K+ Occupancy of the N-methyl-D-aspartate Receptor Channel Probed by Mg2+ Block

Yongling Zhua and Anthony Auerbacha
a Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214

Correspondence to: Anthony Auerbach, Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, SUNY at Buffalo, 124 Sherman Hall, Buffalo, NY 14214. Fax:(716) 829-2569 E-mail:auerbach{at}buffalo.edu.


  Abstract
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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Acknowledgements
References

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 that is internal to the site of Mg2+ block. The internal site is ~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 Mg2+ in the pore. The occupancy of any one of the three sites by K+ effectively prevents the association of extracellular Mg2+. Occupancy of the internal site also prevents Mg2+ permeation and increases (by approximately sevenfold) the rate constant for Mg2+ dissociation back to the extracellular solution. Under physiological intracellular ionic conditions and at -60 mV, there is ~1,400-fold apparent decrease in the affinity of the channel for extracellular Mg2+ and ~2-fold enhancement of the apparent voltage dependence of Mg2+ block caused by the voltage dependence of K+ occupancy of the external and internal sites.

Key Words: ion binding sites, magnesium, channel block, permeation, selectivity


  INTRODUCTION
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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Acknowledgements
References

The N-methyl-D-aspartate receptor (NMDAR)1 channel is permeable to Na+, K+, and Ca2+, and is blocked by Zn2+ and Mg2+. Numerous studies have addressed the position and nature of binding sites for Na+, Ca2+, Zn2+, and Mg2+ in the NMDAR ion permeation pathway (Burnashev et al. 1992 ; Mori et al. 1992 ; Premkumar and Auerbach 1996 ; Sharma and Stevens 1996a , Sharma and Stevens 1996b ; Wollmuth et al. 1998 ; Antonov and Johnson 1999 ; Fayyazuddin et al. 2000 ; see Zhu and Auerbach 2001 , in this issue). However, interactions between K+ and the NMDAR channel have not been examined in detail, even though this ion is highly permeable and is normally present in the intracellular milieu at a high concentration. In this paper, we present information regarding the location and affinity of K+ binding sites in recombinant NR1-NR2A NMDARs, inferred from the kinetics of Mg2+ block as a function of the extra- and intracellular concentrations of K+.

Previous studies have demonstrated that Na+ binds to two sites that are external to the Mg2+ binding site and that are accessible from both the intra- and extracellular solutions. Occupancy of these sites by Na+ prevents the movement of extracellular Mg2+ between the extracellular compartment and the pore (i.e., blocks the association and "locks in" Mg2+). We find that in addition to interacting with these external sites, K+ binds to a site that is near the intracellular entrance of the permeation pathway. Occupancy of this internal site by K+ reduces the rate constants for extracellular Mg2+ association and permeation, and increases the rate constant of Mg2+ dissociation back to the extracellular solution. Under physiological conditions, the interactions between K+ and Mg2+ in the NMDA pore affect the affinity and voltage dependence of Mg2+ blockade.


  MATERIALS AND METHODS
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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Acknowledgements
References

Wild-type rat cRNAs for the rat NR1 and NR2A subunits were expressed in Xenopus oocytes. Single-channel currents were recorded from outside-out patches. A detailed description of the molecular biology, expression protocols, electrophysiology, solutions, signal processing, kinetic analysis, and fitting procedures is given in Zhu and Auerbach 2001 (in this issue).


  RESULTS
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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Acknowledgements
References

The results are presented in four sections: first, the effects of intracellular K+ ([K+]in) on Mg2+ dissociation and permeation; second, the effects of [K+]in on Mg2+ association; third, the effects of extracellular K+ ([K+]ex) on Mg2+ association; and fourth, the effects of [K+]ex on Mg2+ dissociation and permeation.

Intracellular K+ Increases the Mg2+ Dissociation Rate Constant and Decreases the Mg2+ Permeation Rate Constant
Increasing intracellular [K+] shortens the duration of the gaps arising from Mg2+ block (Fig 1 A); i.e., Mg2+ is released from the NMDAR pore more rapidly when [K+]in is elevated. The apparent Mg2+ release rate (koff) is the sum of a dissociation rate constant back to the extracellular solution (k-Mg) and a permeation rate constant (kpMg). These two rate constants have opposite voltage dependencies: the former decreasing and the latter increasing with hyperpolarization. Fig 1 B shows that between -60 and -120 mV, koff increases with increasing [K+]in, as does its voltage dependence.



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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 Equation 1. The best-fit parameters are shown in Table 1. (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 Equation 2 and Equation 3. The solid lines are the predicted curves from the model with the best fit parameters (Table 2).

