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Original Article

^a Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214
Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, SUNY at Buffalo, 124 Sherman Hall, Buffalo, NY 14214.(716) 829-2569

auerbach{at}buffalo.edu

	ABSTRACT

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The effect of extracellular and intracellular Na⁺ on the single-channel kinetics of Mg²⁺ block was studied in recombinant NR1-NR2B NMDA receptor channels. Na⁺ prevents Mg²⁺ 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, Mg²⁺ binds rapidly (>10⁸ M^–1s^–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 Mg²⁺ dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the Mg²⁺ dissociation rate constant is >10 times greater than the Mg²⁺ permeation rate constant, and the Mg²⁺ equilibrium dissociation constant is 12 µM. Physiological concentrations of extracellular Na⁺ reduce the Mg²⁺ association rate constant 40-fold but, because of the "lock-in" effect, reduce the Mg²⁺ equilibrium dissociation constant only 18-fold.

Key Words: ion binding sites • magnesium • channel blockade • permeation • selectivity

	INTRODUCTION

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At synapses, the channel domain of the N-methyl-D-aspartate receptor (NMDAR) interacts with several different metal cations, including Na⁺, K⁺, Ca²⁺, and Mg²⁺. These interactions have important physiological consequences. Activation of NMDARs depolarizes dendrites, with Na⁺ and K⁺ carrying the bulk of the current. Ca²⁺ entry into dendrites via NMDARs regulates synaptic strength and plasticity (Maren and Baudry 1995; Asztely and Gustafsson 1996). Voltage-dependent Mg²⁺ block of NMDARs may allow these receptors to respond specifically to contemporaneous excitatory inputs and, thus, serve as a substrate for Hebbian learning. Given such critical functions, it is likely that the NMDAR has specific structures that govern the passage of Na⁺, K⁺, Ca²⁺, and Mg²⁺ through its ion permeation pathway.

These four cations interact with the NMDAR protein over time scales that span more than four orders of magnitude. Na⁺ and K⁺ are highly permeable and pass through the pore rapidly. In symmetric, 100-mM divalent cation-free solutions, the NMDAR conductance is 70 pS for both of these ions, which means that at –60 mV, Na⁺ and K⁺ interact with the channel for <0.04 µs. The contact between these ions and the protein is too brief to be resolved as discrete events in the single-channel record. Instead, the interaction of Na⁺ and K⁺ with the NMDAR pore has been probed mainly using current-voltage relationships (Mayer et al. 1984; Nowak et al. 1984; Cull-Candy and Usowicz 1987) and by measuring their effects on channel block (Chen and Lipton 1997; Antonov et al. 1998; Antonov and Johnson 1999).

Under physiological conditions, 10–15% of the NMDAR current is carried by Ca²⁺ (Burnashev et al. 1995). This divalent cation moves through the wild-type (NR1-NR2B) channel 100 times more slowly than Na⁺, i.e., with a rate constant of 10⁶ s^–1 (Premkumar and Auerbach 1996). Thus, Ca²⁺ resides in the permeation pathway for 1 µs. Information regarding Ca²⁺ transmission has been derived mainly from measurements of macroscopic currents (Mayer and Westbrook 1987; Iino et al. 1990; Jahr and Stevens 1993; Sharma and Stevens 1996b), single-channel currents (Iino et al. 1997), or fluxes (Burnashev et al. 1995). However, certain mutations reduce the Ca²⁺ permeation rate constant to such an extent that its binding and unbinding are manifest as excess open channel noise (Premkumar and Auerbach 1996).

Mg²⁺ is the slowpoke of the group. This divalent cation typically dwells in the pore for >100 µs (Ascher and Nowak 1988; Jahr and Stevens 1990). Occupancy of the channel by one Mg²⁺ eliminates conduction by other ions and generates a discrete gap in the single-channel current record. Therefore, the microscopic rate constants for Mg²⁺ entry and exit from the pore can be determined readily using single-channel kinetic techniques (Ascher and Nowak 1988).

The compositions, characteristics, and locations of the important sites of ion interaction in the NMDAR permeation pathway are not completely understood. Mg²⁺ binds to a site that is (in electric distance) about half way through the channel (Wollmuth et al. 1998; Antonov and Johnson 1999). This site is formed, in part, by an asparagine residue in the M2 (pore-forming) segment of the NR2 subunit (Wollmuth et al. 1998), although mutations of other M2 residues also influence Mg²⁺ blockade (Burnashev et al. 1992; Sharma and Stevens 1996a). Ca²⁺ binds to a site that is distinct from the Mg²⁺ binding site and that may lie at the extracellular margin of the electric field of the membrane (Premkumar and Auerbach 1996; Sharma and Stevens 1996b). Na⁺ interacts with two sites that are also located at the extracellular limit of the electric field (Antonov et al. 1998). The residues that constitute the Ca²⁺ and Na⁺ binding sites have not been clearly identified. In addition, almost nothing is known about the key sites of interaction for K⁺ in the NMDAR channel.

