LOSS OF MG²⁺ BLOCK IN GLUN2A-N615S NMDA RECEPTORS RESHAPES INHIBITION BY MEMANTINE AND K2060

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory neurotransmission, synaptic plasticity and are regulated by voltage-dependent pore block by extracellular Mg²⁺. Because Mg²⁺ occupies the permeation pathway, it can functionally compete with open-channel antagonists and thereby reduce their apparent potency. We tested how impaired Mg²⁺ block influences antagonist inhibition using the epilepsy-associated GluN2A-N615S variant. Recombinant GluN1/GluN2A wild-type receptors (WT) and GluN1/GluN2A receptors carrying the GluN2A-N615S substitution (N615S) were expressed in heterologous cells and examined by whole-cell patch clamp to compare inhibition by memantine and the dizocilpine derivative K2060. In WT receptors, 1 mM Mg²⁺ reduced apparent drug potency and altered inhibition kinetics, slowing onset and accelerating recovery, consistent with Mg²⁺ limiting blocker occupancy in the pore. In N615S receptors, Mg²⁺-dependent modulation was largely absent, and both antagonists retained strong inhibition in 1 mM Mg²⁺ (memantine IC₅₀ ≈ 2.0 µM; K2060 IC₅₀ ≈ 0.55 µM). Kinetic analysis showed Mg²⁺-dependent changes in onset and offset rates in WT but not in N615S receptors; notably, K2060 exhibited relatively slow onset (~1.5 s) and prolonged recovery (~15 s), consistent with high-affinity yet reversible trapping. Both antagonists also produced membrane-to-channel inhibition, most prominently for K2060, and this component was enhanced in N615S receptors, suggesting that Mg²⁺ normally restricts membrane-mediated access to the pore. Together, these results indicate that reduced Mg²⁺ block preserves effective open-channel inhibition by minimizing Mg²⁺–drug competition and highlight K2060 as a potent, reversible inhibitor under Mg²⁺-insensitive conditions relevant to GRIN-related disorders.