[K.sup.+] Occupancy of the N-methyl-D-aspartate Receptor Channel Probed by [Mg.sub.2+] Block

The single-channel kinetics of extracellular [Mg.sup.2+] block was used to probe [K.sup.+] binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels. [K.sup.+] binds to three sites: two that are external and one that is internal to the site of [Mg.sup.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.sup.+] is 304 mM at 0 mV and with [Mg.sup.2+] in the pore. The occupancy of any one of the three sites by K+ effectively prevents the association of extracellular [Mg.sup.2+]. Occupancy of the internal site also prevents [Mg.sup.2+] permeation and increases (by approximately sevenfold) the rate constant for [Mg.sup.2+] dissociation back to the extracellular solution. Under physiological intracellular ionic conditions and at -60 mV, there is ~l,400-fold apparent decrease in the affinity of the channel for extracellular [Mg.sup.2+] and ~2-fold enhancement of the apparent voltage dependence of [Mg.sup.2+] block caused by the voltage dependence of [K.sup.+] occupancy of the external and internal sites. KEY WORDS: ion binding sites * magnesium * channel block * permeation * selectivity