Spatial coupling tunes NMDA receptor responses via Ca²⁺Diffusion

In the CNS, NMDA receptors generate large and highly regulated Ca ²⁺signals, which are critical for synaptic development and plasticity. They are highly clustered at postsynaptic sites and along dendritic arbors, but whether this spatial arrangement affects their output is unknown. Synaptic NMDA receptor currents are subject to Ca ²⁺-dependent inactivation (CDI), a type of activity-dependent inhibition that requires intracellular Ca ²⁺and calmodulin (CaM). We asked whether Ca ²⁺influx through a single NMDA receptor influences the activity of nearby NMDA receptors, as a possible coupling mechanism. Using cell-attached unitary current recordings from GluN1-2a/GluN2A receptors expressed in human HEK293 cells and from NMDA receptors native to hippocampal neurons from male and female rats, we recorded unitary currents from multichannel patches and used a coupled Markov model to determine the extent of signal coupling (κ). In the absence of extracellular Ca ²⁺, we observed no cooperativity (κ 0.1), whereas in 1.8 mM external Ca ²⁺, both recombinant and native channels showed substantial negative cooperativity (κ 0.27). Intracellular Ca ²⁺chelation or overexpression of a Ca ²⁺-insensitive CaM mutant, reduced coupling, which is consistent with CDI representing the coupling mechanism. In contrast, cooperativity increased substantially (κ 0.68) when overexpressing the postsynaptic scaffolding protein PSD-95, which increased receptor clustering. Together, these new results demonstrate that NMDA receptor currents are negatively coupled through CDI, and the degree of coupling can be tuned by the distance between receptors. Therefore, channel clustering can influence the activity-dependent reduction in NMDA receptor currents.