K(+) Occupancy of the N-Methyl-d-Aspartate Receptor Channel Probed by Mg(2+) Block

The single-channel kinetics of extracellular Mg(2+) 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 Mg(2+) 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 Mg(2+) in the pore. The occupancy of any one of the three sites by K(+) effectively prevents the association of extracellular Mg(2+). Occupancy of the internal site also prevents Mg(2+) permeation and increases (by approximately sevenfold) the rate constant for Mg(2+) 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 Mg(2+) and ∼2-fold enhancement of the apparent voltage dependence of Mg(2+) block caused by the voltage dependence of K(+) occupancy of the external and internal sites.