PMC3934797_01
Identification of a GRIN2A missense mutation in a boy with intractable seizures and epileptic encephalopathy. (a) A schematic linear representation of GluN2A with portions of the polypeptide chain that fold into semiautonomous domains labeled is shown. The position of the patient's mutation is indicated by an asterisk. Leu812 is conserved through all GluN1 and GluN2 subunits. ATD (amino terminal domain), S1 and S2 (agonist binding domains), M1-4 (transmembrane domains 1-4), CTD (carboxy terminal domain). (b) A homology model of the GluN1/GluN2A receptor built from the GluA2 crystallographic data is shown as ribbon superimposed on a space-filled representation of the receptor. The position of the alteration p. L812M is indicated by magenta color in the linker region between ligand-binding and transmembrane domains. (c) An expanded view of the location of residue Leu812 (cyan color; upper panel) and the possible interaction between L812M (magenta color; lower panel) and transmembrane domain M3 of GluN1 subunit and a GluN1 pre-M1 helix are predicted from the homomeric GluA2 structure.
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GluN1 and all GluN2 NMDAR subunits (Fig. 1a), indicating a possible critical role in NMDAR function. GluN2A Leu812 resides in the linker region between the lower portion of the agonist-binding domain (S2) and the transmembrane domain (M4) (Fig. 1a, b). M3 transmembrane helix and the pre-M1 helix (Fig. 1c). The data in Supplementary Fig. 1 show that L812M has no detectable effect on the relative Ca2+ permeability.
The L812M mutation changes the pharmacology of NMDARs. (a) and (b) Composite concentration-response curves for agonists were determined by TEVC recordings from Xenopus oocytes expressing di-heteromeric GluN1/GluN2A (WT 2A) or GluN1/GluN2A-L812M (L812M) receptors and tri-heteromeric receptors with identity of each GluN2A in the tetrameric complex controlled (e.g. GluN1/GluN2A/GluN2A (2A/2A), GluN1/GluN2A-L812M/GluN2A (L812M/2A), and GluN1/GluN2A-L812M/GluN2A-L812M (L812M/L812M)) (see Methods and Supplemental Figure S2). a (left) and b (left) show the composite glutamate (in the presence of 100 muM glycine) concentration-response curves for di-heteromeric receptors and tri-heteromeric receptors. a (right) and b (right) show the composite glycine (in the presence of 100 muM glutamate) concentration-response curves for di-heteromeric receptors and tri-heteromeric receptors. (c) Mg2+ current voltage (I-V) curves reveal a decreased Mg2+-inhibition for L812M di-heteromeric receptors. (d) Mg2+ current-voltage (I-V) curves reveal a dominant decreased Mg2+-inhibition in the receptors containing a single copy of L812M. (e) Mg2+ concentration-response curves for di-heteromeric receptors are shown. (f) Composite concentration-response curves for proton (left) and zinc (right) inhibition show a decreased inhibition for L812M in di-heteromeric receptors.
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The mutation L812M increased the glutamate potency, as measured by reduction in the half-maximally effective concentration of agonist (EC50) from 3.5 muM to 0.41 muM (Fig. 2a, left; Table 1). Table 1) and glycine potency (3.6-fold decrease in EC50 to 0.20 muM from 0.72 muM for 2A/2A; Fig. 2b, right; Table 1). Thus, NMDAR activation was enhanced at lower extracellular concentrations of agonist even for receptors with a single copy of the mutation. Table 1) shows that nearly twice as much current (60% vs. 33% of WT) will flow in the mutant receptors at negative holding potentials (-30 mV). The concentration-response curves for Mg2+ block (Fig. 2e; Table 1) show that this mutation increased the Mg2+ IC50 value to 43 muM from 17 muM for WT receptors at the holding potential of -60 mV; similar decreases in potency were observed at other holding potentials (WT vs. The current-voltage relationship (Fig. 2d; Table 1) shows that more current flows in the single copy of mutant receptors at the negative holding potentials (e.g. 55% vs. 35% of 2A/2A at -30 mV). Figure 4a shows 2A/2A single channel currents that have a complex bursting behavior with one predominant conductance state (84 pS), and an open probability of 0.21. Use of this approach to estimate open probability yielded the same result as data from one active channel in excised outside-out patches (Supplementary Fig. 2), confirming a dominant effect on channel gating by a single copy of the mutant receptor.
The L812M mutation prolongs deactivation time course. (a) and (b) The L812M mutation prolongs deactivation time course of di-heteromeric GluN2A receptors. (a) The normalized representative current response of di-heteromeric NMDARs (WT 2A and L812M) to 1 mM glutamate (Left: long application (1 sec) is shown; Right: brief application (5 msec); 50 muM glycine in all solutions) (VHOLD -60 mV). L812M (black) prolongs deactivation time course compared with the WT 2A receptors (gray). (b) The normalized representative current response of di-heteromeric NMDARs to 1 mM glycine is shown (Left: long application; Right: brief application; 30 muM glutamate in all solutions) (VHOLD -60 mV). (c, d) The L812M mutation prolongs deactivation time course of tri-heteromeric GluN2A receptors. (c) A representative current response is shown for tri-heteromeric NMDARs with 0, 1, or 2 copies of the L812M mutation in each complex (2A/2A, L812M/2A, and L812M/L812M) to 1 mM glutamate (Left: long application; Right: brief application; 50 muM glycine in all solutions). (d) The normalized representative current response is shown for tri-heteromeric NMDA receptors to 1 mM glycine (Left: long application; Right: brief application; 30 muM glutamate in all solutions) (VHOLD -60 mV).
