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Specific inhibition of N-methyl-D-aspartate receptor function in rat hippocampal neurons by L-phenylalanine at concentrations observed during phenylketonuria

Abstract

Hippocampal N-methyl-D-aspartate receptors (NMDARs) are thought to be involved in the regulation of memory formation and learning. Investigation of NMDAR function during experimental conditions known to be associated with impaired cognition in vivo may provide new insights into the role of NMDARs in learning and memory. Specifically, the mechanism whereby high concentrations of L-phenylalanine (L-Phe) during phenylketonuria (>1.2 mM) cause mental retardation remains unknown. Therefore, the effects of L-Phe on NMDA-activated currents (INMDA) were studied in cultured hippocampal neurons from newborn rats using the patch-clamp technique. L-Phe specifically and reversibly attenuated INMDA in a concentration-dependent manner (IC50 = 1.71 ± 0.24 mM). In contrast, L-tyrosine (L-Tyr), an amino acid synthesized from L-Phe in normal subjects, did not significantly change INMDA. Although the L-Phe-INMDA concentration-response relationship was independent of the concentration of NMDA, it was shifted rightward by increasing the concentration of glycine. Consistent with an effect of L-Phe on the NMDAR glycine-binding site, L-Phe (1 mM) did not attenuate INMDA in the presence of D-alanine (10 μM). Furthermore, L-Phe significantly attenuated neither glutamate-activated current in the presence of MK-801, nor current activated by AMPA. The finding that L-Phe inhibits specifically NMDAR current in hippocampal neurons by competing for the glycine-binding site suggests a role for impaired NMDAR function in the development of mental retardation during phenylketonuria and accordingly an important role for NMDARs in memory formation and learning.

Introduction

Glutamate activates three major types of ionotropic receptors in the central nervous system: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors. In contrast to AMPA and kainate receptors, modulation of NMDA receptor function occurs at a number of sites that are distinct from the glutamate-binding site. One of these is the strychnine-insensitive glycine-binding site where glycine allosterically increases the probability of the open state of NMDA receptor channels.1,2,3,4 Other amino acids such as serine and alanine also bind to the glycine-binding site and can modulate NMDA receptor function.1,5,6

NMDA receptor activation is thought to play an important role in the formation of neural networks during development and in synaptic plasticity, physical correlates of memory and learning. For example, pharmacological blockade of NMDA receptors or knockout of the NMDA receptor gene in hippocampal CA1 pyramidal neurons significantly disrupted some types of memory and learning in animals.7,8,9 Likewise, ketamine significantly disturbed verbal memory in humans by inhibiting NMDA receptors in the hippocampal CA1 area.10

The investigation of NMDA receptor function at experimental conditions known to be associated with impaired memory formation and learning in vivo may provide further insight into the role of NMDA receptors in these processes. Specifically, phenylketonuria (PKU), an autosomal recessive disorder marked by extremely high blood concentrations of the amino acid L-phenylalanine (>1200 μM vs 55–60 μM in healthy subjects), is characterized by severe neurological deficits such as mental retardation and postnatal brain damage.11,12 The neurological manifestations of phenylketonuric subjects develop rapidly within the first months of life.11,13,14,15 While it is well established that hyperphenylalaninemia is caused by mutations of the gene encoding phenylalanine hydroxylase, an enzyme that catalyzes the conversion of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr),12,16,17 the mechanism whereby hyperphenylalaninemia results in mental retardation is not understood. If hyperphenylalaninemia specifically impairs NMDA receptor function then, PKU as well as its associated animal models such as the genetic murine model of PKU (Pahenu2)18,19 can be used to study the role of NMDA receptors in learning and memory.

Therefore, to examine whether impairment of NMDA receptor function in the hippocampus could be involved in the development of mental retardation associated with PKU, we investigated the effect of L-Phe on the NMDA-activated current (INMDA) in cultured hippocampal neurons from newborn rats.

