A GLUN2A-N615S PORE MUTATION MODEL REVEALS HOW MG²⁺ CONTROLS KETAMINE INHIBITION OF NMDA RECEPTORS

NMDA receptors (NMDARs) are essential for neurodevelopment and synaptic plasticity, and de novo missense variants in NMDAR subunits cause GRIN-related encephalopathies. The pore-forming transmembrane domain contains the M2 loop, which contributes to ion selectivity and, together with M3, shapes the binding environment for open-channel blockers such as Mg²⁺ and ketamine. Because Mg²⁺ and channel blockers can interact within the pore, pathogenic variants that weaken Mg²⁺ block may reshape antagonist pharmacology.
We investigated how loss of Mg²⁺ gating affects ketamine inhibition in wild-type (WT) GluN1/GluN2A receptors and receptors carrying the pathogenic GluN2A-N615S substitution, which markedly reduces Mg²⁺ affinity and is associated with epilepsy and developmental delay. Using whole-cell patch-clamp recordings in HEK293 cells and acute hippocampal slices (CA1-evoked EPSCs), we quantified ketamine potency, inhibition kinetics, and a membrane-to-channel inhibition (MCI) component reflecting membrane-mediated access to the pore.
In HEK293 cells, N615S abolished Mg²⁺-dependent modulation of ketamine block. Whereas 1 mM Mg²⁺ substantially reduced ketamine potency at WT receptors, GluN2A-N615S receptors remained highly sensitive (IC₅₀ ≈ 0.5 µM) even in 1 mM Mg²⁺. In 0 Mg²⁺, the onset of inhibition was similar for WT and N615S; in 1 mM Mg²⁺, onset slowed in WT but not in N615S. Slice recordings recapitulated both onset and equilibrium effects, supporting relevance in native tissue. Unexpectedly, we detected a measurable ketamine MCI component that was suppressed by Mg²⁺ and enhanced by N615S, suggesting Mg²⁺ normally restricts membrane-mediated access. These findings provide a mechanistic framework for altered ketamine responses in conditions with reduced Mg²⁺ block.