Zhu Y and Auerbach A (2001), K(+) occupancy of the N-methyl-d-aspartate rece...

XB-ART-9505

J Gen Physiol 2001 Mar 01;1173:287-98.

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K(+) occupancy of the N-methyl-d-aspartate receptor channel probed by Mg(2+) block.

Zhu Y , Auerbach A .

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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 approximately 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 approximately 1,400-fold apparent decrease in the affinity of the channel for extracellular Mg(2+) and approximately 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.

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Species referenced: Xenopus
Genes referenced: grin1 grin2a grin2b tbx2

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	Figure 2. Effects of intracellular K+ on the Mg2+ association rate constant. (A) Single-channel currents showing Mg2+ block at different intracellular K+ concentrations. 100 mM Na+ and 54 μM Mg2+ were present in the extracellular solution, and the membrane potential was −55 mV. Open times are longer in high [K+]in. (B) The inverse of open channel lifetime (τo) plotted as a function of the extracellular Mg2+ concentration. The decreased slope with the increased [K+]in indicates that intracellular K+ reduces the Mg2+ association rate constant. Where not shown, the SD is smaller than the symbol. (C) A global fit of experimental Mg2+ association rate constants using a model that allows intracellular K+ to bind only to the two external monovalent cation sites () does a poor job of describing the experimental results (Model Selection Criterion = 2.3). (D) A global fit of the same experimental data by a model that allows intracellular K+ to bind to one internal site as well as the two external sites () describes the experimental results (Model Selection Criterion = 4.3). The parameters for the best fit are shown in Table .
	Figure 3. Effects of extracellular K+ on the Mg2+ association rate constant. (A) Single-channel currents at different extracellular K+ concentrations (2 μM Mg2+ in the extracellular solution; the intracellular solution contained 150 mM Na+; V = −100 mV). (B) The inverse of open channel lifetime plotted as a function of Mg2+ concentration. The decreased slope with increasing [K+]in indicates that extracellular K+ reduces the Mg2+ association rate constant. (C) Fits of the experimental Mg2+ association rate constants using a model where extracellular K+ binds only to the two external sites. (see from Zhu and Auerbach 2001, in this issue; Model Selection Criterion = 4.6). (D) Fits of the experimental Mg2+ association rate constants using a model where extracellular K+ binds to two external sites and one internal site (; Model Selection Criterion = 5.4). Parameters for the best fit are shown in Table . The model with two external and one internal binding site for extracellular K+ is superior. The SDs are all smaller than the symbol.
	Figure 1. Effects of intracellular K+ on Mg2+ release from the pore. (A) Single-channel currents showing Mg2+ block at different intracellular K+ concentrations (50 mM Na+ and 3 μM Mg2+ in the extracellular solution, V = −80 mV). The current amplitude is larger at lower [K+]in because of a positive shift in the reversal potential. (Bottom) Closed interval duration histograms. The aggregate release rate of Mg2+ from the pore (koff) increases with [K+]in. (B) Separating koff into Mg2+ dissociation and permeation rate constants. The symbols are mean ± SD (usually, the SD was smaller than the symbol and is not visible) and were fitted using . The best-fit parameters are shown in Table . (C) Model-based analysis of the effects of intracellular K+ on Mg2+ dissociation and permeation. The two sets of data were simultaneously fitted by the sum of and . The solid lines are the predicted curves from the model with the best fit parameters (Table ).
	Figure 4. Effects of extracellular K+ on the Mg2+ off rate constant. (A) Single-channel currents showing Mg2+ block at different extracellular K+ concentrations (3 μM extracellular Mg2+, 100 mM intracellular Na+; V = −140 mV). Closed interval duration histograms are shown to the right. koff increases with increasing [K+]ex, and is larger in equivalent concentrations of extracellular K+ compared with Na+. (B) Separating koff into the Mg2+ dissociation and permeation rate constants. Solid lines are fits by . The best-fit parameters are shown in Table . (C) Model-based analyses of the Mg2+ dissociation and permeation rate constants as a function of [K+]ex. The koff values from both K+ concentrations were simultaneously fitted by the sum of and . The best-fit parameters are shown in Table (n = 2).
	Figure 5. The distinct effects of Na+ and K+ on the voltage dependence of Mg2+ association reflect the locations of the monovalent cation-binding sites. (A) The inhibition of k+Mg by [Na+]in is voltage-dependent because intracellular Na+ must cross the entire electric field to occupy the external sites. (B) The inhibition of k+Mg by [K+]in is only weakly voltage-dependent between −140 and −80 mV because, in this range, intracellular K+ blocks Mg2+ association via the occupancy of the internal site. The large inhibition at −55 mV arises from K+ occupancy of the external site. (C) The inhibition of k+Mg by [Na+]ex is not voltage-dependent because extracellular Na+ does not have to enter the entire electric field to occupy the external sites. (D) The inhibition of k+Mg by [K+]ex is voltage-dependent because extracellular K+ can cross the entire electric field to occupy the internal site. Its occupancy of the external sites is significant, but voltage-independent.
	Figure 6. A representation of the ion binding sites in the NMDA receptor channel. The protein is a modified structure of KcsA (Doyle, et al. 1998), drawn upside down (Wood et al. 1995) and with a wide extracellular entrance. The large intra- and extracellular domain of the NMDAR are not shown. The amino acid sequence is that of the NR1 subunit; the homologous region (N to C) is TVGYGD in KcsA and GluR0, and NSPVPQ in NR2A. The four regions where Na+, K+, and Mg2+ linger during their passage through the channel are drawn as circles. There are two monovalent cation-binding sites in the external portion of the permeation pathway. The location of one of these external sites (indicated by a question mark) is undetermined, and could be either beyond the extracellular margin or deep within of the electric field. Under physiological conditions, these sites are occupied both by Na+ (mainly from the extracellular solution) and K+ (mainly from the intracellular solution). There is a Mg2+ binding site located 0.60 through the electric field from the extracellular solution. The equilibrium dissociation constant of this site for Mg2+ (in the absence of competing ions and with no membrane potential) is 12 μM. Extracellular Mg2+ associates rapidly to this site (∼5 × 108 M−1s−1), thus, motivating the wide extracellular entrance. There is an internal, K+-selective site located 0.16 through the electric field from the intracellular solution. Access to the Mg2+ site from the extracellular solution is reduced by monovalent cation occupancy of either the internal site or the external sites. Occupancy of one external site (by Na+ or K+) prevents Mg2+ dissociation, and occupancy of the internal site (by K+) prevents Mg2+ permeation and increases the rate constant of Mg2+ dissociation back to the extracellular solution. The apparent voltage dependence of Mg2+ blockade strongly depends on the occupancies of the three monovalent cation-binding sites. Under standard conditions (140 mM [Na+]ex, 5 mM [Na+]in, 2 mM [K+]ex, and 140 mM [K+]in; V = −60 mV, 23°C, no extracellular Mg2+), the external sites are occupied by at least one monovalent cation with P = 0.978, and the internal site is occupied by K+ with P = 0.806.

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