Materials and methods

Cell preparation

Hippocampi were dissected from newborn rats and treated with 0.25% trypsin to dissociate cells, using the exact procedures that were employed for preparation of cortical cultures.20 Dissociated cells were resuspended in Neurobasal Medium containing B-27 serum-free supplement (Invitrogen Life Technologies, Carlsbad, CA, USA), and were plated in poly-L-lysine coated 35-mm Nunc plastic tissue culture dishes (1.5 × 106 cells per dish per 2 ml media). Cells were cultured in an atmosphere of 5% CO2/95% air, and 50% of the media was replaced every 3 days. Neurons were used for electrophysiological recordings 7–20 days after plating.

Electrophysiological recordings

Electrophysiological recordings of INMDA in cultured rat hippocampal neurons were made in the whole cell configuration of the patch-clamp technique21 using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Patch microelectrodes were pulled from 1.5-mm borosilicate glass tubing using a two-stage vertical pipette puller (Narishige, East Meadow, NY, USA). When filled with recording solution, patch microelectrodes had a resistance of 3–5 MΩ. For rapid application of agonist-containing solutions to neurons, the SF-77B system (Warner Instrument Corp, Hamden, CT, USA) was used. Current data were digitized on-line using a DigiData 1200A analog-to-digital board and stored on the hard disc of an IBM compatible Pentium computer (GP7–600 MHz, Gateway Computer, Sioux City, ND, USA). Voltage-clamp experimental protocols and off-line data analysis were performed using the software program pCLAMP7 (Axon Instruments). The experiments were performed at room temperature (22–23°C).

In order to examine whether impairment of NMDA receptor function in the hippocampus can be potentially involved in the development of mental retardation associated with PKU, we investigated the effect of L-Phe on INMDA in rat hippocampal neurons at conditions similar to those at which NMDA receptors are active in vivo. Therefore, experiments were performed at a VH of −30 mV and the extracellular solutions contained Mg2+. INMDA was recorded in response to a 6-s period of NMDA application in the presence of glycine (0.1 μM), unless stated otherwise.

Solution and drugs

The extracellular control solution contained (in mM): NaCl 140, KCl 4, CaCl2 2, MgCl2 1, CsCl 3, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, and glucose 11. The pH of the extracellular solution was adjusted to 7.4 using NaOH. Tetrodotoxin (TTX, 0.3–1 μM) was added to the external solution to block voltage-gated Na+ channels. The solution for filling the patch electrodes contained (in mM): CsF 135, NaCl 5, KCl 10, MgCl2 1, CaCl2 1, ethyleneglycoltetraacetic acid (EGTA) 11, HEPES 10. The pH of the intracellular solution was adjusted to 7.4 using CsOH. Various concentrations of AMPA, NMDA, glycine, L-Phe, L-Tyr, D-alanine, aspartate, dizocilpine (MK-801; (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,b)cyclohepten-5,10-imine) were added to the extracellular solution according to the protocols described below. All compounds were purchased from Sigma Chemical Co, St Louis, MO, USA.

General data analysis

Values are reported as mean ± SEM. Prior to parametric testing, the assumption of normality was validated using the Kolmogorov–Smirnov test with Lilliefor's correction (SSPS v10, SPSS, Inc, Chicago, IL, USA). Multiple comparisons among groups were analyzed using ANOVA (two- or one-way repeated measures with two or one-way replication where appropriate) followed by Student–Newman–Keuls testing. Single comparisons were analyzed using a 2-tailed Student's t-test. A P < 0.05 was considered significant.

Results

NMDA (100 μM) activated a rapidly rising inward current (ie, INMDA) that decayed from a peak value of 796 ± 45 pA to a steady state value of 344 ± 25 pA (n = 56). High concentrations of L-Phe (≥1 mM) significantly attenuated INMDA (Figure 1a). L-Phe (1 mM) decreased the peak and steady-state values of INMDA to 549 ± 36 pA (67.9 ± 1.1% of control values) and 294 ± 21 pA (85.8 ± 1.1% of control values), respectively (n = 56, P < 0.001). L-Phe was found to inhibit the peak component of INMDA in all neurons tested, whereas it did not significantly change the steady-state component of INMDA in eight neurons of 56 studied. The effect of L-Phe on INMDA was reversible upon washout of the drug from the extracellular solution.