The vast difference in residence times for Na⁺ and K⁺ versus Mg²⁺ in the NMDAR channel is such that there are on the order of 10⁴ monovalent cation-binding/unbinding events for each Mg²⁺ binding event. Accordingly, the occupancy by monovalent cations is in steady state on the time scale of Mg²⁺ blockade. The locations and affinities of the binding sites for these mobile ions can be probed by measuring the effect of the extra- and intracellular concentrations of Na⁺ and K⁺ on the kinetics of Mg²⁺ block. This approach has been used to probe permeant ion binding sites in native NMDARs (Antonov et al. 1998; Antonov and Johnson 1999).

In this and in the companion paper (see Zhu and Auerbach 2001, in this issue), we present a single-channel analysis of the effects of extra- and intracellular Na⁺ and K⁺ on the kinetics of Mg²⁺ block of recombinant NR1-NR2A NMDAR. The results suggest that Na⁺ mainly interacts with two sites that are located external to the site of Mg²⁺ blockade, whereas K⁺ interacts with these sites plus an additional site located near the intracellular margin of the electric field. We extrapolate the results to estimate the kinetics, affinity, and voltage dependence of Mg²⁺ block in the absence of competing ions.

	MATERIALS AND METHODS

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Expression of NMDAR in Xenopus Oocytes
Wild-type rat cDNA for the NR1 (splice variant 1) and NR2A subunits were provided by Dr. Thomas Kuner and Dr. Peter Seeburg (Max-Planck Institute for Medical Research, Heidelberg, Germany). These two subunits were coexpressed in Xenopus oocytes by injection of 50 nl each of cRNA (1 µg/ml). Electrophysiology experiments were performed 3–10 d after injection. A more detailed description of the molecular biology and expression protocols is given in Premkumar and Auerbach 1996.

Electrophysiology and Solutions
Single-channel currents were recorded from outside-out patches. Recording pipets were pulled from borosilicate glass (World Precision Instruments) and were coated with Sylgard (Dow Corning). The pipet resistance was 10–15 M. Patch pipets were filled with the following reagents (in mM): 5–100 NaCl, 2 K₂ATP, 1 BAPTA, 0.25 GTP, and 10 HEPES, pH adjusted to 7.3. The extracellular solution contained 50 µM NMDA, 10 µM glycine, 2.5 mM KCl, 5 mM HEPES, and 1.5 mM EDTA (pH adjusted to 7.3) plus added NaCl, KCl, and MgCl₂ (ultrapure grade from Johnson Mattey). BAPTA, EDTA, HEPES, and all other salts were obtained from Sigma-Aldrich. Without compensation by other ions, the amount of NaCl or KCl was adjusted to achieve the desired Na⁺ or K⁺ concentration. Using the parameters estimated by the program MAXC, the desired free Mg²⁺ concentrations were established by adding the calculated amount of MgCl₂ to solutions buffered with EDTA (1.5 mM) as the Mg²⁺ chelator. Here, we report [Mg²⁺] as a concentration rather than an activity. Glucose was added to the extracellular solution or pipet solution to balance the osmolarity. The junction potentials between the pipet solution and the extracellular solution were calculated and the membrane potential was corrected accordingly (Barry and Lynch 1991). All experiments were performed at 22–25°C. Upon excision of patches in the outside-out configuration, an ALA BPS-4 perfusion system (ALA Scientific Instruments) controlled the exchange of experimental and control solutions.

Signal Processing
The currents were recorded using a patch-clamp amplifier (model EPC-7; Medical-Systems-List). The currents were low-pass filtered at 10 kHz, sampled at 94 kHz using a data recorder (model VR-10B; Instrutech Corp.), and stored on videotape. Recorded currents were stored on a PC at sampling frequency of 94 kHz using a VR-111 interface (Instrutech Corp.).

Kinetic Analysis
To study the kinetic properties of Mg²⁺ block, it was necessary to distinguish the blocked state from the other nonconducting (closed) states. In the absence of Mg²⁺, NR1-NR2A receptors have two main conductance levels: open and closed (Fig. 1 A, top trace). There were usually two components in the open interval lifetime distribution (0.1 ms, 29%; and 5 ms, 71%; data not shown) and at least three components in the closed interval lifetime distribution (0.5 ms, 22%; 7 ms, 12%; and a duration >40 ms that varied with the agonist concentration; data not shown). The addition of Mg²⁺ to the extracellular solution induces frequent, brief gaps (Fig. 1 A, middle and bottom traces) that reflect the binding and unbinding of Mg²⁺ to the NMDAR channel.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   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 –80 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 s–1, and the inverse of the mean block time was 901 s–1, 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 (o) 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 µM–1s–1; mean ± SD).

We used the following kinetic model (Fig. 1) to describe the transitions between open and blocked states:

View larger version (4K): [in this window] [in a new window] [Download PPT slide]   Scheme S1

where is the intrinsic channel closing rate constant. Thus, the inverse of the channel open lifetime is a linear function of [Mg²⁺]_ex, with a slope equal to k_+Mg (Fig. 1 C). Mg²⁺ unbinding, either by dissociation back to the extracellular solution or permeation through the channel, relieves the block. We define the sum of k_–Mg and k_pMg to be k_off, which is the net rate for Mg²⁺ release from the pore.

According to this model, the equilibrium dissociation constant for Mg²⁺ (K_{d, Mg}) is defined as the ratio of the "off" rate and the association rate constant:

In the following studies, k_+Mg, k_–Mg, and k_pMg were estimated as a function of the concentration of permeant ions (Na⁺ or K⁺). Throughout, we use k to represent the apparent rate constant for Mg⁺² block in the presence of permeant ions, and the Greek letter for the corresponding rate constant in the absence of competing ions.

Fitting, Simulations, and Statistics
Fits and simulations were made using Microcal Origin (version 4.0; Microcal Software) and ScientisT (version 2.0; MicroMath Scientific Software). In ScientisT, the comparison of the models having different numbers of free parameters was carried out using the Model Selection Criterion (MSC):

where n is the number of points, p is the number of free parameters, and _obs is the mean of the observed data. The MSC will give the same ranking as the Akaike Information Criterion, but is normalized so that it is independent of the scaling of the data points. The most appropriate model, regardless of the number of free parameters, is the one with the largest MSC.

The number of intervals in each open or closed duration histogram was 1,000. Each symbol in the figures is the mean ± SD of from three to seven patches. In most cases, the SD was smaller than the size of the symbol and is not visible.

	RESULTS

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Extracellular Na⁺ Decreases the Mg²⁺ Association Rate Constant in a Voltage-independent Manner
Fig. 2 A illustrates the effects of extracellular Na⁺ on Mg²⁺ association. At higher extracellular Na⁺ concentrations ([Na⁺]_ex) openings are longer, indicating a reduced rate of Mg²⁺ association. In Fig. 2 B the inverse open channel lifetime is plotted as a function of [Mg²⁺]_ex for different [Na⁺]_ex. The slope of this relationship, which is the apparent Mg²⁺ association rate constant, is nearly five times slower in 150 mM [Na⁺]_ex compared with 50 mM [Na⁺]_ex.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   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 = –80 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/o) 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 µM–1s–1 in 50 mM [Na+]ex to 109.6 ± 6.9 µM–1s–1 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.

(1)

where k^V_+Mg is the apparent Mg²⁺ association rate constant at membrane potential V, k⁰_+Mg is this rate constant in the absence of a membrane potential, and is the apparent voltage dependence of this rate constant (i.e., the inverse of the voltage that elicits an e-fold change). The voltage dependence of k^V_+Mgwas the same at two different [Na⁺]_ex (Fig. 2 C), which indicates that the inhibition of the apparent Mg²⁺ association rate constant by extracellular [Na⁺] is not voltage-dependent.

Intracellular Na⁺ Decreases the Mg²⁺ Association Rate Constant in a Voltage-dependent Manner
Fig. 3 A shows the effects of intracellular Na⁺ on Mg²⁺ association. The open channel lifetime is longer at higher [Na⁺]_in, indicating a reduced k_+Mg. Fig. 3 B shows that the reduction in k_+Mg by [Na⁺]_in decreases with hyperpolarization, i.e., that [Na⁺]_in inhibits Mg²⁺ association in a voltage-dependent manner. Using , the apparent voltage dependence of Mg²⁺ association decreases from 44 ± 1 mV per e-fold change in 5 mM [Na⁺]_in, to 32 ± 2 mV per e-fold change in 100 mM [Na⁺]_in.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   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 –80 mV. (A, right) Open interval duration histograms. Because intracellular Na+ slows the association of extracellular Mg2+, the inverse of open channel lifetime (o) 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.

We start with a simple "one-site" scheme to describe the effects of Na⁺ on Mg²⁺ association. It is assumed that the Na⁺ site is external to, or at the same location as, the Mg²⁺ site, and that extracellular Mg²⁺ can enter and block the channel only when the Na⁺ site is empty. Accordingly (see ), the apparent Mg²⁺ association rate constant (i.e., in the presence of Na⁺), k^V_+Mg, is a function of three experimental variables ([Na⁺]_ex, [Na⁺]_in, and V) and five free parameters (⁰_+Mg, K_Naex, K_Nain, , and ):

(2)

k^V_+Mg is the Mg²⁺ association rate constant in the absence of competing ions at membrane potential V, and K_Naex and K⁰_Nain are dissociation constants (k_off/k_on) for [Na⁺]_ex and [Na⁺]_in, respectively, with no membrane potential. Note that because Na⁺ is a permeant species, these are not equilibrium constants. The two apparent fractional electrical distances in are (between the extracellular compartment to the peak of the entry barrier for Mg²⁺) and (between the Na⁺ binding site and the intracellular compartment). k_B is Boltzmann's constant, and T is the absolute temperature (k_BT = 25.3 mV).

Fig. 4 A shows the result of fitting simultaneously (using ) two groups of measurements of k^V_+Mg as a function of membrane potential. The first group (Fig. 4 A, left) was obtained from five different [Na⁺]_ex (25–150 mM) at a single, high [Na⁺]_in (100 mM). The second group (Fig. 4 A, right) was obtained from two different [Na⁺]_ex (50 and 150 mM) at a single, low [Na⁺]_in (5 mM). The curves drawn according to the best fit by clearly do not describe the experimental data.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   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 "n-site" 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).

(3)

where n is a free parameter. Fig. 4 B illustrates the fit of the same two groups of experimental data by . The results are given in Table . A two-site scheme is able to describe the experimental results across Na⁺ concentrations and voltages.

(4)

, with eight free parameters, could not be fit to the data (i.e., the SD of the parameters became large). To reduce the number of free parameters, we imposed the constraint of a single voltage dependence term for intracellular Na⁺ occupancy (i.e., ₂ = ₁). The results are shown in Table . The fit using the constrained (MSC = 5.9) was better than using (MSC = 5.5), indicating that the two Na⁺ sites probably are not identical. The apparent dissociation constants differed by 7-fold for extracellular Na⁺ (113 ± 35 vs. 15 ± 5 mM) and 3.6-fold for intracellular Na⁺ (7.2 ± 5.5 vs. 2.0 ± 0.8 mM). The intrinsic Mg²⁺ association rate constant and fractional electrical distance were similar with and .

The results indicate that there are at least two Na⁺ binding sites in the external portion of the ion permeation pathway that must be empty for Mg²⁺ to associate with the NMDAR pore. Extracellular Na⁺ occupies these sites in a nearly voltage-independent manner, whereas intracellular Na⁺ occupies these sites in a highly voltage-dependent manner. Na⁺ permeates through the channel, thus, this voltage dependence cannot be directly related to a location of the external sites in the electric field.

This analysis provides information on the intrinsic rate constant of Mg²⁺ association in pure water (i.e., in the absence of competing ions). The association rate constant for extracellular Mg²⁺ is very high (>10⁸ M^–1s^–1), even in the absence of a membrane potential. The peak of the barrier of the Mg²⁺ association rate constant is located 25% through the electric field from the extracellular surface.

Extracellular Na⁺ Reduces the Mg²⁺ Dissociation Rate Constant
A bound Mg²⁺ has two routes by which it can exit the channel. It can either dissociate back into the extracellular compartment (rate constant k^V_–Mg) or it can permeate into the intracellular compartment (rate constant k^V_pMg). Both of these processes may be influenced by the presence of Na⁺ in the permeation pathway, and such effects are of interest insofar as they provide information about the location of the Na⁺ binding sites with respect to the Mg²⁺ site.

Only the sum of the exit rate constants, k^V_off, can be measured directly from the duration of the blocking gaps in the single-channel current record. However, because the exit routes require Mg²⁺ to move in opposite directions in the electric field, k^V_–Mg and k^V_pMg will have opposite voltage dependencies, the former decreasing and the latter increasing with hyperpolarization. Using the standard barrier framework, the apparent exit rate constants can be estimated using the relationships:

(5)

where k⁰_–Mg and k⁰_pMg are the apparent exit rate constants in the absence of a membrane potential, is the fractional electrical distance from the Mg²⁺ binding site to the peak of the dissociation barrier, and is the fractional electrical distance from the binding site to the peak of the permeation barrier (see Fig. 6).

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   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 pertains to the Mg2+ entry barrier (= 0.25), pertains to the Mg2+ dissociation (= 0.35), and pertains to the Mg2+ permeation barrier (= 0.03). The Mg2+ binding site is 0.60 through the electric field from the extracellular solution.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   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 = –80 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 0–Mg = 8,944 ± 205 s–1, = 0.36 (fixed), k0pMg = 624 ± 5 s–1, = 0.03 (fixed), and KNaex = 251 ± 12 mM. Additional results are summarized in Table and Fig. 6.

(6)

n is the number of (independent and identical) sites that must be occupied to lock-in the Mg²⁺, ^V_–Mg is the Mg²⁺ dissociation rate constant when all of the salient external Na⁺ sites are empty at membrane potential V, and J_d,Naex is the equilibrium dissociation constant of [Na⁺]_ex for each Na⁺ site, and is assumed to be independent of the membrane potential. Note that J_d,Naex is a true equilibrium dissociation constant for Na⁺ when there is a Mg²⁺ in the pore, whereas K_Naex is an apparent dissociation constant for extracellular Na⁺ when the pore does not contain a Mg²⁺.

Four sets of experimental data were fitted by , either with n as a fixed parameter (equal to 1 or 2) or as a free parameter. and were fixed at their previously determined values (0.36 and 0.03, respectively), leaving only ⁰_–Mg, J_d,Naex, and k⁰_pMg as free parameters. The predicted curves match the experimental results (Fig. 5 C), with the best-fit parameters shown in Table . The results indicate that a model having a single Na⁺ site (with an equilibrium dissociation constant of 88 mM), or one with two independent Na⁺ sites (each with an equilibrium dissociation constant of 251 mM) can account for the results. In pure water, the Mg²⁺ dissociation rate constant is 9,000 s^–1, and the Mg⁺² permeation rate constant is 624 s^–1.

	DISCUSSION

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Changes in the Mg²⁺ parameters as a function of the intra- and extracellular Na⁺ concentrations were interpreted as arising from occupancy of specific sites in the ion permeation pathway by the monovalent ion. Because the ionic strength was not constant, it is also possible that some of the effects can be attributed to different degrees of surface charge. Several results lead us to suspect that charge screening was not a major factor. First, the magnitudes of the observed changes in k_+Mg are greater than those predicted by charge-screening effects alone. In native hippocampal NMDAR, a reduction in extracellular Cs⁺ from 150 to 10 mM creates an excess local negative potential of 6.5 mV (Zarei and Dani 1994), which would be expected to increase k_+Mg only e^6.5/12.5 = 1.7-fold. We observe a 4.6-fold increase in this rate constant between 150 and 50 mM extracellular Na⁺ (Fig. 2). Second, increasing the intracellular Na⁺ concentration reduces the apparent association rate constant for extracellular Mg²⁺ in a voltage-dependent manner, which is not predicted by a simple charge-screening mechanism. Third, the difference in the apparent affinity of the external sites for extracellular Na⁺ versus K⁺ (see Zhu and Auerbach 2001, in this issue) suggests specific binding rather than a nonspecific surface charge effect. Fourth, the ability of extracellular Na⁺ to prevent Mg²⁺ dissociation (the lock-in effect; Table ) is more consistent with occupancy of a binding site than with a charge screening mechanism. Overall, the results suggest that monovalent cations exert their effects on Mg²⁺ association and dissociation predominantly by binding to specific sites in the ion permeation pathway rather than by acting via a nonspecific electrostatic shielding mechanism.

The Na⁺ Binding Sites
The results suggest that occupancy of either of the two external sites by Na⁺ (arising from the extracellular or the intracellular compartment) slows or prevents Mg²⁺ entry into the channel from the extracellular solution. When the pore is free from Mg²⁺, extracellular Na⁺ binding is voltage-independent, whereas intracellular Na⁺ binding is strongly voltage-dependent. When the pore is blocked by Mg²⁺, a voltage-independent occupancy of a single external site by Na⁺ is sufficient to prevent Mg²⁺ dissociation to the extracellular compartment, but is without effect on Mg²⁺ permeation.

In unblocked NMDAR, the voltage sensitivities of the Na⁺ effect cannot be used to pinpoint the locations of the external sites in the electric field because we do not know the extent to which Na⁺ release is determined by its dissociation back to the extracellular solution versus permeation. If dissociation dominates, then the voltage independence of the dissociation constant for extracellular Na⁺ would indicate that both sites lie near or beyond the external margin of the electric field. However, if Na⁺ release is predominantly determined by its permeation to the intracellular compartment, the voltage sensitivities would suggest that at least one of the external sites is located deep in the pore. Under this condition, Na⁺ occupancy of a deep site would show a reduced voltage dependence because the on and off rates to the site will change in the same direction with a change in the membrane potential. The apparent voltage dependence of occupancy would disappear if the electrical distance from the site to the peak of the permeation barrier was similar to that from the extracellular solution to the entry barrier (i.e., , for Na⁺). At the same time, intracellular Na⁺ occupancy of that site would exhibit a steep voltage dependence because the on and off rates would change in opposite directions with voltage.

