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 t hat is internal to the site of Mg2+ block. The internal site is similar to0 .84 through the electric field from the extracellular surface. The equilibr ium dissociation constant for this site for K+ is 304 mM at 0 mV and with M g2+ in the pore. The occupancy of any one of the three sites by K+ effectiv ely prevents the association of extracellular Mg2+. Occupancy of the intern al site also prevents Mg2+ permeation and increases (by approximately seven fold) the rate constant for Mg2+ dissociation back to the extracellular sol ution. Under physiological intracellular ionic conditions and at -60 mV, th ere is similar to1,400-fold apparent decrease in the affinity of the channe l for extracellular Mg2+ and similar to2-fold enhancement of the apparent v oltage dependence of Mg2+ block caused by the voltage dependence of K+ occu pancy of the external and internal sites.