Figure 1
figure1

Effects of L-phenylalanine (L-Phe) and L-tyrosine (L-Tyr) on NMDA-activated currents (INMDA) in rat cultured hippocampal neurons. Examples of INMDA before, during, and after application of L-Phe (1 mM, panel a) or L-Tyr (1 mM, panel b) are shown. Horizontal bars denote NMDA (100 μM) application. Application of L-Phe or L-Tyr was begun 45 s before the initiation of NMDA application. (Panel c) Time-dependence of the inhibitory effect of L-Phe on INMDA in a single cultured hippocampal neuron. INMDA was recorded in the absence of L-Phe (Control) and in the presence of L-Phe either during simultaneous application of NMDA and L-Phe (L-Phe no pretreatment), or after pretreatment with L-Phe for 45 s (L-Phe pretreatment). (Panel d) Summary data for four experiments shown in Panel c. Peak INMDA in the presence of L-Phe was normalized to control values and plotted as mean ± SEM against the time delay of the beginning of NMDA application after application of L-Phe. *P < 0.01 compared to control. The concentration of glycine was 0.1 μM whereas the membrane voltage (Vm) was −30 mV.

In contrast to L-Phe, L-Tyr, the amino acid produced by hydroxylation of L-Phe, did not significantly change INMDA at concentrations up to 1 mM (Figure 1b). In the presence of L-Tyr (1 mM), the steady state and peak component values of INMDA were 96.0 ± 1.8% (n = 6) and 97.2 ± 1.1% (n = 6) of control values, respectively.

The attenuation of INMDA caused by L-Phe was dependent on the time of L-Phe application. When L-Phe was applied simultaneously with NMDA, attenuation of the steady state but not peak component of INMDA occurred (Figure 1c, d). In contrast, pre-incubation of neurons with L-Phe, prior to the application of NMDA, caused a significant attenuation of both current components of INMDA with a greater attenuation of the peak current (Figure 1c). Pre-treatment of neurons with L-Phe for 10 s, prior to application of NMDA, was sufficient to cause a maximal inhibitory effect. Prolonging the time of L-Phe pre-treatment beyond 10 s did not further increase the inhibitory effect of L-Phe on INMDA.

L-Phe inhibited both the outward and inward components of INMDA. Although the inhibitory effect of L-Phe was also observed on the outward component of INMDA, recorded at positive membrane potentials, the attenuation of the outward component was smaller than that of the inward component (Figure 2). L-Phe (1 mM) decreased the peak components of INMDA by 20.7 ± 3.4% and 32.1 ± 1.1% (n = 4, P < 0.01) at membrane potentials of +30 mV and −30 mV, respectively.

Figure 2
figure2

L-Phenylalanine-induced changes in the current-voltage relation for the NMDA-activated current. (Panel a) Example of individual current traces recorded from the same neuron at different membrane potentials in the absence and presence of L-Phe (1 mM). Holding membrane potential was changed from −80 mV to +50 mV in steps of 10 mV. (Panel b) Current-voltage relations for the peak component of INMDA in the absence and presence of L-Phe (1 mM). The same neuron as in Panel a.

The attenuation of INMDA caused by L-Phe was concentration-dependent with the threshold effect occurring at concentrations greater than 200 μM (Figure 3a,b). Analysis of the concentration-response curve for the effect of L-Phe on INMDA using a non-linear logistic regression technique showed that the concentration of L-Phe required to cause half-maximal inhibition of INMDA (IC50) was 1.71 ± 0.24 mM, whereas the Hill coefficient was 0.94 ± 0.11 (ie, a value not significantly different from unity, P = 0.61). The concentration-response relationship for L-Phe to attenuate the steady-state component of INMDA lies to the right of that to attenuate the peak component of INMDA. The effect of L-Phe on INMDA was reversible at all concentrations of L-Phe studied.