Our results do not allow us to unequivocally pinpoint the locations of the Na⁺ sites. Some results point to a separation of the Na⁺ sites, with one external Na⁺ site located near or beyond the extracellular margin of the electrical field and the other being close to, or perhaps the same as, the Mg²⁺ site (which is 0.6 though the electric field). First, the higher apparent affinity of the external sites for intracellular (compared with extracellular) Na⁺ suggests that the barrier to Na⁺ dissociation to the external compartment is higher than the Na⁺ permeation barrier. Thus, Na⁺ release is likely to be dominated by permeation. Second, although occupancy of either of two external sites prevents Mg²⁺ association, the results are consistent with a scheme in which only a single site must be occupied to prevent Mg²⁺ dissociation to the extracellular solution. The occupancy of this lock-in site for Na⁺ is not voltage-dependent. Because this affinity constant is determined under equilibrium conditions (i.e., no permeation), this indicates that this site lies near or beyond the extracellular boundary of the electric field. These arguments are not definitive, and the existence of two nonidentical Na⁺ sites near or beyond the extracellular limit of the electric field remains a possibility.

Mg²⁺ Block in Pure Water
The intrinsic parameters for Mg²⁺ binding to the NMDAR pore, i.e., in the absence of competing permeant ions, are shown in Fig. 6. In pure water and no membrane potential, the association rate constant for extracellular Mg²⁺ is 7.8 x 10⁸ M^–1s^–1. This is 100 times faster than the apparent association rate constant in 140 mM extracellular Na⁺. The large magnitude of this rate constant indicates that, in the absence of competing ions, Mg²⁺ has unhindered access to its blocking site in the channel.

In pure water and no membrane potential, Mg²⁺ exits the NMDAR pore mainly by dissociating to the extracellular solution (at 9,000 s^–1), but it can also permeate into the intracellular compartment (at 62 s^–1). Thus, under these conditions, Mg²⁺ stays in the pore 0.1 ms and 6.5% of the blocking events results in the permeation of a Mg²⁺. The Mg²⁺ permeation rate constant is slow and predicts that this ion can carry only a tiny current (0.2 fA) that would normally be an insignificant fraction of the total single-channel current. From the rate constants, we estimate that in the absence of competing ions and at zero membrane potential, the intrinsic equilibrium dissociation constant of the NMDAR for Mg²⁺ is 12 µM.

The main barrier to Mg²⁺ association is located 0.25 through the electric field (i.e., this process increases e-fold with a 46-mV hyperpolarization). At –60 mV, the Mg²⁺ association rate constant in pure water is 2.2 x 10⁹ M^–1s^–1. The entry barrier appears to be nearly symmetric, as a bound Mg²⁺ must traverse 0.35 of the field to return to the extracellular compartment (35 mV for an e-fold change). The sum of these two positional parameters indicates that the Mg²⁺ binding site is located 0.6 through the electric field from the extracellular solution. In contrast, the barrier to Mg²⁺ permeation is very steep, as the ion, once bound, must traverse only 0.03 of the field to reach the intracellular compartment. Thus, hyperpolarization has very little effect on Mg²⁺ permeation, per se, whereas it significantly speeds the association from, and slows the dissociation of Mg²⁺ to, the extracellular solution.

In the absence of competing ions, equilibrium block by Mg²⁺ increases e-fold with a hyperpolarization of 21 mV, so that at –60 mV, the Mg²⁺ equilibrium dissociation constant is only 0.7 µM. Under these conditions, almost one out of every three Mg²⁺ that binds permeates through the channel.

Comparison with Native NMDARs
There have been several excellent studies of the effects of extracellular Na⁺ and intracellular Cs⁺ on block of native NMDAR (embryonic rat cortical neurons, probably composed of a mixture of NR2A and B subunits) by adamantine derivatives (Antonov et al. 1998) and Mg²⁺ (Antonov and Johnson 1999). For the most part, our results using recombinant NMDARs are in good agreement with these studies. Both sets of results show that extracellular Na⁺ binds to two sites (average K_Naex = 40 mM) that are in the external portion of the channel to thereby prevent the entry of Mg²⁺ into the pore. In addition, both sets of results show a lock-in effect, i.e., that the occupancy of either site prevents Mg²⁺ dissociation back to the extracellular solution. Our results also agree with those of Antonov and Johnson 1999 with respect to the intrinsic parameters for Mg²⁺ association. Both sets of results indicate a similar electrical distance of the association barrier from the extracellular solution (0.25 vs. 0.23) and a large association rate constant at zero potential in pure water (8 vs. 11 x 10⁸ M^–1s^–1).