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Table 2). The mutant L812M receptors significantly prolonged the glutamate deactivation time course, which was fitted by two exponential components with a tauW of 443 ms (n=10; Fig. 3a, left; Table 2). As with the long pulse of agonist, the mutant receptors deactivated with a slower time course compared to WT NMDARs (tauW 425 ms vs. 44 ms of WT; Fig. 3a, right). Table 2). The mutant GluN2A-L812M receptors showed a slower glycine deactivation time course, which was fitted by two exponential components with a tauW of 691 ms (n=5; Fig. 3b; Table 2). As expected, the glutamate and glycine deactivation time constants of receptors with two copies of mutant GluN2A (L812M/L812M) and receptors with no mutation (2A/2A) were comparable to those determined for GluN2A-L812M and WT GluN2A, respectively (Fig. 3c; Table 2). Table 2). The NMDAR current response upon briefly moving the cell into the glutamate solution to mimic the synaptic activation showed a similar tendency of the GluN2A-L812M mutation to slow the deactivation time course (Fig. 3c, right). The data from experiments moving the cell briefly into the glycine solution show a similar intermediate effect of the GluN2A-L812M mutation on slowing the glycine deactivation time course (Fig. 3d, right). These data show that even a single copy of the mutant subunit prolong the deactivation time course for both agonists, and thus the time course of the NMDAR component of the EPSC. Figure 4d-f and Table 3 summarize the pooled data describing single channel properties from patches that contained one active channel with GluN2 subunit composition of 2A/2A, L812M/2A and L812M/L812M.
The L812M mutation alters the single channel properties. Steady-state recordings from an outside-out patch containing a single active NMDAR with two wild type leucine residues at GluN2A position 812 (2A/2A; a), a single copy of mutation L812M (L812M/2A; b), and two copies of L812M (L812M/L812M; c). Unitary currents were activated in these excised patches by 1 mM glutamate and 50 muM glycine, data are shown on two different time scales. (d, e, f) The pooled open duration and shut duration histogram are shown for 4 single channel recordings. Open duration histograms were made from 42302 openings for 2A/2A, 80303 openings for L812M/2A, 74138 openings for L812M/L812M. Closed duration histograms were made from 42299 events for 2A/2A, 80301 events for L812M/2A, 74134 events for L812M/L812M.
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Figure 4a shows 2A/2A single channel currents that have a complex bursting behavior with one predominant conductance state (84 pS), and an open probability of 0.21. A parallel set of recordings for GluN1 co-expressed with L812M/2A (Fig. 4b) and L812M/L812M (Fig. 4c) showed identical single channel conductance (L812M/2A 85 pS; L812M/L812M 85 pS), but significantly more channel openings with prolonged open periods and shortened shut periods, resulting in an increased open probability. Figure 4d-f and Table 3 summarize the pooled data describing single channel properties from patches that contained one active channel with GluN2 subunit composition of 2A/2A, L812M/2A and L812M/L812M.
The L812M mutation changes agonist potency of other NMDAR subunits. Composite concentration-response curves were determined by TEVC recordings from Xenopus oocytes expressing different GluN subunits-containing receptors (a and e, GluN1/GluN2A and GluN1-L808M/GluN2A; b and f, GluN1/GluN2B, GluN1/GluN2B-L813M, and GluN1-L808M/GluN2B; c and g, GluN1/GluN2C and GluN1/GluN2C-L810M; d and h, GluN1/GluN2D and GluN1/GluN2D-L840M). (a, b, c, d) show the composite glutamate concentration-response curves determined in the presence of 100 muM glycine. (e, f, g, h) show the composite glycine concentration-response curves determined in the presence of 100 muM glutamate. (i, j) show the composite glutamate (i, in the presence of 100 muM glycine) and glycine (j, in the presence of 100 muM glutamate) concentration-response curves of GluN2A-L812G, -L812S, -L812C, -L812A, -L812Q, -L812E constructs.
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GluN1-L808M mutation coexpressed with GluN2A increased glutamate potency by 9.2-fold (EC50: 0.39 muM vs. 3.6 muM for WT 2A) and glycine potency by 9.1-fold (EC50: 0.12 muM vs. 1.1 muM for WT) (Fig. 5a, e; Table 4). GluN1-L808M when coexpressed with GluN2B similarly increased glutamate potency by 6.8-fold (Fig. 5b; 0.19 muM vs. 1.3 muM for WT 2B).