The effect of [K+]in on Mg2+ dissociation and permeation was quantified by fitting koff at different membrane potentials by:

(1)

where V is the membrane potential, the superscript 0 indicates the salient rate constant at 0 mV, kB is Boltzmann's constant, T is the absolute temperature (under our conditions, kBT = 25.3 mV), and {varepsilon} is the fractional electrical distance from the Mg2+ binding site to the top of the dissociation energy barrier. Results regarding Na+ interactions with Mg2+ indicate that kpMg is essentially voltage-independent (see Zhu and Auerbach 2001 , in this issue). Therefore, in Equation 1, the fractional electrical distance from the Mg2+ binding site to the top of the permeation barrier was assumed to be zero.

The result of fitting the experimental koff values using Equation 1 are shown in Table 1. At 0 mV, k-Mg is larger and kpMg is smaller in 150 mM compared with 5 mM [K+]in; i.e., elevating intracellular [K+] enhances Mg2+ dissociation back to the extracellular space, but reduces Mg2+ permeation to the intracellular space.


 
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Table 1. The Effects of Intracellular [K+] on the Apparent Rates of Mg2+ Dissociation and Permeation

The NMDAR channel has external binding sites that are able to bind Cs+ and Na+ (Antonov and Johnson 1999 ). The effects of [K+]in on the Mg2+ dissociation and permeation rate constants cannot, however, be explained by K+ occupancy of these external sites because intracellular K+ does not have access to the external sites when Mg2+ blocks the channel. Therefore, intracellular K+ must be interacting with regions of the protein that are internal to the Mg2+ binding site. We assume the reduction in kpMg with increasing [K+]in occurs because the occupancy of this internal site by K+ blocks the pathway for Mg2+ permeation into the intracellular compartment. Moreover, we speculate that the enhancement of k-Mg with increasing [K+]in may be caused by an electrostatic repulsion between the two ions.

To further quantify the effects of [K+]in on Mg2+ dissociation, we used a model having one external Na+ site and one internal K+ site. The model assumes that Mg2+ cannot dissociate if the external site is occupied, and that Mg2+ dissociates at two different rate constants depending on whether or not the internal site is occupied. Accordingly, the apparent dissociation rate constant (kV-Mg) is described by:

(2)

Jd,Kin,internal is the equilibrium dissociation constant of the internal site for intracellular K+ (with Mg2+ in the pore); k0-Mg1 and k0-Mg2 are the Mg2+ dissociation rate constants (at zero voltage and in the presence of extracellular [Na+]) without and with a K+ at the internal site, respectively; ß is the fractional electrical distance from the intracellular solution to the internal K+ binding site; and Jd,Naex is the equilibrium dissociation constant of the lone external site for extracellular Na+ when the pore is occupied by Mg2+. The three experimental variables in Equation 2 are [K+]in, [Na+]ex, and V.

Because extracellular Na+ does not alter Mg2+ permeation (see Zhu and Auerbach 2001 , in this issue), this process can be described simply by:

(3)

where {kappa}0pMg1 is the intrinsic rate constant for Mg2+ permeation (with the internal site empty and no membrane potential), and kVpMg is the net Mg2+ permeation rate constant, which is a function of only two experimental variables, [K+]in and V.

The experimental values of kVoff were fitted by the sum of Equation 2 and Equation 3, which has four free parameters (Table 2). Fig 1 C shows that the predicted curves match the experimental data, indicating that a single internal K+ binding site is sufficient to explain the effects of [K+]in on Mg2+ release from the pore. The Mg2+ dissociation rate constant is approximately seven times greater when there is a K+ at the internal site compared with when this site is empty. The affinity of the internal site for K+ when there is a Mg2+ in the pore is low, perhaps because of electrostatic repulsion between the ions. The internal K+ binding site is ~84% through the electric field from the extracellular surface, and is ~24% deeper in the electric field than the Mg2+ binding site.


 
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Table 2. Intracellular K+ at the Internal Site: Equilibrium Dissociation Constant and Rate Constants for Mg2+ Dissociation and Permeation.

Intracellular K+ Reduces the Mg2+ Association Rate Constant
In this section, we address the Mg2+ association rate constant as a function of intracellular [K+]. Fig 2 A shows that the open channel lifetimes are longer (i.e., the Mg2+ association rate constant is slower; Fig 2 B) when [K+]in is elevated. We first considered whether the apparent reduction in the Mg2+ association rate constant is caused exclusively by the binding of intracellular K+ to the external monovalent cation sites. We made the simplifying assumptions that the two external sites are independent and identical, and that the occupancy of these sites by extracellular Na+ is not voltage-dependent. Three sets of data, obtained at different [K+]in (25, 50, and 100 mM) and at l00 mM [Na+]ex, were fitted simultaneously by:

(4)

where {kappa}+Mg0is the intrinsic Mg2+ association rate constant (i.e., in the absence of competing ions and with no membrane potential), KNaex and KKin are the apparent dissociation constants for [Na+]ex and [K+]in at the external sites, respectively, (without Mg2+ in the pore), and {alpha} is the fractional electrical distance between the external binding sites and the intracellular compartment. Because Na+ and K+ can permeate, KNaex and KKin are not true equilibrium constants. As can be seen in Fig 2 C, the predicted curves provide a poor description of the experimental data. We conclude that in addition to the two external sites, there are other K+ binding sites involved in the inhibition of the Mg2+ association rate constant.



