In all glutamate-activated channels, the outward portion of the M3 transmembrane helix consists of a conserved nine amino acid residue stretch (lurcher motif): SYTANALAAF. Substitution of position 8 alanine with threonine (A8T, the lurcher position) in a wide range of glutamate receptors result in constitutively active channels. Scanning mutagenesis and cysteine modification experiments, implicated the position 7 alanine (A7) of GluN1 and GluN2 subunits as critical elements in gating, perhaps the equivalent of the ‘bundle crossing’ in potassium-selective channels. To investigate how A7 and A8 residues affect NMDA receptor gating, we used single channel recordings and kinetic models of receptor activity from HEK 293 cells. We found that substitutions at these positions in GluN1 or GluN2A resulted indeed in channels with modified gating. Compared to wild-type receptors (PO = 0.51 ± 0.05, MOT = 8.6 ± 1 ms, MCT = 7.5 ± 0.7 ms), A7C and A8T resulted in distinct gating patterns depending on the subunit in which they were introduced. GluN1(A7C) had shorter openings (Po = 0.02 ± 0.01, MOT= 3.2 ± 0.5 ms, MCT = 143 ± 17 ms) but GluN2A A7C) had significantly prolonged openings (PO = 0.17 ± 0.02, MOT = 13 ± 1 ms, MCT = 65 ± 7 ms). Further, A8T prolonged openings in GluN1 (PO = 0.41 ± 0.07, MOT = 21 ± 4 ms, MCT = 30 ± 6.4 ms) but had no effect on gating when introduced in GluN2A (PO = 0.57 ± 0.08, MOT = 11.2 ± 1 ms, MCT = 9.5 ± 2.6 ms). The implication is that the A7 and A8 positions play distinct gating roles in GluN1 vs. GluN2A subunits, supporting the view that in NMDA receptors, subunits are staggered along M3 into the channel's external vestibule.