The opening and closing kinetics of NMDA receptors (NMDARs) contribute to their unique role in synaptic computation. NMDARs are heterotetramers composed of the obligate GluN1 and typically some combination of GluN2 subunits. The initial step in ion channel opening is agonist binding, which generates free energy that propagates to the ion channel to open a canonical gate at the M3 helix bundle crossing. A conserved glycine in the outer most transmembrane helices, the M4 helix, regulates NMDAR function. Here we find that the GluN1 glycine mainly regulates single channel events within a cluster or bursts of activity, whereas the GluN2A glycine mainly regulates entry and exit from clusters. Molecular dynamic simulations suggest that, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates the gate at the M3 helix bundle crossing, the GluN1 glycine regulates a ‘gate’ at the M2 loop. Subsequent functional experiments support this interpretation. Thus, the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation.