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The Journal of Neuroscience, April 1, 2002, 22(7):2550-2560
Glutamate But Not Glycine Agonist Affinity for NMDA Receptors Is Influenced by Small Cations
Rinat Nahum-Levy*, Eyal Tam*, Sara Shavit, and Morris Benveniste Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Israel
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
NMDA receptor currents desensitize in an agonist-dependent manner when either the glutamate or glycine agonist is subsaturating. This may result from a conformational change in the NMDA receptor protein that lowers glutamate and glycine binding site affinity induced by co-agonist binding, channel opening, or ion permeation. We have used whole-cell voltage clamp of cultured hippocampal neurons with agonist paired-pulse protocols to demonstrate that glutamate and glycine dissociate 7.9- and 6.8-fold slower in the absence of their respective co-agonists than when their co-agonists are present. Paired-pulse and desensitization protocols were used to show that co-agonist binding and channel opening are sufficient to cause a reduction in glycine affinity, but extracellular sodium or magnesium binding was required in addition to conformational changes leading to channel opening to reduce glutamate binding-site affinity. Use of cesium or potassium as the major extracellular cation prevented the reduction of glutamate affinity. In addition, the use of choline-, sodium-, or cesium-based intracellular solutions did not alter desensitization characteristics, indicating that the site responsible for reduction of glutamate affinity is not in the intracellular domain. The fact that the reduction of glutamate affinity is dependent on certain small extracellular cations whereas the reduction of glycine affinity is insensitive to such cations indicates that conformational changes induced by the binding of glutamate are not completely paralleled by the conformational changes induced by glycine. Although glutamate and glycine are essential co-agonists, these data suggest that they have differential roles in the process of NMDA receptor activation.
Key words: glutamate; glycine; NMDA receptor; ion permeation; desensitization; channel gating; channel activation
INTRODUCTION
Activation of NMDA receptors can cause both membrane depolarization and calcium influx into the postsynaptic neuron. Such a response can initiate changes in cellular physiology that may include induction of intracellular cascades, gene activation, and possible changes in synaptic strength (for review, see Ozawa et al., 1998
; Dingledine et al., 1999
In whole-cell voltage-clamp experiments, sustained glutamate and glycine agonist applications of constant concentration can result in a decay or "desensitization" of the receptor response. One type of NMDA receptor desensitization can be observed at saturating glutamate and glycine agonist concentrations and may be sensitive (Legendre et al., 1993
High activity of glutamate and glycine transporters near the synaptic cleft of many excitatory synapses may reduce glutamate and glycine concentrations to subsaturating levels (Bergles et al., 1999
MATERIALS AND METHODS
Dissociated neuronal cultures. Sprague Dawley postnatal day 1 rat pups were decapitated, and 14 hippocampal hemispheres were removed. The tissue was digested with papain (100 U; Sigma, St. Louis, MO) for 20 min, triturated to a single-cell suspension, and plated at a density of 150,000 cells per milliliter on a substrate of bovine collagen type IV and 100 µg/ml poly-L-lysine in 35 mm dishes. The culture medium consisted of Modified Eagle's Medium containing 5% horse serum (Biological Industries, Beit HaEmek, Israel), B-27 neuronal supplement (Invitrogen, Carlsbad, CA), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. D-Glucose was supplemented to a final concentration of 6 gm/l. Cytosine-1-
-D-arabinofuranoside (5 µM) was added after ~5 d to arrest glial cell division. All cultures were maintained at 36°C in humidified air containing 5% CO2.
Electrophysiology and agonist application. Whole-cell voltage-clamp experiments were conducted using an Axopatch 200A amplifier (Axon Instruments, Union City, CA) at room temperature between 1 and 2 weeks after neurons were plated. The holding potential was
60 mV unless indicated otherwise. Data were acquired with a Macintosh PPC 7600 computer equipped with an ITC-16 analog-to-digital converter (Instrutech Corp., Port Washington, NY) using a free Macintosh-based electrophysiological software package (Synapse, Synergistic Systems, Bromma, Sweden).
The standard extracellular control solution consisted of 160 mM NaCl, 2.5 mM KCl, 0.2 mM CaCl2, 10 mM glucose, 10 mM HEPES, 400 nM tetrodotoxin, 5 µM bicuculline methochloride, and 10 µg/ml phenol red and was adjusted to pH 7.3 and 325 mOsm. In some cases, 160 mM choline chloride, CsCl, or KCl was substituted for NaCl. Unless indicated otherwise, the standard intracellular solution consisted of 125 mM CsMeSO3, 15 mM CsCl, 0.5 mM CaCl2, 3 mM MgCl2, 5 mM Cs4BAPTA, and 2 mM Na2ATP and was adjusted to pH 7.2 and 305 mOsm. In some cases, 140 mM NaCl replaced CsMeSO3 and CsCl, and BAPTA-free acid was used instead of its cesium salt and titrated with NaOH. In other cases, 140 mM choline chloride replaced CsMeSO3 and CsCl, and BAPTA-free acid was used instead of its cesium salt and titrated with Trizma base; and Tris-ATP was used instead of its sodium salt.
