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Na(+) occupancy and Mg(2+) block of the n-methyl-d-aspartate receptor channel.
Zhu Y
,
Auerbach A
.
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The effect of extracellular and intracellular Na(+) on the single-channel kinetics of Mg(2+) block was studied in recombinant NR1-NR2B NMDA receptor channels. Na(+) prevents Mg(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(+) is voltage-dependent. In the absence of competing ions, Mg(2+) binds rapidly (>10(8) M(-1)s(-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(+) may be sufficient to prevent Mg(2+) dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the Mg(2+) dissociation rate constant is >10 times greater than the Mg(2+) permeation rate constant, and the Mg(2+) equilibrium dissociation constant is approximately 12 microM. Physiological concentrations of extracellular Na(+) reduce the Mg(2+) association rate constant approximately 40-fold but, because of the "lock-in" effect, reduce the Mg(2+) equilibrium dissociation constant only approximately 18-fold.
Figure 1. Block of NR1-NR2A NMDA receptors by extracellular Mg2+. (A) Single-channel currents from NR1-NR2A NMDA receptors recorded from outside-out patches in the absence and presence of extracellular Mg2+. Currents were activated by 50 μM NMDA and 10 μM glycine, and the membrane potential was â80 mV. Both the extracellular and intracellular solutions contained 100 mM Na+. Openings are shown as downward deflections. (B) Interval duration histograms (9 μM Mg2+). In this example, the inverse of the mean open time was 917 sâ1, and the inverse of the mean block time was 901 sâ1, which is the net rate of Mg2+ release from the pore (koff) either by dissociation back to the extracellular solution or permeation into the intracellular solution. (C) The inverse of the open interval lifetime (Ïo) versus the extracellular [Mg2+]. Each point is the mean ± SD from at least three patches (in some cases, the SD is smaller than symbol). The Mg2+ association rate constant is the slope of this relationship (89.0 ± 2.2 μMâ1sâ1; mean ± SD).
Figure 2. Effects of extracellular Na+ on the Mg2+ association rate constant. (A, left) Single-channel currents recorded at different [Na+]ex ([Mg2+]ex = 3 μM, [Na+]in = 5 mM; V = â80 mV). (A, right) Open interval duration distributions. Open intervals are longer (i.e., block by Mg2+ is slower) at higher [Na+]ex. (B) The inverse open channel lifetime (1/Ïo) versus [Mg2+]ex. Increasing extracellular [Na+] reduces the slope (which is equal to the Mg2+ association rate constant) about fivefold, from 500.0 ± 35.6 μMâ1sâ1 in 50 mM [Na+]ex to 109.6 ± 6.9 μMâ1sâ1 in 150 mM [Na+]ex. (C) The Mg2+ association rate constant versus membrane potential, at different [Na+]ex. The solid line is the fit by . The apparent voltage dependence of the Mg2+ association rate constant is the same in 50 mM and 150 mM [Na+]ex (43 ± 2 mV and 44 ± 1 mV per e-fold change, respectively). In B and C, the SD is smaller than the symbols for all except one point.
Figure 3. Effects of intracellular Na+ on the Mg2+ association rate constant. (A, left) Single-channel currents recorded with different intracellular Na+ concentrations. The extracellular solution contained 150 mM Na+ and 7 μM Mg2+, and the membrane potential was â80 mV. (A, right) Open interval duration histograms. Because intracellular Na+ slows the association of extracellular Mg2+, the inverse of open channel lifetime (Ïo) is faster in 5 mM [Na+]in (top histogram) than in 100 mM [Na+]in (bottom histogram). (B) The voltage dependence of the effect of intracellular Na+ on the Mg2+ association rate constant. The apparent Mg2+ association rate is plotted against the membrane potential for different [Na+]in. The solid lines are fits by . The inhibition of Mg2+ association by [Na+]in is enhanced by depolarization.
Figure 4. Number and affinities of Na+ binding sites. The Mg2+ association rate constant was used to probe the occupancy of the channel by Na+. Data obtained in 100 mM [Na+]in are shown to the left, and data obtained in 5 mM [Na+]in are shown to the right. (A) Fits of experimental Mg2+ association rate constants assuming one Na+ binding site (). The apparent Mg2+ association rate constant obtained from different [Na+]ex is plotted as a function of the membrane potential. A model with a single Na+ binding site does not provide an adequate description of the experimental results. (B) Fits of the same experimental data by an ân-siteâ scheme (). The solid lines are the predicted curves from the model (parameters for the best fit are shown in Table ). The model with n = 2 independent Na+ sites is better (MSC = 5.5) than the model with a single Na+ site (MSC = 4.0).
Figure 5. Effect of extracellular Na+ on the Mg2+ off rate constant. (A) Single-channel currents of Mg2+ block recorded with different extracellular Na+ concentrations (5 μM extracellular Mg2+, 5 mM [Na+]in, V = â80 mV). Closed interval duration histograms are shown below. The Mg2+ off rate decreases with increasing [Na+]ex. (B) Separating the Mg2+ off rate into a dissociation rate constant and a permeation rate constant. The solid lines are the fits using . Each point is the mean ± SD of at least three patches (SD may be smaller than the symbol). (C) Analyses of the inhibition of the Mg2+ dissociation rate constant by extracellular Na+. The same four sets of data as in B were fitted simultaneously by . The solid lines are the predicted curves from the model. The parameters are κ0âMg = 8,944 ± 205 sâ1, É = 0.36 (fixed), k0pMg = 624 ± 5 sâ1, λ = 0.03 (fixed), and KNaex = 251 ± 12 mM. Additional results are summarized in Table and Fig. 6.
Figure 6. Energy profile for Mg2+ block and permeation. The rate constants pertain to extracellular Mg2+ in pure water (no competing ions) and with no membrane potential. The fractional electrical distances are indicated below, where δ pertains to the Mg2+ entry barrier (= 0.25), É pertains to the Mg2+ dissociation (= 0.35), and λ pertains to the Mg2+ permeation barrier (= 0.03). The Mg2+ binding site is 0.60 through the electric field from the extracellular solution.
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