Figure 3
figure3

Concentration-dependent attenuation of NMDA receptor current by L-Phe. (Panel a) NMDA (100 μM) receptor currents (INMDA) were recorded from the same neuron exposed to different concentrations of L-Phe (noted in figure) that were applied at intervals of 1 min. L-Phe exposure was initiated 45 s before the start of NMDA application. Horizontal bar denotes NMDA application. (Panel b) Peak and steady-state (plateau) INMDA was normalized to control values and plotted against the concentration of L-Phe. Data expressed as mean ± SEM of 4–7 cells. *P < 0.01 compared to control. Curve fitting and estimation of values for the peak component of INMDA was made according to a 4-parameter logistic equation. The IC50 estimated from the concentration-response relationship curve for the effect of L-Phe on the steady-state component was not determined because inhibition of this component of INMDA caused by L-Phe did not achieve a lower plateau even at L-Phe concentrations up to 30 mM. Some error bars fell with the radius of a symbol. (Panel c) The inhibitory effect of L-Phe on NMDA receptor current does not depend on the concentration of NMDA. Peak values of INMDA evoked by NMDA (200 μM) were normalized to control values and plotted against the concentration of L-Phe. Values are mean ± SEM of 4–11 cells. *P < 0.01 compared to control. The nearly superimposed, dashed curve represents the concentration-dependence of the attenuation caused by L-Phe of INMDA activated by 100 μM NMDA (from Figure 3b). (Panel d) The inhibitory effect of L-Phe on NMDA receptor current depends on the concentration of glycine. Peak and steady-state (plateau) values of INMDA were normalized to control levels and plotted against the concentration of L-Phe. Data expressed as mean ± SEM of 4–9 cells. *P < 0.01 compared to control. The IC50 values for the effect of L-Phe on peak or steady state INMDA were not determined because inhibition of INMDA caused by L-Phe in the presence of glycine (1 μM) did not achieve a plateau at concentrations even up to 30 mM L-Phe. The dashed curve represents the concentration-dependence of the attenuation of the peak component of INMDA caused by L-Phe recorded in the presence of lower concentration of glycine, 0.1 μM (Figure 3b).

To determine whether the effect of L-Phe on INMDA depends on the concentration of NMDA, INMDA was measured at the same concentration of L-Phe (1 mM) and different concentrations of NMDA. An increase in the concentration of NMDA from 100 μM to 1000 μM did not significantly change the attenuation caused by L-Phe of either peak or steady-state components of INMDA. Likewise, a 2-fold increase in the concentration of NMDA did not shift the L-Phe-INMDA concentration-response curve (Figure 3c). The IC50 values of L-Phe in the presence of 200 μM and 100 μM NMDA were 1.80 ± 0.18 mM and 1.71 ± 0.24 mM, respectively. These results indicate that L-Phe does not act as a competitive inhibitor of the glutamate-binding site on the NMDA receptor.

In order to assess if L-Phe inhibits INMDA by allosterically interacting with the glycine-binding site of the NMDA receptor, the effects of L-Phe on INMDA were studied at a constant concentration of NMDA (100 μM) in combination with a higher concentration of glycine (1 μM vs 0.1 μM). Increasing the concentration of glycine from 0.1 to 1 μM significantly increased the amplitude of INMDA. The peak and steady-state components of INMDA in the presence of glycine (1 μM) were 138.9 ± 7.8% (n = 7, P = 0.01) and 188.2 ± 8.6% (n = 7, P = 0.01), respectively, of those measured in the presence of glycine (0.1 μM). The increase in the concentration of glycine significantly decreased the inhibitory effect of L-Phe on INMDA, and markedly shifted the concentration-response relationship rightward (Figure 3d). Similar to the effect of L-Phe on INMDA in the presence of a lower concentration of glycine, L-Phe was more effective at inhibiting the INMDA peak component than its steady-state component. At a glycine concentration of 1 μM L-Phe (1 mM) decreased the peak and steady-state component of INMDA to 88.9 ± 1.0% (P = 0.002) and 94.1 ± 1.0% (P = 0.038, n = 9) of control values, respectively. Likewise, a higher concentration of glycine increased the threshold concentration of L-Phe required to significantly inhibit INMDA from 200 μM to 1200 μM. In these experiments, it was not possible to determine the IC50 and Hill coefficient values from the L-Phe-INMDA concentration-response relations because the inhibitory effect of L-Phe on INMDA did not attain a plateau value, even at concentrations of L-Phe up to 30 mM.

