K⁺ occupancy of the N-methyl-D-aspartate receptor channel probed by Mg²⁺ block

The single-channel kinetics of extracellular Mg²⁺ block was used to probe K⁺ binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels. K⁺ binds to three sites: two that are external and one that is internal to the site of Mg²⁺ block. The internal site is ∼0.84 through the electric field from the extracellular surface. The equilibrium dissociation constant for this site for K⁺ is 304 mM at 0 mV and with Mg²⁺ in the pore. The occupancy of any one of the three sites by K⁺ effectively prevents the association of extracellular Mg²⁺. Occupancy of the internal site also prevents Mg²⁺ permeation and increases (by approximately sevenfold) the rate constant for Mg²⁺ dissociation back to the extracellular solution. Under physiological intracellular ionic conditions and at -60 mV, there is ∼1,400-fold apparent decrease in the affinity of the channel for extracellular Mg²⁺ and ∼2-fold enhancement of the apparent voltage dependence of Mg²⁺ block caused by the voltage dependence of K⁺ occupancy of the external and internal sites.