Experimental solutions were perfused continuously over the whole neuron from a flow pipe consisting of an array of eight glass barrels (
400 µm outer diameter) that was positioned ~100 µm away from the neuronal soma as described previously (Nahum-Levy et al., 1999
Four different agonists were used in this study. In general for desensitization experiments, the background agonist present before NMDA receptor-channel activation had a relatively high affinity such that desensitization kinetics could be observed at subsaturating concentrations [for more details, see Nahum-Levy et al. (2001)
Two different treatments were used to reduce the level of NMDA receptor activation resulting from the application of glutamate agonists and low concentrations of endogenous glycine agonists present in our hippocampal cultures. For experiments in which 1 mM L-alanine was the pulsing agonist, solutions lacking L-alanine contained 10 mM 5-methyl-indole-2-carboxylic acid (5MeI2CA), a low-affinity glycine site competitive antagonist (Huettner, 1989
RESULTS
Glutamate and glycine bind with high affinity in the absence of co-agonist
Previously, it has been estimated from analysis of desensitization experiments that a three- to eightfold difference exists between low- and high-affinity agonist binding states (Vyklicky et al., 1990
Testing for changes in apparent agonist affinity in the absence of the respective co-agonist cannot be measured at equilibrium, because electrophysiological experiments cannot directly measure channel states other than those that allow ion conduction, and no NMDA receptor-mediated ion conduction can occur in the absence of either the glutamate or the glycine co-agonist (Kleckner and Dingledine, 1988
To determine whether a high-affinity glutamate binding state exists in the absence of a glycine co-agonist, a paired-pulse protocol was used as shown in Figure 1A. A control response was elicited by application of 10 µM glutamate and 1 mM L-alanine. L-alanine was then removed, and after a 2.5 sec re-equilibration period, glutamate was removed. After a delay period of ~25 msec, a test pulse of L-alanine (in the absence of exogenously applied glutamate) revealed the population of channels still bound with glutamate. The experiment was then repeated at various delay periods (Fig. 1A). Analysis of the peak amplitudes resulting from the L-alanine test pulses yielded a single exponential decay time constant of 1.3 ± 0.1 sec (n = 7 cells), which reflects the apparent dissociation of glutamate in the absence of a glycine co-agonist (Fig. 1B, Table 1). Current decay during the L-alanine test pulse should reflect the apparent glutamate dissociation rate when both types of agonist are bound (Fig. 1C). The exponential current decay time constant measured during the L-alanine test pulse of the first paired-pulse record was significantly faster than the time constant measured from the peak test pulse amplitudes in Figure 1B (Table 1). The average current decay time constant measured for the test L-alanine pulse of the first paired-pulse record was
= 171.3 ± 38.1 msec (n = 13 cells). For comparison, apparent glutamate dissociation time constants were also measured by pulsing 10 µM glutamate in the continual presence of 1 mM L-alanine. Decays of the glutamate-elicited current could only be measured with the sum of two exponentials (Table 1). The weighted average of these exponentials,
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Figure 1. Glutamate dissociates slowly in the absence of a glycine co-agonist. A, Overlay of 18 paired-pulse records from a single hippocampal neuron. Inward currents were elicited when 10 µM glutamate (striped bar) was applied simultaneously with a 600 msec control pulse of 1 mM L-alanine (open bars). A re-equilibration period of 2.5 sec followed the L-alanine control pulse after which the exogenous glutamate was removed. To reduce activation of NMDA receptors by glutamate and endogenous glycine, 10 mM 5MeI2CA was included in all solutions lacking L-alanine. An L-alanine test pulse then followed at various delay times after the removal of the glutamate. The current observed reflects receptors bound with L-alanine that still have glutamate bound. The
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Table 1. Exponential decay times resulting from the removal of exogenous glutamate under various conditions To determine whether a high-affinity glycine binding state exists in the absence of a glutamate co-agonist, the converse experiment was conducted (Fig. 2A). NMDA (300 µM), a low-affinity glutamate binding site agonist, was applied for 600 msec in the presence of 10 µM glycine. Exogenous glycine was removed after a re-equilibration period of 2.5 sec, and an NMDA test pulse was applied at various times after the removal of glycine. Analysis of the peak responses versus time after exogenous glycine removal yielded an exponential time constant of 0.9 ± 0.2 sec (n = 11 cells; data not shown). The peaks of the test pulses decayed to an offset of 46.1 ± 3.3%, indicating that peak responses to NMDA in the presence of endogenous glycine were significant. To lower the response resulting from the presence of endogenous glycine, 0.5 µM 5,7-diClKyn was added to all solutions. Paired-pulse experiments conducted in the presence of 5,7-diClKyn (Fig. 2A) lowered the offset to 20.2 ± 3.3% and slowed the time constant measured from the plot of test pulse peak amplitudes (Fig. 2B) to 1.5 ± 0.2 sec (n = 7 cells,). The average decay time constant of current during the test pulse of NMDA in the paired-pulse protocol should reflect apparent dissociation of glycine in the presence of NMDA (Fig. 2C). This value was 219.7 ± 60.2 msec (n = 8 cells) and approximates earlier measurements of glycine apparent dissociation kinetics (Benveniste et al., 1990b