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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 ({tau}o) 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 (Equation 4) 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 (Equation 5) describes the experimental results (Model Selection Criterion = 4.3). The parameters for the best fit are shown in Table 3.

As described above, intracellular K+ increases the Mg2+ dissociation rate constant and decreases the Mg2+ permeation rate constant because it occupies an internal binding site that is close (in electrical distance) to the Mg2+ binding site. Therefore, we speculated that Mg2+ binds to the NMDAR pore only when both of the external sites and the internal site are empty. That is, we hypothesized that when K+ occupies the internal site, the association rate constant for extracellular Mg2+ is significantly reduced.

To quantify the observations, we used a scheme that had four ion binding sites: two external sites that bind Na+ or K+; one intermediate site that is selective for Mg2+; and one internal site that is selective for K+. For simplicity, we assumed that occupancy of any one of the three monovalent cation binding sites completely prevents the association of extracellular Mg2+. (Although the results given in Table 2 suggest that intracellular K+ can bind to the internal site when the channel is blocked by Mg2+, in the following analysis, we made the simplifying assumption that Mg2+ association is effectively eliminated when K+ occupies the internal site.) A 12-state model is required to account for the effects of intracellular K+ and extracellular Na+ on the Mg2+ association rate constant, with six external site configurations (two Na+, two K+, one Na+ and one K+, one Na+, one K+, and empty) and two internal site configurations (one K+ and empty).

We assume that the apparent association rate constant for Mg2+ is a function of the probability of all three of the monovalent cation sited being empty:

where Pexternaleand Pinternaleare the probabilities of the external sites and the internal site being empty. The apparent Mg2+ association rate constant is related to the experimental variables [Na+]ex, [K+]in, and V by:

(5)

The first term in parentheses is the inhibition of Mg2+ association because of Na+ and K+ occupancy of the two external sites and the second term in parentheses is the inhibition because of K+ occupancy of the single internal site. K0Kin,internal is the apparent dissociation constant for intracellular K+ at the internal site with no membrane potential. In the case of association, Mg2+ is not present in the pore yet and the monovalent ions are free to permeate. As stated before, the apparent affinities are not true dissociation equilibrium constants.

Fig 2 D shows the results of fitting the experimental data using Equation 5, with {kappa}0+Mg, K0Kin and K0Kin,internal as the only free parameters. The predicted curves match the experimental data. We conclude that a model having two external monovalent cation-binding sites and one internal K+-selective site accounts for the effects of intracellular [K+] on Mg2+ association. The parameters for the best fit are shown in Table 3.


 
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Table 3. Intracellular K+ at the Monovalent Cation Sites: Apparent Dissociation Constants and the Rate Constant for Mg2+ Association

Extracellular K+ Decreases the Mg2+ Association Rate Constant
We next investigated the effects of extracellular K+ on the Mg2+ association rate constant. Fig 3 A illustrates single-channel currents recorded at two different extracellular K+ concentrations ([K+]ex, 25 and 150 mM) in the presence of 100 mM intracellular Na+. The channel open lifetime is longer at the higher [K+]ex, indicating that the Mg2+ association rate constant decreases with an increase in [K+]ex.



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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 Equation 3 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 (Equation 6; Model Selection Criterion = 5.4). Parameters for the best fit are shown in Table 4. The model with two external and one internal binding site for extracellular K+ is superior. The SDs are all smaller than the symbol.

We again used the two-external, one-internal site model to quantify the results. We assumed that all three sites can bind extracellular K+, and that occupancy of any one of these sites by K+ effectively eliminates Mg2+ association. We also assumed that that Na+ does not bind to the internal site.

For this model, we used an expression that relates the apparent association rate constant (kV+Mg) to [K+]ex, [Na+]in, and V:

(6)

Fig 3 D shows experimental kV+Mg values obtained at three different [K+]ex as a function of the membrane potential, fitted simultaneously by Equation 6. The fitting results are given in Table 4. The fitted curves provide a good description of the experimental data, indicating that a model with two external sites that can bind either Na+ or K+, and with one internal site that is K+-selective, accounts for the effects of extracellular Na+ and K+ on the Mg2+ association rate constant.


