LRRC8A constitutively inhibits pain hypersensitivity in rodent models by restraining NMDA receptor activity at spinal cord synapses

Amplification of nociceptive transmission due to aberrant N-methyl-d-aspartate receptor (NMDAR) hyperactivity in the spinal cord is a key characteristic of neuropathic pain. However, under normal conditions, both presynaptic and postsynaptic NMDARs in the spinal dorsal horn are largely inactive. The mechanisms restraining synaptic NMDAR activity remain enigmatic. Leucine-rich repeat-containing protein 8A (LRRC8A or SWELL1) is an essential component of volume-regulated anion channels typically involved in regulating cell volume. Here, we report that LRRC8A was highly expressed in dorsal root ganglion (DRG) and spinal dorsal horn neurons of rats. Nerve injury persistently reduced LRRC8A expression in the DRG. siRNA-mediated Lrrc8a knockdown in rats or conditional Lrrc8a knockout in DRG neurons in mice consistently caused a pain hypersensitivity phenotype that was readily reversed by NMDAR antagonists. Correspondingly, Lrrc8a knockdown or conditional Lrrc8a knockout in DRG neurons markedly augmented synaptic localization and activity of NMDARs in the spinal cord. LRRC8A interacted with NMDARs in both rat and human spinal cord tissues primarily through its C-terminal LRR domain, restricting the synaptic trafficking and activity of NMDARs. Furthermore, Lrrc8a^ebo/ebo mutant mice, which lack the LRR domain, exhibited NMDAR-dependent pain hypersensitivity and synaptic NMDAR hyperactivity in the spinal cord. Additionally, intrathecal Lrrc8a gene delivery eliminated nerve injury-induced pain hypersensitivity and synaptic NMDAR hyperactivity in rats. These findings reveal that LRRC8A physically interacts with NMDARs and constitutively restricts their synaptic expression in the spinal cord. Reduced LRRC8A-NMDAR interactions increase synaptic expression of "unleashed" NMDARs, contributing to NMDAR hyperactivity and neuropathic pain in rodent models.