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Figure 2. Glycine dissociates slowly in the absence of a glutamate co-agonist. A, Overlay of 12 paired-pulse records from a single hippocampal neuron. Inward currents were elicited when 100 µM glycine (open bar) was applied simultaneously with a 600 msec control pulse of 300 µM NMDA (striped bar). 5,7DiClKyn (0.5 µM) was present continuously throughout the experiment to limit activation by NMDA with endogenous glycine. A re-equilibration period of 2.5 sec followed the NMDA control pulse, after which the exogenous glycine was removed. An NMDA test pulse then followed at various delay times after the removal of the glutamate. The current observed reflects receptors bound with NMDA that still have glycine bound. The
Permeant ions may reduce the affinity for glutamate but not for glycine
Figures 1 and 2 suggest that agonist binding is in an apparent high-affinity state in the absence of co-agonist when the channel is in a nonactivated state, and agonist binding is in an apparent low-affinity state when the co-agonist is bound and the channel is activated. We measure receptor activation by current flow through open channels. This depends on (1) glutamate and glycine agonist binding, (2) conformational changes in the protein allowing channel opening once the agonists are bound, and (3) ion permeation through the open channel. Each of these three events could trigger a change in agonist binding from a high- to a low-affinity state.
To examine whether ion permeation through open NMDA channels leads to a transition in glutamate apparent affinity from a high- to low-affinity state, paired-pulse experiments were conducted in which neurons were bathed with 10 µM glutamate and 1 mM L-alanine in the presence of nonpermeating 160 mM choline chloride. The control pulse was initiated by switching the choline chloride to NaCl for 600 msec after which choline again replaced sodium as the main extracellular cation. After a 2.5 sec re-equilibration time in the presence of glutamate, L-alanine, and choline chloride, exogenous glutamate was removed while L-alanine remained. Test pulses of NaCl were elicited at various delay times after the removal of glutamate (Fig. 3A). A plot of the peak amplitudes of the second sodium pulse as a function of the delay time between glutamate removal and the peak of the second sodium pulse may serve as a measure of glutamate apparent dissociation from the NMDA receptor in the absence of permeant ions but in the presence of co-agonist L-alanine (Fig. 3B). This plot could not be well fit with a single exponential (Fig. 3B). Thus, these data were fit with a sum of two exponentials, and the parameters are presented in Table 1. The weighted average for apparent glutamate dissociation in the absence of permeant ions (
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Figure 3. Glutamate but not glycine dissociation is slowed in the absence of ion permeation through NMDA channels. A, Overlay of 12 paired-pulse records from a single hippocampal neuron. Inward currents were elicited when a sodium-based extracellular solution (black bars) replaced a choline-based extracellular solution (gray bars) in the presence of 10 µM glutamate (striped bar). L-alanine (1 mM) (open bar) was present continuously throughout the experiment. The initial sodium-based control pulse was 600 msec followed by a re-equilibration period of 2.5 sec in choline-based extracellular solution after which the exogenous glutamate was removed. A test pulse of sodium-based extracellular solution then followed at various delay times after the removal of the glutamate. The current observed reflects receptors bound with L-alanine that still have glutamate bound. B, Plot of peak current amplitudes elicited from the sodium-based extracellular solution test pulse normalized to the amplitude measured near the end of the sodium-based extracellular solution control pulse plotted against the delay time between exogenous glutamate removal and the peak of the test pulse for each record. Peak measurements are indicative of the rate of glutamate dissociation in the presence of L-alanine but in the absence of permeant ions. Each symbol type represents the data from a different cell. The continuous line indicates a fit to a sum of two exponentials with the weighted average time constant (
Our extracellular solutions routinely contained 0.2 mM calcium. We have conducted paired-pulse experiments like those depicted in Figure 3A in which calcium was omitted from the extracellular solution and 10 mM EGTA was added. Under these conditions, the decay of peak amplitudes elicited by the test sodium pulse with respect to the time after glutamate removal yielded a single exponential time constant of 561.2 ± 156.2 msec (n = 5 cells). This is consistent with our results obtained under routine conditions (Fig. 3, Table 1), suggesting that 0.2 mM calcium does not cause the transition of the glutamate site to a low-affinity state.