 
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Table 4. Extracellular K+ at the Monovalent Cation Sites: Apparent Dissociation Constants and the Rate Constant for Mg2+ Association

In the absence of a membrane potential, the internal site has an extremely low apparent affinity for extracellular K+. To evaluate the significance of extracellular K+ occupancy of the internal site with regard to Mg2+ association, we fitted the same experimental data using a model that allowed K+ to bind only to the external sites. The equations for this fit were the same as were used to describe the effects of extracellular Na+ on Mg2+ association (see Zhu and Auerbach 2001 , in this issue). The results show that the fit using this scheme (Fig 3 C; Model Selection Criterion = 4.6) is significantly worse than the fit by Equation 6 (Fig 3 D; Model Selection Criterion = 5.4). Without the incorporation of an internal K+ binding site, the predicted curves deviate from the experimental results because of an overestimation of the voltage dependence of kV+Mg. The small apparent voltage dependence of kV+Mg can be attributed to voltage-dependent binding of extracellular K+ to the internal sites, which are deep within the electric field. Hyperpolarization increases Mg2+ association, but also enhances the occupancy of the internal site by extracellular K+, which in turn serves to reduce the Mg2+ association rate constant. Thus, the results support the conclusion that extracellular K+ binds to both the external sites and the internal site.

Extracellular K+ Reduces the Mg2+ Dissociation Rate Constant but Increases the Mg2+ Permeation Rate Constant
Extracellular K+ and Na+ have different effects on the kinetics of Mg2+ unbinding. [Na+]ex reduces the rate of Mg2+ release from the channel. In contrast, [K+]ex shortens the gaps in the single-channel record that reflect sojourns of Mg2+ in the channel (Fig 4 A), indicating that extracellular K+ increases the rate of Mg2+ release. In addition, the blocking gaps are longer-lived in equivalent concentrations of Na+ versus K+, which further highlights the distinct effects of these two cations.



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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 Equation 1. The best-fit parameters are shown in Table 5. (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 Equation 7 and Equation 9. The best-fit parameters are shown in Table 3 (n = 2).

Fig 4 B shows koff measured at different [K+]ex over a wide range of membrane potentials. (In these experiments, there was no extracellular [Na+]). koff increases with increasing [K+]ex between -100 and -140 mV. However, the difference between koff in 25 vs. 150 mM [K+]ex becomes smaller as the membrane is depolarized, until it disappears entirely at about -80 mV.

We assume that extracellular K+ binding to the external sites is voltage-independent. The apparent Mg2+ dissociation and permeation rate constants were obtained by fitting with Equation 1. The results are shown as solid lines in Fig 4 B, using the parameters in Table 5. k0-Mg is substantially lower in high [K+]ex (a "lock-in" effect), whereas k0pMg is slightly higher in high [K+]ex (a "kick-out" effect).


 
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Table 5. The Effects of Extracellular [K+] on the Apparent Rate Constants for Mg2+ Dissociation and Permeation

We again analyzed the results using schemes in which extracellular K+ can bind to two external sites and one internal site. Accordingly, only the external sites are involved in the modulation of Mg2+ unbinding because the presence of a bound Mg2+ prevents the access of extracellular ions to the internal site. With Na+, the data could be described assuming that only one external site was available when Mg2+ was present in the pore. For the K+ experiments, we used models (having either one or two external sites) that assumed that Mg2+ dissociates back to the extracellular solution only when all of the external sites are vacant:

(7)

The quantitative analysis of the effects of [K+]ex on Mg2+ permeation is more complex because this process could be differentially affected in the case of zero, one, or two K+ bound to the external sites. We use three different models to fit the data.

First, we assumed that only one extracellular K+ binds when the pore is blocked by Mg2+. Thus, the observed Mg2+ permeation rate constant (kVpMg) is the weighted average of only two components, the permeation rate constant without (kV0,pMg) and with (kV1,pMg) a bound K+:

(8)

Note that we have assumed that the voltage dependence of Mg2+ permeation (given by the electrical distance parameter, {lambda}) is the same regardless of the occupancy status of the external site. The observed net Mg2+ release rate (kVoff) as a function of [K+]ex, and V was fitted by the sum of Equation 7 (with n = 1) and Equation 8. The best-fit parameters (Table 6) indicate that, with this scheme, the Mg2+ permeation rate constant is approximately three times greater when there is a K+ at the external site compared with that when this site is empty.


 
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Table 6. Extracellular K+ at the External Cation Site: The Equilibrium Dissociation Constant and the Rate Constants for Mg2+ Dissociation and Permeation

The second model assumed that extracellular K+ occupies either of two external sites, but that the occupancy of only one influences Mg2+ permeation:

(9)

The observed Mg2+ release rate (kVoff) as a function of [K+]ex, and V was again fitted simultaneously by the sum of Equation 7 (with n = 2) and 9. The best-fit parameters (Table 6) indicate that, with this scheme, the Mg2+ permeation rate constant again is more than two times greater when there is a K+ at the external site compared with that when this site is empty.

