Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors

Abstract

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).

Fig. 1.

Single amino acid substitutions in the NR2A subunit generated functional NR1/NR2A receptors. A, Amino acid sequence alignment of rat NR1 (Moriyoshi et al., 1991) and NR2 subunits (Monyer et al., 1992; Ishii et al., 1993) with the bacterial periplasmic binding proteins LAOBP (Oh et al., 1993) and HISJ (Oh et al., 1994). Residues that reduced glycine potency when mutated in NR1 are marked by an arrow above the sequence alignment. NR2A residues that were mutated in this study are markedbelow the sequence alignments by an arrowif the mutation reduced potency by more than a factor of 2; otherwise they are indicated by a minus sign.Numbers indicate the first residue of the alignment; numbering is for mature proteins without signal peptides.B, Glutamate-activated currents recorded in the two-electrode voltage-clamp configuration from an oocyte expressing wild-type receptors (left) and an oocyte expressing T671A mutant receptors (right). Increasing concentrations of glutamate were applied in the presence of 30 μm glycine.

Fig. 2.

Mean normalized dose–response relationships for glutamate and glycine. A, Glutamate-activated currents were evoked in the presence of 30 μm glycine. Mean dose–response curves for wild-type (filled squares, mean of fitted I_max = 0.41 μA), N463A (open diamonds, 1.3 μA), K465E (filled pentagons, 1.6 μA), H466A (filled diamonds, 1.4 μA), H466F (filled circles, 1.8 μA), G664A (open triangles, 1.1 μA), T665A (open squares, 0.42 μA), V666A (plus signs, 1.7 μA), G669A (open circles, 3.7 μA), S670A (open hexagons, 0.38 μA), and T671A (filled hexagons, 2.0 μA) receptors were fitted with the Hill equation as described in Materials and Methods. Error bars have been omitted for clarity. The points represent the means of two to eight observations. B, Glycine-activated currents were evoked in the presence of a saturating concentration of glutamate (30 μm to 10 mm depending on glutamate potency for each receptor). Dose–response curves were constructed as inA. Symbols represent the same as inA.

Fig. 3.

Schild analysis of the competitive antagonism of the glutamate binding site by APV. A, Partial, low-concentration, glutamate dose–response curves for one oocyte expressing wild-type receptors in the absence (open squares) and presence of 3 μm(filled diamonds), 10 μm(open pentagons), 30 μm(filled hexagons), and 100 μm(open circles) APV. Dashed linesrepresent free fits of the power function (see Materials and Methods) to the data, and solid lines show fits of the same function but with slopes constrained to be the same for all curves. Fitted lines have been extrapolated for display purposes.B, Partial, low-concentration, glutamate dose–response curves for one oocyte expressing T671A mutant receptors in the absence (filled pentagons) and presence of 300 μm (open diamonds) and 1 mm (filled triangles) APV. Data were fitted as in A. C, Schild plot for antagonism of wild-type (filled circles) and T671A (filled squares) receptors by APV using dose ratios estimated from results such as those in Aand B. The points represent the means of five dose ratios. The dashed lines are free fits with slopes that are not exactly 1; the solid lines are fits of the Schild equation (both have slope = 1). Fitted lines have been extrapolated for display purposes.

Fig. 4.

Wild-type and T671A single-channel amplitudes.A, Single-channel currents recorded in an outside-out patch from an oocyte expressing T671A mutant receptors in the presence of 60 μm glutamate and 20 μm glycine. At −100 mV a main level (∼5 pA) and a sublevel (∼4 pA) are clearly visible. B, Wild-type single-channel currents adapted from Béhé et al. (1995). Transitions between the main level (∼5 pA) and sublevel (∼4 pA) are evident. C, Amplitude histogram of 791 events of duration greater than 2.5 filter rise times, for the patch illustrated in A. The distribution was fitted with two Gaussian components with means of 5.18 and 4.15 pA D, Amplitude histogram for the same patch as in B reanalyzed from Béhé et al. (1995). The distribution of 1048 events greater than 2.5 rise times was fitted with two Gaussian components with means of 5.52 and 4.35 pA.

Fig. 5.

Open period distribution for T671A mutant and wild-type channels. A, Distribution of the length of 1947 apparent open periods evoked by 200 μm glutamate and 20 μm glycine in a patch containing T671A mutant channels. The distribution was fitted with a mixture of three exponential densities with parameters as shown. B, Distribution of 949 apparent open periods evoked by 30 nmglutamate (+20 μm glycine) in a patch excised from an oocyte expressing wild-type NR1/NR2A receptors. The distribution was fitted with a mixture of three exponential components.

Fig. 6.

Fast concentration jumps on the T671A mutant receptor. A, Two individual responses obtained from an outside-out patch after a 100 msec pulse of 10 mm glutamate (in the presence of 20 μm glycine). Single-channel currents can be seen clearly in these example traces. The timing and duration of the glutamate application are indicatedabove each pulse. B, The average of 25 such jumps. The deactivation of this ensemble current is rapid and can be reasonably well described by a single exponential with a time constant of 16 msec, shown as a white line.