The influence of extracellular choline on glycine apparent dissociation was also examined in an analogous paired-pulse experiment. Neurons were bathed with 300 µM NMDA and 10 µM glycine in the presence of choline chloride. Control currents were elicited by replacing the choline chloride with NaCl for 600 msec. Exogenous glycine was removed 2.5 sec after the end of the 160 mM NaCl control pulse. Test pulses of sodium were then elicited at various delay times after the removal of exogenous glycine. A plot of the peaks of the second sodium pulse as a function of the delay time between glycine removal and the peak of the normalized second sodium pulse serves as a measurement of glycine apparent dissociation from the NMDA receptor in the absence of permeant ions (Fig. 3C). A single exponential decay time constant of 346.1 ± 49.0 msec was measured from six cells. Analysis of the current decay from the test pulse from the first paired-pulse record yielded a
Changes in extracellular sodium concentration can cause changes in electrogenic transporter and exchanger activity that could influence peak current amplitudes in our paired-pulse assay. Potential effects resulting from electrogenic activity could be removed by conducting experiments in the presence and absence of glutamate binding site competitive antagonist, 2-amino-5-phosphonopentanoic acid (AP5). By subtracting the traces acquired in the presence of 200 µM AP5 from those acquired in the absence of AP5, NMDA receptor-mediated currents could be isolated from other electrogenic activity. However, acquisition of agonist dissociation data with paired-pulse protocols in the presence and absence of AP5 required a large number of traces in the same cell. This was technically very difficult. Therefore, we designed a desensitization protocol in which desensitization is modulated by subsaturating concentrations of either glutamate or glycine (Fig. 4). This type of desensitization results from agonist re-equilibration that takes place after a decrease in agonist apparent affinity on channel activation (Nahum-Levy et al., 2001
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Figure 4. Ion permeation can cause glutamate- but not glycine-sensitive desensitization. A, Assay for glutamate-sensitive desensitization in a hippocampal neuron. The left trace shows current activated by a pulse of 1 mM L-alanine (open bars) with a background concentration of 0.1 µM glutamate (striped bars) in a 160 mM sodium-based extracellular solution (black bars). The inward current that was elicited peaks and desensitizes 69.0%. The right trace shows current activated by switching a 160 mM choline-based extracellular solution (gray bars) for a 160 mM sodium-based solution (black bar) in the continual presence of 1 mM L-alanine and 0.1 µM glutamate in the same cell. The peak amplitude of this current is 382% of the peak amplitude of the left trace. The current in the right panel desensitizes 92.4% and reaches a steady-state level similar to that of the left trace. Note that all solutions not containing L-alanine contained 10 mM 5MeI2CA to limit activation of NMDA receptors by glutamate with endogenous glycine. B, Assay for glycine-sensitive desensitization in a hippocampal neuron. The left trace shows current activated by a pulse of 1 mM NMDA (striped bars) with a background concentration of 0.1 µM glycine (open bars) in a 160 mM sodium-based extracellular solution (black bars). The inward current that was elicited peaks and desensitizes 66.1%. The right trace shows current activated by switching a 160 mM choline-based extracellular solution (gray bars) for a 160 mM sodium-based solution in the continual presence of 1 mM NMDA and 0.1 µM glycine in the same cell. Although the steady-state levels are similar in both the left and right traces, no peak is observed by current elicited by permeant ions (right panel). To isolate NMDA receptor-mediated currents from other electrogenic activity, the protocols depicted in A and B were acquired in the presence of AP5 and subtracted from those acquired in the absence of AP5. C, Summary of the degree of glutamate-sensitive desensitization for three different intracellular solutions in which cesium (black bars), sodium (striped bars), or choline (gray bars) is the major cation. The left three bars summarize results from currents elicited by a 1 mM L-alanine test pulse (L-Ala) in the continual presence of 0.1 µM glutamate in a sodium-based extracellular solution (A, left panel). The right three bars summarize results from currents elicited by the switch from a choline-based extracellular to a sodium-based extracellular solution (Na) in the continual presence of 0.1 µM glutamate and 1 mM L-alanine (A, right panel). D, Summary of the degree of glycine-sensitive desensitization for three different intracellular solutions in which cesium (black bars), sodium (striped bars), or choline (gray bars) is the major cation. The left three bars summarize results from currents elicited by a 1 mM NMDA test pulse (NMDA), in the continual presence of 0.1 µM glycine in a sodium-based extracellular solution (B, left panel). The right three bars summarize results from currents elicited by the switch from a choline-based extracellular to a sodium-based extracellular solution (Na), in the continual presence of 0.1 µM glycine and 1 mM NMDA (B, right panel). E, Summary of the degree of desensitization elicited by changes of various extracellu- lar solutions containing 0.1 µM glutamate and 1 mM L-alanine using a cesium-based intracellular solution. Currents were elicited by a switch from either a choline-based (black bars) or sucrose-based (striped bars) extracellular solution to solutions in which the major extracellular permeant cation was sodium (Na), potassium (K), or cesium (Cs). Note that only the switch to a sodium-based extracellular solution exhibits high degrees of desensitization.