Finally, we attempted to use a model where the occupancy of two external sites influences both dissociation and permeation, so that the observed Mg2+ permeation rate constant is a weighted average of three components:

(10)

The fit by the sum of Equation 7 (with n = 2) and Equation 10 to the observed Mg2+ "off" rates would not converge. There was a large SD in the estimated value of k02,pMg even after constraining k00,pMg.

In summary, the occupancy of the external site(s) by K+ increases the Mg2+ permeation rate constant by about a factor of three. The analysis does not allow us to distinguish if there are one or two such sites, and, in the case of two sites, if double occupancy alters Mg2+ permeation to a different extent than single occupancy.

A Qualitative Assessment of the Affinity and Selectivity of the Internal Site
In contrast to the external sites, we hypothesize that the internal site specifically binds K+. The low relative affinity of the internal site for intracellular Na+ versus K+ is immediately apparent in Fig 5, which shows the voltage dependence of k+Mg. At -140 mV, k+Mg is the same in 100 and 5 mM [Na+]in (Fig 5 A). This is because there is very little binding of intracellular Na+ to either the external or internal sites at this hyperpolarized potential. (The external site has an apparent dissociation constant of 532 mM for intracellular Na+ at -140 mV; see Zhu and Auerbach 2001 , in this issue) However, at –80 mV, k+Mg is 1.6 times smaller in 100 mM vs. 5 mM [Na+]in. This is because, upon depolarization, intracellular Na+ increasingly occupies the external sites (after crossing the entire electric field) and increasingly inhibits Mg2+ association. The significantly higher voltage dependence in high intracellular Na+ is evidence that intracellular Na+ binds mainly to the external sites, and that the affinity of the internal site for Na+ is so low that it can be ignored.



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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.

In contrast, the internal site has a relatively high affinity for K+. Fig 5 B shows that [K+]in inhibits Mg2+ association at hyperpolarized potentials, and that this inhibition does not show a strong voltage dependence. This is a reflection of the weak voltage dependence of the occupancy of the internal site by intracellular K+. (The internal site apparent affinity for intracellular K+ increases only from 23 mM at 0 mV to 55 mM at -140 mV). We conclude that intracellular Na+ binds mainly to the external sites, whereas intracellular K+ binds to a significant extent to both the internal and the external sites.

Fig 5 also illustrates that the internal site also selects for K+ over Na+ when these ions originate from the extracellular compartment. The inhibition of k+Mg by extracellular Na+ shows only a slight voltage dependence (Fig 5 C), whereas the inhibition by extracellular K+ shows a strong voltage dependence (Fig 5 D). This is consistent with the interpretation that extracellular Na+ binds mainly to the external sites, whereas extracellular K+ binds both to the external sites as well as to the internal site, which lies deep in the electric field.


  DISCUSSION
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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Acknowledgements
References

Ion Binding Sites in the NMDAR Permeation Pathway
Fig 6 shows a fanciful representation of the ion binding sites in the NMDAR pore, motivated by the close structural (Wood et al. 1995 ) and evolutionary (Chen et al. 1999 ) relationship between K+ channels (which are shaped like an inverted teepee; Doyle et al. 1998 ) and cation-selective glutamate receptor channels. When the channel is free of Mg2+, Na+, and K+ interact with two sites that are located in the external portion of the permeation pathway. Either both monovalent cation sites are located outside the electric field, or one is outside and the other is about midway through the electric field (see Zhu and Auerbach 2001 , in this issue), perhaps in a central cavity. K+ also lingers at an additional site that is in the internal portion of the permeation pathway. As a fraction of the electric field (from the extracellular solution), the Mg2+ site is at ~0.60, and the internal, K+-selective site is at ~0.84.



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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 x 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.

Although we can estimate the locations of the ion-binding sites in terms of their electrical distance, we can only guess at their physical locations in the protein. The amino acid sequences in the vicinity of the selectivity filter for representative glutamate receptor and K+ channel subunits are as follows:

KcsA TTVGYGDL

GluR0 TTVGYGDR

NR1 LNSGIGEG

NR2A NNSVPVQN

In KcsA, Ba2+ binds to the channel at the juncture of the selectivity filter and the central cavity (Jiang and MacKinnon 2000 ) near a threonine (Doyle et al. 1998 ). In the NMDAR NR1 subunit, the homologous residue in the sequence is an asparagine, the mutation of which has only a modest effect on Mg2+ block but substantially alters Ca2+ permeability (Burnashev et al. 1992 ; Wollmuth et al. 1998 ). The sequence of the NR2A subunit is not conserved, but mutation of the second of the vicinal asparagines has a strong inhibitory effect on Mg2+ binding (Mori et al. 1992 ; Sharma and Stevens 1996a ; Wollmuth et al. 1998 ). In terms of electrical distances, the Ba2+ site of potassium channels is ~30% from the internal solution, whereas the Mg2+ site of the NMDAR is ~60% from the external solution.