To obtain additional evidence that L-Phe inhibits INMDA by competing with glycine for the glycine-binding site on the NMDA receptor, the effect of L-Phe on INMDA was studied in the presence of D-alanine. The amino acids alanine and serine bind to the glycine-binding site and potentiate the glutamate responses of NMDA receptors. The D-isomers of alanine and serine are 10 to 100-fold more potent than their L-isomers.6 As expected, the addition of D-alanine (10 μM) to the extracellular solution significantly increased the amplitude of INMDA. D-alanine increased the peak and steady-state components of INMDA to 119.1 ± 1.5 and 126.5 ± 4.9% of control value, respectively. In the presence of D-alanine, L-Phe (1 mM) had no significant effect on INMDA. The peak and steady-state components of INMDA were 116.9 ± 2.3 and 124.8 ± 5.5% (n = 4) of control value, respectively. However, L-Phe markedly inhibited INMDA in the same neurons after washout of D-alanine. Representative records of INMDA recorded from the same neuron in control, in the presence of L-Phe (1 mM), and in the presence of D-alanine (10 μM) with or without L-Phe (1 mM) are shown in Figure 4a.

Figure 4
figure4

Inhibitory effect of L-Phe at different experimental conditions on currents induced by glutamate receptor agonists. (Panel a) The inhibitory effect of L-Phe on NMDA receptor current is decreased by D-alanine. Representative records of NMDA-activated currents (INMDA) recorded from the same neuron in control, in the presence of L-Phe (1 mM), and in the presence of D-alanine (10 μM) with or without L-Phe (1 mM). Horizontal bars denote the time of NMDA (100 μM) application. (Panel b) The effects of L-Phe on ionic currents evoked by the glutamate receptor agonists L-aspartate, AMPA and L-glutamate. (Panel b, A) Representative records from one neuron of the ionic currents recorded in response to application of either L-Asp (300 μM) or AMPA (100 μM) in the absence and presence of L-Phe (2.5 mM). Horizontal bars denote the time of application of L-Asp or AMPA. (Panel b,B) Examples of glutamate-evoked currents recorded from one neuron in the absence and presence of L-Phe (2.5 mM). Horizontal bars denote the time of application of L-Glu (300 μM) or MK-801 (10 μM). (Panel c) The effect of L-Phe on NMDA receptor current is not modified by L-Tyr. Examples of NMDA-activated currents (INMDA) recorded from the same neuron in control, in the presence of L-Phe (50 μM) and L-Tyr (100 μM), and in the presence of L-Phe (1 mM) and L-Tyr (100 μM). Horizontal bars denote the time of NMDA (100 μM) application.