When incubating neurons with a subsaturating background concentration of glutamate (0.1 µM) and pulsing with 1 mM L-alanine in an extracellular solution containing sodium as the predominating cation, a peak current is observed (Fig. 4A, left panel) that desensitizes 76.1 ± 14.3% (n = 13 cells). This desensitization is similar to that observed previously for subsaturating concentrations of glutamate and is diminished with increasing background concentrations of glutamate (Nahum-Levy et al., 2001
A similar strategy was used to test whether ion permeation could cause a decrease in glycine binding site apparent affinity resulting in glycine-sensitive desensitization. Figure 4B (left panel) shows that when 1 mM NMDA is pulsed in the presence of a background concentration of subsaturating glycine (0.1 µM) in a sodium-based extracellular solution, a peak current is observed that desensitizes 65.7 ± 3.9% (n = 12 cells) in accordance with previous observations (Benveniste et al., 1990a
Reduction in glutamate affinity by permeant ions is sodium specific
We can use the desensitization protocol with different intracellular and extracellular solutions to determine whether the transition from high to low apparent affinity glutamate or glycine binding states is specific to a particular monovalent cation. In addition, such experiments might indicate whether the cation modulation site is located in the intracellular or extracellular domains. Experiments similar to those done with a cesium-based intracellular solution (Fig. 4A,B), were conducted with sodium-based and choline-based intracellular solutions. Peak responses elicited by the switch from a choline-based to a sodium-based extracellular solution in the presence of a background concentration of 0.1 µM glutamate (Fig. 4A, right panel) were 248.0 ± 88.0 and 229.3 ± 71.6% of the peak elicited by an L-alanine pulse (Fig. 4A, left panel) for experiments done with sodium-based or choline-based intracellular solutions, respectively. These responses desensitized to similar degrees regardless of the intracellular solution used (Fig. 4C). The extent of desensitization observed when a background concentration of 0.1 µM glycine was used was also not dependent on whether a sodium-based or choline-based intracellular solution was used (Fig. 4D).
We also checked the specificity of extracellular cations in their ability to reduce glutamate apparent affinity. Using a desensitization protocol, extracellular solutions were switched from a choline-based extracellular solution to a sodium-, potassium-, or cesium-based extracellular solution in the continual presence of 0.1 µM glutamate and 1 mM L-alanine. High degrees of desensitization (>75%) were observed only when the main extracellular cation was sodium (Fig. 4E), suggesting that reduction of the glutamate apparent affinity may be sodium specific.
Extracellular sucrose was also substituted for the choline cation to reduce ion permeation in some experiments. Degrees of desensitization for solution switches from sucrose to sodium or potassium were similar to their choline counterparts (Fig. 4E), indicating that reversion of the glutamate site to a high apparent affinity state does not result from an interaction of choline with open NMDA channels.