The location of the superficial external monovalent cation site is more difficult to pinpoint. It may be formed by the NH2-terminal domain up to M1 and the M3-M4 linker (Beck et al. 1999 ), and may perhaps relate to external Zn2+-binding residues (Fayyazuddin et al. 2000 ). The lack of voltage dependence in the occupancy of this site is different from that of the internal lock-in site of K+ channels, which are ~30% through the field from the intracellular solution (Neyton and Miller 1988 ).

The internal K+ site of NMDAR appears to be homologous to the external lock-in site of potassium channels, as both are ~15% through the field from the closest bulk solution. In NMDAR, this site may be located in the filter or in an inner vestibule formed by M2 residues (Kuner et al. 1996 ). Mutation of the second glycine in the NR1 sequence, and the final asparagine in the NR2A sequence, reduces the channel conductance for outward current carried by Cs+ (Kupper et al. 1996 ), thus these residues are candidates for the internal site. Mutation of the glutamate in NR1 and the glutamine in NR2A (to lysine) also decreases block by internal Mg2+, but these residues are less attractive candidates because they are not accessible to intracellular sulfhydryl reagents (Kuner et al. 1996 ). A tryptophan residue in both NR1 and NR2 subunits modulates Mg2+ block (Williams et al. 1998 ) and may also influence the internal K+ site. In NMDAR, as in K+ channels, this lock-in site is more selective than the corresponding one on the other face of the permeation pathway.

Although the results and analyses clearly indicate that there are at least three K+ binding sites in the NMDAR channel (two external and one internal), there are certain inconsistencies in the parameter values that suggest that the situation is more complex. First, there is a substantial spread in the estimated rate constant for Mg2+ association in the absence of a membrane potential and competing ions. The value obtained from varying [K+]ex was 2.0 ± 0.1 x 108 M-1s-1 (Table 4), whereas that obtained from varying [Na+]ex was 7.8 ± 2.4 x 108 M-1s-1 (see Table 1 in Zhu and Auerbach 2001 , in this issue). Second, for both Na+ and K+, the apparent affinities of the external sites are higher for intra- versus extracellular ions. However, the parameters indicate that intracellular Na+ associates 8 times faster, whereas K+ associates 18 times faster than its extracellular counterpart, even though these two ions have similar reversal potentials and conductances (Zhu, Y., and A. Auerbach, unpublished observations). Third, the results show that whereas intracellular K+ has ready access to the external sites (K0Kin = 8.3 mM; Table 3), extracellular K+ has an extremely low affinity for the internal site (K0Kex,internal = 16 M; Table 4). We cannot give specific reasons for these inconsistencies. It is possible that Na+ and K+ can differentially alter the shape and/or properties of the permeation pathway via a direct interaction with the protein, as has been proposed for K+ channels (Immke et al. 1999 ). In addition, it is likely that at least some of the basic assumptions of the analysis, e.g., discrete barriers, ion independence and single-filing, are not accurate in detail.

Comparison with Previous Results
Our results agree with those of Antonov et al. 1998 and Antonov and Johnson 1999 with regard to the number and the relative locations in the electric field of the NMDAR external monovalent ion-binding sites, and the effect of occupancy of these sites on the kinetics of Mg2+ blockade. One difference is that we observe that occupancy of an internal site by intracellular K+ accelerates Mg2+ dissociation limits Mg2+ permeation, whereas Antonov and Johnson 1999 did not observe any effect of intracellular Cs+ on the Mg2+ net unbinding rate constant. This difference can perhaps be traced to the difficulty in detecting the effect of intracellular monovalent ions on the kinetics of Mg2+ blockade. First, the increased rate of Mg2+ dissociation and decreased rate of Mg2+ permeation offset, to some extent. That is, the effect of intracellular permeant ions on the net Mg2+ release rate is small. Second, electrostatic repulsion between the bound Mg2+ and the ion at the internal site decreases the affinity of internal site. As a consequence, this site has a very low affinity for intracellular ions. Third, extracellular Na+ reduces Mg2+ dissociation by binding to the external site. Thus, in the presence of a high concentration of extracellular Na+, the enhancement of Mg2+ dissociation by the intracellular permeant ion is obscured. In our experiments, [Na+]ex was low (50 mM), specifically to minimize this effect. Antonov and Johnson 1999 used a high [Na+]ex (140 mM), which is perhaps the main reason why a change in the Mg2+ unbinding rate in different [Cs+]in was not observed. Finally, given the high selectivity of the internal site for K+ over Na+, it is possible that this site has a low affinity for Cs+.

