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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

The effect of extracellular and intracellular [Na.sup.+] on the single-channel kinetics of [Mg.sup.2+] block was studied in recombinant NR1-NR2B NMDA receptor channels. [Na.sup.+] prevents [Mg.sup.2+] access to its blocking site by occupying two sites in the external portion of the permeation pathway. The occupancy of these sites by intracellular, but not extracellular, [Na.sup.+] is voltage-dependent. In the absence of competing ions, [Mg.sup.2+] binds rapidly ([is greater than] [10.sup.8] [M.sup.-1][s.sup.-1], with no membrane potential) to a site that is located 0.60 through the electric field from the extracellular surface. Occupancy of one of the external sites by [Na.sup.+] may be sufficient to prevent [Mg.sup.2+] dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the [Mg.sup.2+] dissociation rate constant is [is greater than] 10 times greater than the [Mg.sup.2+] permeation rate constant, and the [Mg.sup.2+] equilibrium dissociation constant is ~12 [micro]M. Physiological concentrations of extracellular [Na.sup.+] reduce the [Mg.sup.2+] association rate constant ~40-fold but, because of the 'lock-in' effect, reduce the [Mg.sup.2+] equilibrium dissociation constant only ~18-fold. KEY WORDS: ion binding sites magnesium * channel blockade * permeation selectivity

Investigates the potassium (K)-binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels through the use of single-channel kinetics of extracellular magnesium (Mg) block. Effects of intracellular K on the apparent rates of Mg dissociation and permeation; Equilibrium dissociation constant and rate constants for Mg dissociation and permeation; Qualitative assessment of the affinity and selectivity of the internal site.

Investigates the effect of extracellular and intracellular sodium (Na) on the single-channel kinetics of magnesium (Mg) block in recombinant NR1-NR2B NMDA receptor channels. Association rate constant of Mg; Locations and apparent affinities of the Na-binding sites; Dissociation rate constant of Mg.