Extracellular cesium and potassium do not cause large degrees of desensitization when switching from a choline-based extracellular solution in the continual presence of 0.1 µM glutamate and 1 mM L-alanine (Figs. 4E, 5A, right panel), suggesting that the glutamate binding site remains in an apparent high-affinity state when extracellular cesium and potassium are the main extracellular cations. This enabled us to determine whether re-equilibration with the high apparent affinity state of the glutamate binding site could be observed. Neurons were incubated with 0.1 µM glutamate and 1 mM L-alanine in a sodium-based extracellular solution. This should cause the glutamate binding site of NMDA receptors to revert to their low apparent affinity state. Indeed, only a minimal amount of inward current is initially observed in Figure 5A (left panel), because 0.1 µM glutamate is well below the steady-state EC50 for glutamate (Nahum-Levy et al., 2001
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Figure 5. Extracellular sodium reduces glutamate affinity. A, A neuron was incubated in either a sodium-based (left panel) or choline-based (right panel) extracellular solution (gray bars) in the continual presence of 1 mM L-alanine. A minimal amount of inward current was elicited after addition of 0.1 µM glutamate (striped bar). After 1 sec, the extracellular solution was replaced with a cesium-based solution (black bar) containing the same agonist concentrations for 3 sec. Inward current increased rapidly for the choline-to-cesium solution transition and did not desensitize appreciably (right panel). In contrast, the sodium-to-cesium solution transition caused a slow increase in inward current until the same steady state was reached (left panel). After changing the cesium-based solution back to sodium (left panel), a rapid increase in inward current was observed that then desensitized. Shown are records in which the leak current has been subtracted by repeating each protocol in the presence of 200 µM AP5. B, Plot of current relaxation rates with increasing concentrations of glutamate in the presence of extracellular cesium (black bars) or potassium (striped bar) measured from experiments like those in A (left panel). Changes in current result from glutamate re-equilibration with a high-affinity receptor state after the switch from a sodium-based extracellular solution to either a cesium-based or potassium-based extracellular solution. C, I-V relationship for NMDA receptor-mediated currents elicited by 10 µM glutamate and 1 mM L-alanine in the presence of a sodium-based (Na), potassium-based (K), or cesium-based (Cs) extracellular solution. Currents depicted have been leak subtracted by eliciting voltage ramps in the presence of AP5 and in the absence of glutamate. D, Relative conductance of NMDA receptors in different extracellular solutions from whole-cell recordings. In each cell, I-V relationships were measured for either sodium-based and cesium-based extracellular solutions or potassium-based and cesium-based extracellular solutions. The reversal potential was determined for each condition, in each cell, and the conductance at
After returning to a sodium-based extracellular solution from either a potassium-based or cesium-based extracellular solution, currents subsequently desensitized with a time course reminiscent of that observed for choline-based to sodium-based extracellular solution changes (Fig. 4A, right panel). This desensitization further indicates that the glutamate binding site reverted to a high apparent affinity state in the presence of extracellular potassium or cesium.
It should also be noted that the switch from a cesium-based or potassium-based extracellular solution to one in which the major cation is sodium induces a rapid rise in inward current (Fig. 5A). Steady-state NMDA receptor-mediated currents in the presence of either extracellular potassium or cesium were 45.7 ± 10.5% (n = 5 cells) or 42.7 ± 14.7% (n = 8 cells) of their peak currents in the presence of the sodium-based extracellular solution (Fig. 5A, left panel). This change in current amplitude may reflect differences between the permeabilities of the different extracellular monovalent cations or may indicate that channel gating changes when sodium is present. Current-voltage (I-V) relationships were measured for NMDA receptor channels activated with 10 µM glutamate and 1 mM L-alanine in which the intracellular solution was cesium-based and the extracellular solution was sodium-, potassium-, or cesium-based. Comparison of the relative amplitudes of NMDA currents in the presence of different extracellular solutions revealed that currents measured when using a sodium-based extracellular solution were more than threefold larger than when potassium or cesium extracellular solutions were used (Fig. 5C). Outward rectification observed at hyperpolarized potentials probably results from contamination of solutions with micromolar amounts of magnesium.
Reversal potentials for NMDA currents elicited in extracellular sodium-based and cesium-based solutions were 0.7 ± 3.0 mV (n = 9 cells) and 1.5 ± 3.7 mV (n = 13 cells), respectively, indicating a similarity in the extracellular cation relative permeabilities. The potassium-based extracellular solution yielded NMDA receptor-mediated currents with a slightly higher reversal potential of 9.1 ± 3.2 mV (n = 9 cells). We normalized for any differences in driving force by making conductance calculations at ±30 mV. These potentials were chosen because they are in the linear part of the I-V curve. Conductances measured for NMDA receptor mediated currents at ±30 mV in either sodium-based or potassium-based extracellular solutions were then averaged and normalized to similar measurements made during perfusion with the cesium-based extracellular solution on the same cell. Figure 5D indicates that cesium and potassium had similar overall conductances despite the differences observed in their reversal potentials (Fig. 5C). In contrast, the relative conductance of the population of NMDA channels was 3.5-fold higher in a sodium-based extracellular solution in comparison with the cesium-based control. The differences in relative conductance despite the similarity in reversal potentials for sodium-based and cesium-based extracellular solutions suggests that the permeability of sodium and cesium through NMDA channels is similar but that changes in channel gating occur.