The Effect of Physiological Concentrations of Intracellular K+ on the Apparent Parameters of Mg2+ Block
In contrast to the voltage-independent binding of extracellular Na+ to the external sites, the binding of intracellular K+ to both the external sites and the internal site is voltage-dependent. Therefore, under physiological conditions, intracellular K+ will have a significant influence on the apparent voltage dependence of Mg2+ block.

In the presence of 140 mM K+ in the intracellular solution (and without any permeant ions in the extracellular solution), kV+Mg can be described by Equation 5 (with [Na+]ex = 0). Using the values in Table 1, we used this equation to compute apparent association rate constants in 140 mM intracellular K+ (kV+Mg, 140) between -60 to -140 mV). These were fitted by a standard exponential function to estimate the apparent voltage dependence ({delta}140):

(11)

The fitted parameters were k0+Mg,140 = 1.4 x 106 M-1s-1 and {delta}140 = 0.75. This voltage dependence is about threefold greater than the intrinsic voltage dependence of Mg2+ association ({delta} = 0.24; see Zhu and Auerbach 2001 , in this issue). In 140 mM [K+]in, the Mg2+ association rate constant is only ~0.2% of its value in pure water at 0 mV, but it is 2% of this value at -60 mV. Physiological concentrations of intracellular K+ increase the apparent voltage dependence of Mg2+ association primarily as a consequence of voltage-dependent occupancy of the external site.

A similar approach was used to examine the effect of intracellular K+ on Mg2+ dissociation and permeation. The apparent Mg2+ dissociation rate is given by Equation 2 (with [Na+]ex = 0). Using the values in Table 1 and this equation to compute kV-Mg,140 values, and then fitting these by an exponential function (see Equation 11), we estimate k0-Mg,140 = 2.4 x 104 s-1 and {xi}140 = 0.40. Thus, 140 mM [K+]in alone increases the magnitude of the apparent Mg2+ dissociation rate constant approximately threefold (at 0 mV), but does not influence the apparent voltage dependence of this process ({varepsilon} = 0.35; see Zhu and Auerbach 2001 , in this issue).

The effect of extracellular Na+ on Mg2+ permeation is small (see Zhu and Auerbach 2001 , in this issue). However, occupancy of the internal site by K+ prevents Mg2+ permeation, thus, the effect of intracellular K+ on Mg2+ permeation is expected to be significant. The apparent Mg2+ permeation rate constant is given by Equation 3 (with [Na+]ex = 0). Proceeding as above, we estimate k0pMg,140 = 427 s-1 and {xi}140 = 0.05. Thus, 140 mM [K+]in alone decreases the magnitude of the Mg2+ permeation rate by ~30% of its value in pure water (at 0 mV), but has only a small effect on the apparent voltage dependence of this process {lambda} = 0.35; see Zhu and Auerbach, 2001, in this issue).

We combined the apparent values of the Mg2+ block to estimate an apparent Mg2+ equilibrium dissociation constant. The results were Kd0Mg,140 = 17 mM and {xi}140 = 1.13, compared with the intrinsic values of 12 µM and 0.57, respectively (see Zhu and Auerbach 2001 , in this issue). Thus, 140 mM [K+]in alone causes a >1,400-fold increase in the apparent Mg2+ equilibrium dissociation constant (at 0 mV), and approximately doubles the voltage dependence of equilibrium blockade.

The results suggest that the three monovalent cation-binding sites in the NMDAR permeation pathway serve two basic functions. First, they contribute to the selectivity and the conductance of the channel to Na+ and K+ in ways that remain to be quantified. We hope that our results will serve as a guide for future studies of permeant ion movement through the NMDAR pore. Second, the equilibrium occupancies of the external and internal sites have a strong influence on the magnitude and voltage dependence of Mg2+ block. It is possible that fluctuations in the concentrations of Na+ and K+ in the synaptic gap and/or dendrite regulate the kinetics and equilibrium blockade of NMDAR at synapses.


  Footnotes

1 Abbreviation used in this paper: NMDAR, N-methyl-D-aspartate receptor.


  Acknowledgements
Top
Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Acknowledgements
References

We thank Thomas Kuner and Peter Seeburg for the rat NR1 and NR2A subunit cDNAs, and Jon Johnson for insightful comments on the manuscript.

This work was supported by a grant to A. Auerbach (NS-86554.)

Submitted: 26 May 2000
Revised: 24 January 2001
Accepted: 25 January 2001


  References
Top
Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Acknowledgements
References

    Antonov, S.M., Gimiro, V.E., and Johnson, J.W. 1998. Binding sites for permeant ions in the channel of NMDA receptors and their effects on channel block. Nat. Neurosci. 1:451-461[Medline].

