Constitutive KCC2 Cell- and Synapse-Specifically Regulates NMDA Receptor Activity in the Spinal Cord
K+-Cl- cotransporter-2 (KCC2) plays a vital role in regulating chloride levels in neurons and ensuring proper synaptic inhibition by GABA and glycine. Following nerve injury, synaptic inhibition in the spinal cord is reduced due to KCC2 dysfunction. However, the mechanisms by which KCC2 influences nociceptive input to spinal excitatory and inhibitory neurons are not well understood. In this study, we found that the baseline reversal potentials for GABA were significantly more depolarized in inhibitory neurons expressing the vesicular GABA transporter (VGAT) compared to excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2) in the spinal cords of male and female mice. Interestingly, inhibiting KCC2 with VU0463271 enhanced the currents induced by NMDA and increased the frequency of NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) in VGluT2 neurons, but not in VGAT neurons. Importantly, VU0463271 did not affect EPSCs evoked monosynaptically from the dorsal root in VGluT2 neurons. Additionally, VU0463271 increased interactions between α2δ-1 and NMDARs, as well as their protein levels in spinal cord synaptosomes. In Cacna2d1 knockout mice, VU0463271 had no effect on NMDA currents or mEPSC frequency in dorsal horn neurons. Disrupting α2δ-1-NMDAR interactions with a mimicking peptide reduced the potentiation caused by VU0463271 on mEPSC frequency and NMDA currents in VGluT2 neurons. Furthermore, intrathecal administration of VU0463271 lowered mechanical and thermal thresholds in wild-type mice, but not in Cacna2d1 knockout mice. The pain hypersensitivity induced by VU0463271 was mitigated by co-treatment with the NMDAR antagonist pregabalin (an α2δ-1 inhibitory ligand) or the α2δ-1 mimicking peptide. Our results indicate that KCC2 modulates both presynaptic and postsynaptic NMDAR activity specifically in excitatory dorsal horn neurons, and its impairment enhances nociceptive transmission through α2δ-1-bound NMDARs.