Modulation of N-methyl-D-aspartate receptor function by glycine transport

Abstract

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-D-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch-clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 microM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.

Figures

Figure 1

The chemical structure for N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine.

Figure 2

Glycine and GLYT1 antagonist enhanced the PSC evoked by Schaffer collateral stimulation. (A) Glycine (10 μM) increased the amplitude of the evoked PSCs. (B) GLYT1 antagonist (100 nM) also increased the amplitude of the evoked PSCs. Each trace is the average of 5 PSCs (Vh = −60 mV). (C) The histogram shows the percentage of change of the amplitude of the PSC induced by glycine (10 μM) or by GLYT1 antagonist (100 nM).The measurement of the amplitude of the PSC was done 4 msec after the beginning of the PSC.

Figure 3

When APV (A, 50 μM) and 7-Cl-Kyn (B, 5 μM) were added to the medium, the enhancing effect of GLYT1 antagonist was blocked completely, suggesting a selective effect on the NMDA component of the PSC. (C) However, the addition of strychnine (25 μM), an antagonist of the glycine-gated chloride channel, did not block the enhancing effect of GLYT1 antagonist (100 nM). Each trace is the average of five PSCs (Vh = −60 mV). (D) The histogram shows the percentage of change of the amplitude of the PSC induced by APV (50 μM) and APV (50 μM) plus GLYT1 antagonist (100 nM), and 7-Cl-Kyn (5 μM) and 7-Cl-Kyn (5 μM) plus GLYT1 antagonist (100 nM). The effect of strychnine (25 μM) and GLYT1 antagonist (100 nM) was compared with the effect of strychnine alone. The measurement of the amplitude of the PSC was done 4 msec after the beginning of the PSC.

Figure 4

GLYT1 antagonism enhanced the isolated NMDA component of the Schaffer collateral-induced synaptic current. (A, 1) Bicucilline (20 μM) and DNQX (20 μM) were added to the medium with a low-Mg2+ concentration to isolate the NMDA component of the EPSC. (A, 2) GLYT1 antagonist (100 nM) enhanced the amplitude of the EPSCs. The maximal effect, indicated by the arrow, was observed at more depolarizing membrane potentials. (A, 3) This trace shows the recovery. (B) The current–voltage curve of the amplitude of the isolated EPSC shows the voltage sensitivity of the EPSC enhancement by GLYT1 antagonism. The measurement of the amplitude of the EPSC was done 25 msec after the beginning of the EPSC.

Figure 5

GLYT1 antagonism can alter glycine concentration proximal to the NMDAR. (A) Glycine (1 μM) in the bath caused no change on the amplitude of the PSC. However, the simultaneous application of glycine (1 μM) and GLYT1 antagonist (100 nM) to the bath induced an enhancement of the amplitude of the PSC. Each trace is the average of five PSCs (Vh = −60 mV). (B) The histogram illustrates the percentage of change of the amplitude of the PSC induced by glycine and/or GLYT1 antagonist compared with control. Glycine (1 μM) did not change the amplitude of the PSC compared with control. GLYT1 antagonist (100 nM) significantly enhanced the amplitude of the PSC. GLYT1 antagonist (100 nM) and glycine (1 μM) enhanced the amplitude of the PSC to a significantly greater extent. The measurement of the amplitude of the PSC was done 4 msec after the beginning of the PSC.

Figure 6

(A) Glycine (10 μM) induced an increase of the PSC. The simultaneous application of glycine (10 μM) and GLYT1 (100 nM) induced a further enhancement of the amplitude of the PSC over that induced by glycine (10 μM) alone. Each trace is the average of five PSCs (Vh = −60 mV). The measurement of the amplitude of the PSC was done 4 msec after the beginning of the PSC.