Modulation of NMDA channel gating by Ca²⁺ and Cd²⁺ binding to the external pore mouth

NMDA receptor channels are characterized by high Ca²⁺ permeability. It remains unclear whether extracellular Ca²⁺ could directly modulate channel gating and control Ca²⁺ influxes. We demonstrate a pore-blocking site external to the activation gate for extracellular Ca²⁺ and Cd²⁺, which has the same charge and radius as Ca²⁺ but is impermeable to the channel. The apparent affinity of Cd²⁺ or Ca²⁺ is higher toward the activated (a steady-state mixture of the open and desensitized, probably chiefly the latter) than the closed states. The blocking effect of Cd²⁺ is well correlated with the number of charges in the DRPEER motif at the external pore mouth, with coupling coefficients close to 1 in double mutant cycle analyses. The effect of Ca²⁺ and especially Cd²⁺ could be allosterically affected by T647A mutation located just inside the activation gate. A prominent "hook" also develops after wash-off of Cd²⁺ or Ca²⁺, suggesting faster unbinding rates of Cd²⁺ and Ca²⁺ with the mutation. We conclude that extracellular Ca²⁺ or Cd²⁺ directly binds to the DRPEER motif to modify NMDA channel activation (opening as well as desensitization), which seems to involve essential regional conformational changes centered at the bundle crossing point A652 (GluN1)/A651(GluN2).