Ca2+-Dependent Inactivation of GluN2A and GluN2B NMDA Receptors Occurs by a Common Kinetic Mechanism

N-Methyl-d-aspartate (NMDA) receptors are Ca2+-permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca2+-dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca2+, we observed robust CDI of whole-cell GluN2A currents (0.42 +/- 0.05) but no CDI in GluN2B currents (0.0850.07). In contrast, when Ca2+ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 +/- 0.03 and 0.67 +/- 0.02, respectively). To examine how the source of Ca2+ affects CDI, we recorded one-channel Na+ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca2+ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (P-o). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high P-o GluN2B receptors (GluN1(A652Y)/GluN2B). Together, these results show that Ca2+ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel P-o. These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca2+ load during high P-o activation.