Our measurements indicate that the intrinsic Mg²⁺ dissociation rate constant is 10 times slower, and that the Mg²⁺ permeation rate constant is 10 times faster than was reported by Antonov and Johnson 1999. As a consequence, we estimate a higher intrinsic affinity (in pure water and the absence of a membrane potential) of the pore for extracellular Mg²⁺ (12 vs. 101 µM). Moreover, our estimate of the location (in electrical distance) of the Mg²⁺ binding site (0.60) is different from that of Antonov and Johnson (0.47). We doubt that these differences arise from a different subunit composition of recombinant (NR2B) versus native systems (NR2A and NR2B). Rather, we speculate that these differences arise from the fact that they used 130 mM Cs⁺ in their intracellular solution, whereas we used 5 mM Na⁺. Occupancy of an internal monovalent cation-binding site by Cs⁺ will increase the apparent rate constant for Mg²⁺ dissociation and slow the apparent rate constant for Mg²⁺ permeation (see Zhu and Auerbach 2001, in this issue).

Mg²⁺ Block and Unblock as a Function of the Na⁺ Concentrations and the Membrane Potential
and can be combined to describe Mg²⁺ block kinetics as a function of [Na⁺] and the membrane potential (in the absence of other competing ions). Using the parameters shown in Table and Table , at –60 mV and under the conditions [Na⁺]_ex = 140 mM and [Na⁺]_in = 10 mM, the apparent Mg²⁺ block and unblock rates are, respectively, 2 x 10⁸ M^–1s^–1 and 1,270 s^–1, with about equal probabilities of release to the extra- and intracellular compartments, yielding an apparent K_d of 0.6 mM. These conditions are not "physiological" because they do not incorporate the effects of K⁺, which is present at a high concentration in the intracellular compartment. In the companion paper (see Zhu and Auerbach 2001, in this issue) we describe the effects of extra- and intracellular K⁺ on the kinetics of Mg²⁺ blockade.

We assume that (1) there is a single Na⁺ binding site; (2) Mg²⁺ can bind to the channel only when this site is empty; and (3) the Na⁺ association and dissociation rate constants are much faster than the Mg²⁺ association rate constant. Thus,

(A1.1)

where k^V_+Mgis the apparent Mg²⁺ association rate constant at membrane potential V, (i.e., in the presence of Na⁺), k^V_+Mgis the Mg²⁺ association rate constant in pure water at membrane potential V, and P^e_Na is the probability of the Na⁺ site being empty. P^e_Na is a function of the equilibrium dissociation constants and ion concentrations:

(A1.2)

where K_Naex and K^V_Nain are the unidirectional dissociation constants (k_off/k_on) for [Na⁺]_ex and [Na⁺]_in, respectively. k^V_+Mgis related to the membrane potential by:

(A1.3)

where ⁰_+Mgis the Mg²⁺ association rate constant in the absence of a membrane potential, is the fractional electrical distance between the extracellular compartment and the peak of the entry barrier for Mg²⁺, k_B, is the Boltzmann constant, and T is the absolute temperature (under our conditions, k_BT = 25.3 mV). We assume that K_Naex is voltage-independent (2 C), and that K^V_d,Nain is voltage-dependent (3 C) and is related to the membrane potential by:

(A1.4)

where K⁰_Nain is the intracellular Na⁺ equilibrium dissociation constant at zero membrane potential, and is the fractional electrical distance of the Na⁺ site from the intracellular solution. We now generate a description of k^V_+Mg as a function of three experimental variables ([Na⁺]_ex,_, [Na⁺]_in, and V) and five free parameters (⁰_+Mg, K_d,Naex, K_d,Nain, , and ):

(A1.5)

We assume that there are n identical and independent Na⁺ binding sites that must be empty for Mg²⁺ to dissociate back to the extracellular compartment. The apparent Mg²⁺ dissociation rate (i.e., in the presence of extracellular Na⁺) is:

(A2.1)

where ^V_–Mg is the Mg²⁺ dissociation rate constant in pure water at membrane potential V and P^e_Na is the probability that all of the Na⁺ sites are empty. Because intracellular Na⁺ cannot reach the external site(s) when Mg²⁺ is bound, was modified so that P^e_Na depends only on [Na]_ex:

(A2.2)

where J_d,Naex is the equilibrium dissociation constant of [Na⁺]_ex for each Na⁺ site and is assumed to be independent of the membrane potential. Note that J_d,Naex is a true equilibrium dissociation constant for Na⁺ when there is a Mg²⁺ in the pore, whereas K_Naex is an apparent (nonequilibrium) dissociation constant for Na⁺ when the pore does not contain a Mg²⁺.