    Antonov, S.M., and Johnson, J.W. 1999. Permeation ion regulation of N-methyl-D-aspartate receptor channel block by Mg2+. Proc. Natl. Acad. Sci. USA. 96:14571-14576[Abstract/Free Full Text].

    Beck, C., Wollmuth, L.P., Seeburg, P.H., Sakmann, B., and Kuner, T. 1999. NMDAR channel segments forming the extracellular vestibule inferred from the accessibility of substituted cysteines. Neuron. 22:559-570[Medline].

    Burnashev, N., Schoepfer, R., Monyer, H., Ruppersberg, J.P., Gunther, W., Seeburg, P.H., and Sakmann, B. 1992. Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor. Science. 257:1415-1419[Medline].

    Chen, G.Q., Cui, C.H., Mayer, M.L., and Gouaux, E. 1999. Functional characterization of a potassium-selective prokaryotic glutamate receptor. Nature. 402:817-821[Medline].

    Doyle, D.A., Morais Cabral, J.H., Pfuetzner, R.A., Kuo, A., Glubis, J.M., Cohen, S.L., Chait, B.T., and MacKinnon, R. 1998. The structure of the potassium channel: molecular basis of conduction and selectivity. Science. 280:69-77[Abstract/Free Full Text].

    Fayyazuddin, A., Villaroel, A., Le Goff, A., Lerma, J., and Neyton, J. 2000. Four residues of the extracellular N-terminal domain of the NR2A subunit control high-affinity Zn2+ binding to NMDA receptors. Neuron 25:683-694[Medline].

    Immke, D, Wood, M., Kiss, L., and Korn, S.J. 1999. Potassium-dependent changes in the conformation of the Kv2.1 potassium channel pore. J. Gen. Physiol. 113:819-836[Abstract/Free Full Text].

    Jiang, Y., and MacKinnon, R. 2000. The barium site in a potassium channel by X-ray crystallography. J. Gen. Physiol. 115:269-272[Abstract/Free Full Text].

    Kuner, T., Wollmuth, L.P., Karlin, A., Seeburg, P.H., and Sakmann, B. 1996. Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines. Neuron. 17:343-352[Medline].

    Kupper, J., Ascher, P., and Neyton, J. 1996. Probing the pore region of recombinant N-methyl-D-aspartate channels using external and internal magnesium block. Proc. Natl. Acad. Sci. USA. 93:8648-8653[Abstract/Free Full Text].

    Mori, H., Masaki, H., Yamakura, T., and Mishina, M. 1992. Identification by mutagenesis of a Mg2+-block site of the NMDA receptor channel. Nature. 358:673-675[Medline].

    Neyton, J., and Miller, C. 1988. Discrete Ba2+ block as a probe of ion occupancy and pore structure in the high conductance Ca2+-activated K+ channel. J. Gen. Physiol. 92:569-586[Abstract].

    Premkumar, L.S., and Auerbach, A. 1996. Identification of a high affinity divalent cation binding site near the entrance of the NMDA receptor channel. Neuron. 16:869-880[Medline].

    Sharma, G., and Stevens, C.F. 1996a. A mutation that alters magnesium block of N-methyl-D-aspartate receptor channels. Proc. Natl. Acad. Sci. USA. 93:9259-9263[Abstract/Free Full Text].

    Sharma, G., and Stevens, C.F. 1996b. Interactions between two divalent ion binding sites in N-methyl-D-aspartate receptor channels. Proc. Natl. Acad. Sci. USA. 93:14170-14175[Abstract/Free Full Text].

    Williams, K., Pahk, A.J., Kashiwagi, K., Masuko, T., Nguyen, N.D., and Igarashi, K. 1998. The selectivity filter of the N-methyl-D-aspartate receptor: a tryptophan residue controls block and permeation of Mg2+. Mol. Pharm. 52:933-941.

    Wollmuth, L.P., Kuner, T., and Sakmann, B. 1998. Adjacent asparagines in the NR2-subunit of the NMDA receptor channel control the voltage-dependent block by extracellular Mg2+. J. Physiol. 506:13-32[Abstract/Free Full Text].

    Wood, M.W., VanDongen, H.M.A., and Vandongen, A.M.J. 1995. Structural conservation of ion conduction pathways in K channels and glutamate receptors. Proc. Natl. Acad. Sci. USA. 92:4882-4886[Abstract].

    Zhu, Y., and Auerbach, A. 2001. Na+ occupancy and Mg2+ block of the N-methyl-D-aspartate receptor channel. J. Gen. Physiol. 117:275-285[Abstract/Free Full Text].