If L-Phe inhibits INMDA selectively by interacting with the glycine-binding site on the NMDA receptor, then other subtypes of glutamate receptors should be insensitive to this action of L-Phe because they lack glycine-binding sites. To test this hypothesis, we studied the effects of L-Phe on currents activated by: (1) the selective endogenous NMDA receptor agonist L-aspartate,22 and the non-NMDA glutamate receptor agonist AMPA; and (2) glutamate in the absence and presence of the INMDA blocker MK-801.23,24,25 L-aspartate (100–1000 μM) activated an inward current (IL-Asp) similar to that activated by NMDA (Figure 4b, A). At equal concentrations of agonist, the amplitude of IL-Asp was typically greater than that of INMDA. L-Phe inhibited L-aspartate- and NMDA-activated currents to a similar extent. Peak components of IL-Asp and INMDA, measured in the presence of L-Phe (2.5 mM), were 44.5 ± 2.5% (n = 11) and 44.1 ± 2.5% (n = 7) of control values, respectively (P = 0.92). Likewise, L-Phe (2.5 mM) decreased the steady-state components of IL-Asp and INMDA to 56.7 ± 5.1% (n = 11) and 54.9 ± 2.3% (n = 7) of control values, respectively (P = 0.80). In contrast, L-Phe (2.5 mM) failed to attenuate significantly the current activated by AMPA in neurons in which the effects of L-Phe on INMDA and IL-Asp were studied (Figure 4b, A). At the same time, L-Phe attenuated the current activated by L-glutamate (300 μM) (Figure 4b, B). The peak component of the L-glutamate-activated current (IL-Glu) was decreased to 61.1 ± 4.4% in the presence of L-Phe (2.5 mM). Despite a smaller relative inhibition of IL-Glu, in comparison with that of IL-Asp, the absolute values of the L-Phe-inhibited currents were similar. Specifically, the peak components of IL-Glu and IL-Asp were equally attenuated by 614 ± 52 pA (n = 5) and 632 ± 110 pA (n = 5, P = 0.85) from control values of 1678 ± 118 pA (n = 5) and 1122 ± 110 pA (n = 7), respectively. In addition, L-Phe did not significantly affect residual IL-Glu, after exposure of neurons to the NMDA receptor channel blocker MK-801 (5 μM) (Figure 4b, B).

To determine whether concentrations of L-Phe and L-Tyr observed either in healthy subjects or in phenylketonuric patients can interact to modulate NMDA receptor function, INMDA was measured using two different experimental paradigms: (1) L-Phe 50 μM and L-Tyr 100 μM (to simulate healthy subjects); and (2) L-Phe 1000 μM and L-Tyr 100 μM (to simulate hyperphenylalaninemia). The co-application of L-Phe (50 μM) and L-Tyr (100 μM) did not significantly change the amplitude of INMDA relative to that measured in the absence of L-Phe and L-Tyr. Representative records of INMDA recorded from the same neuron in control and in the presence L-Tyr (100 μM) with either 50 or 1000 μM of L-Phe are shown in Figure 4c. The amplitudes of the peak and steady-state components of INMDA in the presence of L-Phe (50 μM) and L-Tyr (100 μM) were 99.9 ± 1.9 and 108.9 ± 1.9% of control values (measured before application of amino acids), respectively. In contrast, the co-application of L-Tyr (100 μM) and the high concentration of L-Phe (1000 μM) reduced peak and steady-state components of INMDA to 70.0 ± 2.70 and 79.3 ± 2.3% of control values, respectively. A similar degree of inhibition of INMDA was caused by L-Phe (1000 μM) alone (69.4 ± 3.0 and 77.7 ± 2.1%, peak and steady-state values of control, respectively, n = 5). These results indicate that the inhibitory effect of high concentrations of L-Phe on INMDA is not dependent upon the presence of physiological concentrations of L-Tyr in the extracellular solution.

Discussion

This is the first study to report that elevated concentrations of L-Phe, similar to those observed in the blood of phenylketonuric patients, can specifically and reversibly antagonize NMDA receptor current in rat cultured hippocampal neurons by competing for the glycine-binding site of the NMDA receptor. Because NMDA receptors are thought to play an important role in the mechanisms of synaptic plasticity and synapse formation that underlie memory, learning and formation of neural networks during development,7,8,9,10 these results may not only help explain the neurologic manifestations of PKU,11,12,13,14,15 but also suggest novel approaches to the study of NMDA receptor function.

