Spatial Coupling Tunes NMDA Receptor Responses via Ca²⁺ Diffusion

In the CNS, NMDA receptors generate large and highly regulated Ca²⁺ 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 Ca²⁺-dependent inactivation (CDI), a type of activity-dependent inhibition that requires intracellular Ca²⁺ and calmodulin (CaM). We asked whether Ca²⁺ 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 Ca²⁺, we observed no cooperativity (κ < 0.1), whereas in 1.8 mm external Ca²⁺, both recombinant and native channels showed substantial negative cooperativity (κ = 0.27). Intracellular Ca²⁺ chelation or overexpression of a Ca²⁺-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.SIGNIFICANCE STATEMENT At central synapses, NMDA receptors are a major class of excitatory glutamate-gated channels and a source of activity-dependent Ca²⁺ influx. In turn, fluxed Ca²⁺ ions bind to calmodulin-primed receptors and reduce further entry, through an autoinhibitory mechanism known as Ca²⁺ -dependent inactivation (CDI). Here, we show that the diffusion of fluxed Ca²⁺ between active channels situated within submicroscopic distances amplified receptor inactivation. Thus, calmodulin-mediated gating modulation, an evolutionarily conserved regulatory mechanism, endows synapses with sensitivity to both the temporal sequence and spatial distribution of Ca²⁺ signals. Perturbations in this mechanism, which coordinates the activity of NMDA receptors within a cluster, may cause signaling alterations that contribute to neuropsychiatric conditions.