NMDAR channel segments forming the extracellular vestibule inferred from the accessibility of substituted cysteines

In NMDA receptor channels, the M2 loop forms the narrow constriction and the cytoplasmic vestibule. The identity of an extracellular vestibule leading toward the constriction remained unresolved. Using the substituted cysteine accessibility method (SCAM), we identified channel-lining residues of the NR1 subunit in the region preceding M1 (preM1), the C-terminal part of M3 (M3(C)), and the N-terminal part of M4 (M4(N)). These residues are located on the extracellular side of the constriction and, with one exception, are exposed to the pore independently of channel activation, suggesting that the gate is at the constriction or further cytoplasmic to it. Permeation of Ca²⁺ ions was decreased by mutations in M3(C) and M4(N), but not by mutations in preM1, suggesting a functionally distinct contribution of the segments to the extracellular vestibule of the NMDA receptor channel.