, following a trend toward recovery by 30 days. We also found an increase in the percent of body weight bearing by the contralateral paw that is not seen in the previously tested behaviors. These results demonstrate that dynamic weight bearing is a reliable measure of non-evoked neuropathic pain and suggest that thermal hyperalgesia, rather than mechanical allodynia, provides a proxy measure for nerve entrapment-induced ongoing pain.

Inhibiting endocytosis in CGRP+ nociceptors attenuates inflammatory pain-like behavior.

The advantage of locally applied anesthetics is that they are not associated with the many adverse effects, including addiction liability, of systemically administered analgesics. This therapeutic approach has two inherent pitfalls: specificity and a short duration of action. Here, we identified nociceptor endocytosis as a promising target for local, specific, and long-lasting treatment of inflammatory pain. We observed preferential expression of AP2α2, an α-subunit isoform of the AP2 complex, within CGRP+/IB4- nociceptors in rodents and in CGRP+ dorsal root ganglion neurons from a human donor. We utilized genetic and pharmacological approaches to inhibit nociceptor endocytosis demonstrating its role in the development and maintenance of acute and chronic inflammatory pain. One-time injection of an AP2 inhibitor peptide significantly reduced acute and chronic pain-like behaviors and provided prolonged analgesia. We evidenced sexually dimorphic recovery responses to this pharmacological approach highlighting the importance of sex differences in pain development and response to analgesics.

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

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 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.

A De Novo Mutation in the Sodium-Activated Potassium Channel KCNT2 Alters Ion Selectivity and Causes Epileptic Encephalopathy.

Early infantile epileptic encephalopathies (EOEE) are a debilitating spectrum of disorders associated with cognitive impairments. We present a clinical report of a KCNT2 mutation in an EOEE patient. The de novo heterozygous variant Phe240Leu SLICK was identified by exome sequencing and confirmed by Sanger sequencing. Phe240Leu rSlick and hSLICK channels were electrophysiologically, heterologously characterized to reveal three significant alterations to channel function. First, [Cl-]i sensitivity was reversed in Phe240Leu channels. Second, predominantly K+-selective WT channels were made to favor Na+ over K+ by Phe240Leu. Third, and consequent to altered ion selectivity, Phe240Leu channels had larger inward conductance. Further, rSlick channels induced membrane hyperexcitability when expressed in primary neurons, resembling the cellular seizure phenotype. Taken together, our results confirm that Phe240Leu is a "change-of-function" KCNT2 mutation, demonstrating unusual altered selectivity in KNa channels. These findings establish pathogenicity of the Phe240Leu KCNT2 mutation in the reported EOEE patient.