Specific effect of L-Phe on INMDA

High concentrations of L-Phe were found to specifically antagonize INMDA in rat cultured hippocampal neurons. Consistent with a selective effect on INMDA, L-Phe did not change the ionic current activated by AMPA, a non-NMDA receptor agonist of glutamate receptors. Likewise, L-Phe had no effect on the current activated by glutamate in the presence of the specific blocker of NMDA receptor channels MK 801. Conversely, in the absence of MK 801, L-Phe caused a similar attenuation of currents (on the basis of absolute values) activated by glutamate, NMDA or the specific NMDA receptor agonist L-aspartate.

The present data demonstrate that at normal blood concentrations, neither L-Phe nor L-Tyr, an aromatic amino acid synthesized from L-Phe by the enzyme phenylalanine hydroxylase, modulate INMDA activity in rat hippocampal neurons. Furthermore, the inhibitory effect of high concentrations of L-Phe does not depend on the presence or absence of L-Tyr in the extracellular solution. L-Tyr did not significantly affect INMDA, even at high concentrations. It should be noted that use of high concentrations of L-Tyr (1 mM) has only theoretical value, because the blood concentration of L-Tyr never exceeds 0.1 mM, either in healthy subjects or in patients with PKU.26 Taken together these results suggest that L-Phe exerts its inhibitory action selectively on the NMDA subtype of ionotropic glutamate receptors.

L-Phe attenuates INMDA by competing for the glycine-binding site

NMDA receptors are unique among glutamate receptors because two co-agonists, L-glutamate and glycine, are required for their activation. Each agonist has a unique binding site within the NMDA receptor complex. Specifically, the binding sites for L-glutamate and glycine are located on two different subunits of the rat NMDA receptor, designated subunits 1 and 2, respectively.27 Glycine significantly increases the probability of NMDA receptor channel opening caused by NMDA receptor agonists without significantly altering the mean open time or single channel conductance.2,3,5,28 At the same time, glycine does not influence the affinity of NMDA for the NMDA receptors.29 Other amino acids such as alanine and serine can also regulate NMDA receptor function by interacting with the glycine-binding site.6

The results of this study provide evidence that L-Phe inhibits INMDA by interacting with the glycine- but not the glutamate-binding site. This suggestion is supported by the finding that changes in the concentration of NMDA (from 100 μM to 1000 μM) did not alter the degree of L-Phe-induced inhibition of INMDA, indicating a non-competitive nature of the L-Phe-NMDA receptor interaction. These results are in agreement with those of a prior study that found L-Phe (1 mM) neither affected NMDA-sensitive nor NMDA-insensitive L-[3H]-glutamate binding in rat brain synaptic membranes.30 On the other hand, a 10-fold increase in the concentration of L-glycine significantly shifted the concentration-response curve for the effect of L-Phe on INMDA rightward. This finding suggests that the glycine-binding site on the NMDA receptor is a possible target for the inhibitory action of L-Phe. In line with this assertion, D-alanine, an agonist of the glycine-binding site, significantly diminished (or prevented) the inhibitory effect of L-Phe on INMDA. The fact that L-Phe selectively antagonized NMDA receptor function but did not influence other glutamate receptor subtypes provides additional indirect evidence that L-Phe is inhibiting INMDA by interacting with the glycine-binding site.

Interaction of L-Phe with the glycine-binding site may explain the rightward shift of the inhibition caused by L-Phe of steady-state INMDA compared to inhibition of peak INMDA. It has been established that the affinity of the glycine-binding site to glycine is decreased following activation of the NMDA-receptor complex.2,31 Because L-Phe also binds to the glycine-binding site of the NMDA receptor, it is possible that the affinity of the glycine-binding site for L-Phe may also decrease upon activation of INMDA. Consequently, a higher concentration of L-Phe will be required to produce the same degree of inhibition of the steady-state component of INMDA in comparison to the concentration of L-Phe required to diminish the peak component of INMDA.