We now describe k^V_–Mg as a function of [Na⁺]_ex and V:

(A2.3)

and k^V_off as a function of the experimental variables [Na⁺]_ex and V:

(A2.4)

	ACKNOWLEDGMENTS

 
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 REFERENCES

 

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

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

Ascher P. & Nowak L.. The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture, J. Physiol, 399, 1988, 247–266.[Abstract/Free Full Text]

Asztely F. & Gustafsson B.. Ionotropic glutamate receptorstheir role in the expression of hippocampal synaptic plasticity, Mol. Neurobiol., 12, 1996, 1–11.[Medline]

Barry P.H. & Lynch J.W.. Liquid junction potentials and small cell effects in patch-clamp analysis, J. Membr. Biol, 121, 1991, 101–117.[Medline]

Burnashev N., Schoepfer R., Monyer H., Ruppersberg J.P., Gunther W., Seeburg P.H. & Sakmann B.. Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor, Science., 257, 1992, 1415–1419.[Abstract/Free Full Text]

Burnashev N., Zhou Z., Neher E. & Sakmann B.. Fractional calcium currents through recombinant GluR channels of the NMDA, AMPA and kainate receptor subtypes, J. Physiol., 485, 1995, 403–418.[Abstract/Free Full Text]

Chen H.S. & Lipton S.A.. Mechanism of memantine block of NMDA-activated channels in rat retinal ganglion cellsuncompetitive antagonism, J. Physiol, 499, 1997, 27–46.[Abstract/Free Full Text]

Chung S.H., Moore J.B., Xia L.G., Premkumar L.S. & Gage P.W.. Characterization of single channel currents using digital signal processing techniques based on hidden Markov models, Proc. R. Soc. Lond. B, 329, 1990, 265–285.

Cull-Candy S.G. & Usowicz M.M.. Multiple-conductance channels activated by excitatory amino acids in cerebellar neurons, Nature., 325, 1987, 525–528.[Medline]

Hille B., Ionic Channels of Excitable Membranes, 2nd edition, 1992, Sinauer Associates, Inc, Sunderland, MApp. 609 pp.

Iino M., Ozawa S. & Tsuzuki K.. Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurones, J. Physiol, 424, 1990, 151–165.[Abstract/Free Full Text]

Iino M, Ciani S., Tsuzuki K., Ozawa S. & Kidokoro Y.. Permeation properties of Na⁺ and Ca²⁺ ions through the mouse epsilon2/zeta1 NMDA receptor channel expressed in Xenopus oocytes, J. Membr. Biol, 155, 1997, 143–156.[Medline]

Jahr C.E. & Stevens C.F.. A quantitative description of NMDA receptor-channel kinetic behavior, J. Neurosci., 10, 1990, 1830–1837.[Abstract]

Jahr C.E. & Stevens C.F.. Calcium permeability of the N-methyl-D-aspartate receptor channel in hippocampal neurons in culture, Proc. Nat. Acad. Sci, USA. 90, 1993, 11573–11577.[Abstract/Free Full Text]

Maren S. & Baudry M.. Properties and mechanisms of long-term synaptic plasticity in the mammalian brainrelationships to learning and memory, Neurobiol. Learn. Mem., 63, 1995, 1–18.[Medline]

Mayer M.L. & Westbrook G.L.. Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cations in mouse cultured central neurones, J. Physiol, 394, 1987, 501–527.[Abstract/Free Full Text]

Mayer M.L., Westbrook G.L. & Guthrie P.B.. Voltage-dependent block by Mg²⁺ of NMDA responses in spinal cord neurones, Nature, 309, 1984, 261–263.[Medline]

Nowak L., Bregestovski P., Ascher P., Herbet A. & Prochiantz A.. Magnesium gates glutamate-activated channels in mouse central neurones, Nature., 307, 1984, 462–465.[Medline]

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

Qin F., Auerbach A. & Sachs F.. Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events, Biophys. J., 70, 1996, 264–280.[Medline]

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

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

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

Zarei M.M. & Dani J.A.. Ionic permeability characteristics of the N-methyl-D-aspartate receptor channel, J. Gen. Physiol., 103, 1994, 231–248.[Abstract/Free Full Text]

Zhu Y. & Auerbach A.. K⁺ occupancy of the N-methyl-D-aspartate receptor channel probed by Mg²⁺ block, J. Gen. Physiol., 117, 2001, 287–297.[Abstract/Free Full Text]

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