Magnesium binding also causes a reduction in glutamate affinity
The data presented thus far are consistent with the possibility that the cation binding site responsible for the reduction of glutamate binding site apparent affinity may also be one of the sites that is involved in ion permeation. Magnesium is known to block ion permeation by binding within the channel pore. Because the key residue required for magnesium binding within the pore also significantly affects permeation properties (Burnashev et al., 1992
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Figure 6. Magnesium binding reduces glutamate affinity. A, Overlay of seven paired-pulse records from a single hippocampal neuron. The cell was incubated with 10 µM glutamate (striped bar), 1 mM L-alanine (open bar), and 2 mM magnesium (gray bar) in a choline-based extracellular solution at a holding potential of
To confirm that magnesium binding can cause the glutamate binding site to revert to a low-affinity state, a desensitization protocol was designed in which a subsaturating concentration of glutamate (0.3 µM) and 10 µM glycine were applied to a neuron in a choline-based extracellular solution in the presence or absence of 2 mM magnesium at a holding potential of
DISCUSSION
Glutamate and glycine binding affinities systematically change during NMDA receptor activation
A plausible model for NMDA channel activation should contain at least three different states for the binding of each agonist (Fig. 7). The existence of an apparent high-affinity glutamate binding state in the absence of a glycine co-agonist has been confirmed by paired-pulse experiments that showed an apparent dissociation constant for glutamate that is 7.9-fold slower in the absence of L-alanine than in its presence (Fig. 1). Similar experiments also showed that the apparent dissociation time constant for glycine in the absence of NMDA was 6.8-fold slower than when NMDA was present and the channel was activated (Fig. 2). These results confirm earlier desensitization studies (Benveniste et al., 1990a
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Figure 7. Model of NMDA receptor activation. Glutamate and glycine binding sites exist in a high-affinity state before any ligand binding. High-affinity states are represented by binding sites with cusps. After binding of glutamate, a conformation change occurs to convert the glycine binding site to low affinity (semicircle without cusp). Note that arrows in this drawing may indicate more than one mechanistic step and thus may appear to violate the law of microscopic reversibility. Exposure of the small cation modulatory site requires the binding of glycine but may also require the binding of glutamate. Sodium or magnesium can then freely bind, causing the glutamate binding site to revert to a low-affinity state (square without cusp). The exact location of the small cation modulatory site should also not be inferred from the drawing. With both glutamate and glycine bound, the channel can now undergo a transition to an open state. Once in the open state, both glutamate and glycine become irreversibly bound.
The conditions required for the reduction of glutamate apparent affinity are somewhat different from those that are required for the reduction of glycine apparent affinity. The process of agonist binding and possibly channel opening is sufficient to cause the transition of glycine binding to an apparent low-affinity state (Figs. 3C, 4B), whereas the reduction in glutamate apparent affinity also requires the presence of extracellular magnesium or sodium (Figs. 3A,B, 4A). When the NMDA channel enters the open state, both glutamate and glycine agonists may enter a third binding state (Fig. 7). Studies conducted with voltage-dependent open channel blocker, 9-aminoacridine, suggest that glutamate and glycine are "locked on" to their binding sites when NMDA channels are in the open state (Benveniste and Mayer, 1995
Nature of the small cation modulatory site that reduces glutamate affinity
Data presented in this paper suggest that a reduction in glutamate apparent affinity requires the binding of the glycine co-agonist and small extracellular cations. Glutamate appears to dissociate slowly from its binding site in the absence of L-alanine (Fig. 1) and in the absence of sodium (Figs. 3A,B). Desensitization assays suggest that glutamate starts to re-equilibrate with its binding site in a low-affinity state only on addition of L-alanine, although extracellular sodium was already present (Fig. 4A, left panel). Yet, re-equilibration of glutamate with its low-affinity binding site occurs on addition of extracellular sodium when L-alanine is already present (Fig. 4A, right panel). Together, these results suggest that dissociation of glutamate from its binding site in a low-affinity state can occur only after a binding site for small cations becomes exposed. These experiments cannot differentiate whether exposure of the cation binding site requires the binding of glycine alone or the binding of both glutamate and glycine agonists (Fig. 7).
The degree of current desensitization measured in the presence of subsaturating glutamate (Figs. 4A,C) did not depend on the major monovalent cation used in the intracellular solution, suggesting that the small cation modulatory site that reduces glutamate affinity is not in the intracellular domain of the protein. However, desensitization under subsaturating glutamate conditions was dependent on the major monovalent extracellular cation. The desensitization observed on switching from a choline-based extracellular solution to a sodium-based extracellular solution (Fig. 4A, right panel) suggests that glutamate binding was in a high-affinity state in the choline-based extracellular solution before sodium exposure.