Because L-Phe likely acts at the glycine-binding site, the question of the effective concentration of glycine at NMDA receptors has critical importance for a possible pathophysiological role of L-Phe-impaired NMDA receptor function in the setting of PKU. The dissociation constant of glycine binding to NMDA receptors in cultured neurons and for most recombinant NMDA receptors varies from 100 to 300 nM.2,15,28 The concentration of glycine in extracellular and cerebrospinal fluid is in the micromolar range (from 1 to 10 μM),32 which is sufficient to saturate the glycine-binding site on the NMDA receptor. If the concentration of glycine near NMDA receptors is sufficient to saturate the glycine-binding site, then L-Phe, at concentrations typically observed in patients with PKU, should have a minimal effect on NMDA receptor function in vivo (see Figure 4). However, recent data indicate that the actual concentration of glycine near the NMDA receptor is markedly reduced by an active glycine transporter.33,34 For example, glycine-transporter expressed in Xenopus oocytes reduced the glycine concentration near the membrane to levels below 1 μM when the bath glycine concentration was 10 μM.35 Likewise, whole-cell patch-clamp experiments from pyramidal neurons in hippocampal slices from 8 to 12 week-old rats demonstrated that a glycine transporter type 1 antagonist selectively increased the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current.34 Glycine transport inhibition increased NMDA receptor function even during superfusion with medium containing 10 μM glycine.34 The additive effects of exogenous glycine and the glycine transporter type 1 antagonist provide evidence that the glycine binding site on the NMDA receptor is not saturated under physiological conditions. Therefore, the interaction of L-Phe with the glycine-binding site may attenuate NMDA receptor function during PKU.

Implications of INMDA regulation by L-Phe

The finding that L-Phe, at concentrations observed during PKU, specifically inhibits NMDA receptor function may have important physiological and pathophysiological implications. Both PKU as well as its associated animal models are characterized by the development of severe cognitive impairments such as a learning deficit.36,37 The mechanism(s) whereby hyperphenylalaninemia produces these acquired mental defects is not known. The results presented above, namely the inhibition of INMDA caused by high concentrations of L-Phe, may provide a cellular mechanism whereby hyperphenylalaninemia contributes to the development of mental retardation during PKU. In support of this contention, it is interesting to note that concentrations of L-Phe (0.4–0.7 mM), which are associated with a lower risk of brain damage during hyperphenylalaninemia,38 only slightly attenuated INMDA. If our hypothesis that impaired NMDA receptor contributes to the development of mental retardation in patients with PKU is correct, it may promote the development of specific therapeutic strategies (ie, use of glycine-binding site agonists or glycine transporter antagonists) aimed at mitigating PKU-related cognitive problems. Furthermore, demonstration of a specific role of NMDA receptor dysfunction as a contributor to deficits of memory formation and learning associated with PKU together with the availability of PKU animal models,36,37 may provide a new approach to study and understand the basic molecular mechanisms of learning and memory under normal conditions.

In conclusion we found that in rat cultured hippocampal neurons L-Phe at concentrations observed in PKU patients specifically and reversibly attenuated NMDA receptor current by competing for the glycine-binding site. These results may have significant impact on understanding of the molecular mechanisms of mental retardation during PKU specifically and the molecular mechanisms of memory formation and learning in general.

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Acknowledgements

This study was supported by funding from the University of Florida McKnight Brain Institute, Howard Hughes Medical Institute and JS Gravenstein Endowed Professorship.

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Correspondence to A E Martynyuk.

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Glushakov, A., Dennis, D., Morey, T. et al. Specific inhibition of N-methyl-D-aspartate receptor function in rat hippocampal neurons by L-phenylalanine at concentrations observed during phenylketonuria. Mol Psychiatry 7, 359–367 (2002). https://doi.org/10.1038/sj.mp.4000976

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Keywords

  • NMDA receptor
  • glycine-binding site
  • hippocampal neurons
  • L-phenylalanine
  • L-tyrosine
  • PKU

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