Although desensitization was observed after switching from choline to sodium in the presence of 0.1 µM glutamate and 1 mM L-alanine, switching the extracellular solution from choline to either cesium or potassium did not cause desensitization (Fig. 5A, right panel). Furthermore, the switch from sodium to either potassium or cesium caused an increase in NMDA receptor responses, the kinetics of which became more rapid as glutamate concentration was raised (Fig. 5A,B). This suggests that although glutamate and glycine agonists are bound, the presence of extracellular sodium causes a weakening of glutamate affinity, whereas extracellular cesium or potassium allows glutamate binding to remain in a high apparent affinity state.
In these same experiments (Fig. 5A), peak currents elicited in the presence of extracellular sodium were larger than the maximal currents elicited in the presence of cesium or potassium. Under saturating agonist conditions, the relative whole-cell conductance observed with a sodium-based extracellular solution was 3.5-fold higher relative to that measured in a cesium-based extracellular solution (Fig. 5C,D). This difference does not result from differences in permeation properties for cesium or sodium as was shown for NR1 N598Q-NR2A recombinant channels (Schneggenburger and Ascher, 1997
It is possible that sodium, cesium, and potassium may generally influence glutamate apparent affinity at an extracellular location not involved with glutamate-dependent desensitization. We tested for this by measuring outward current decays at a holding potential of +60 mV in response to a 10 µM glutamate pulse in the presence of 1 mM L-alanine. The experiment was done with cesium-, potassium-, or sodium-based extracellular solutions with a sodium-based intracellular solution. The single exponential current decay time constants were similar (555.9 ± 138.8, 444.2 ± 72.4, and 523.8 ± 51.0 msec for cesium-, potassium-, and sodium-based extracellular solutions, respectively; n = 3-6 cells for each condition). This indicates that when sodium was the primary monovalent cation flowing outward through the channel, changes in the primary extracellular monovalent ion did not cause an allosteric effect that influenced apparent glutamate dissociation.
Finally, channels exposed to magnesium have a glutamate apparent dissociation rate similar to that of apparent low-affinity dissociation (Fig. 6A, Table 1). In addition, the degree of glutamate-dependent desensitization observed on switching from a choline-based to a sodium-based extracellular solution was reduced when magnesium was present in the choline-based solution (Fig. 6C). This also suggests that glutamate equilibrates with binding site in a low-affinity state in the presence of magnesium. High concentrations of magnesium can potentiate NMDA receptor responses, raise glycine apparent affinity, reduce single-channel current amplitudes, and cause voltage-dependent block of the pore (Paoletti et al., 1995
The fact that sodium and magnesium may permit a reduction of glutamate binding site apparent affinity, whereas potassium and cesium cannot, may indicate that access to the small cation modulatory site might be governed by atomic size. Magnesium and sodium have empirical atomic radii of 1.5 and 1.8 Å, respectively, whereas potassium and cesium have much larger radii of 2.2 and 2.6 Å, respectively.
Physiological implications
Recent evidence suggests that powerful glycine uptake in the synaptic cleft reduces glycine concentrations such that <50% of NMDA channels may be activated during miniature EPSCs at some synapses (Berger et al., 1998
FOOTNOTES Received Sept. 19, 2001; revised Jan. 9, 2002; accepted Jan. 10, 2002.
* R.N.-L. and E.T. contributed equally to this work.
This research was supported by Grant 96-00245 from the United States-Israel Binational Science Foundation, Jerusalem, Israel, and Grant 572/99-16.0 from the Israel Science Foundation. We thank Professor Bernard Attali and Dr. Kathryn Partin for comments on this work.
Correspondence should be addressed to Dr. Morris Benveniste, Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Israel. E-mail: morrisb{at}post.tau.ac.il.
REFERENCES
- Anson LC, Chen PE, Wyllie DJA, Colquhoun D, Schoepfer R (1998) Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors. J Neurosci 18:581-589
[Abstract/Free Full Text] .- Antonov SM, Johnson JW (1999) Permeant ion regulation of N-methyl-D-aspartate receptor channel block by Mg(2+). Proc Natl Acad Sci USA 96:14571-14576
[Abstract/Free Full Text] .- Antonov SM, Gmiro VE, Johnson JW (1998) Binding sites for permeant ions in the channel of NMDA receptors and their effects on channel block. Nat Neurosci 1:451-461[ISI][Medline].
- Armstrong N, Gouaux E (2000) Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 28:165-181[ISI][Medline].
- Benveniste M, Mayer ML (1995) Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9-aminoacridine. J Physiol (Lond) 483:367-384[ISI][Medline].
- Benveniste M, Clements J, Vyklicky Jr L, Mayer ML (1990a) A kinetic analysis of the modulation of N-methyl-D-aspartic ac
