Functional analysis of a de novo GRIN2A missense mutation associated with early-onset epileptic encephalopathy

Abstract

NMDA receptors (NMDARs), ligand-gated ion channels, play important roles in various neurological disorders, including epilepsy. Here we show the functional analysis of a de novo missense mutation (L812M) in a gene encoding NMDAR subunit GluN2A (GRIN2A). The mutation, identified in a patient with early-onset epileptic encephalopathy and profound developmental delay, is located in the linker region between the ligand-binding and transmembrane domains. Electrophysiological recordings revealed that the mutation enhances agonist potency, decreases sensitivity to negative modulators including magnesium, protons and zinc, prolongs the synaptic response time course and increases single-channel open probability. The functional changes of this amino acid apply to all other NMDAR subunits, suggesting an important role of this residue on the function of NMDARs. Taken together, these data suggest that the L812M mutation causes overactivation of NMDARs and drives neuronal hyperexcitability. We hypothesize that this mechanism underlies the patient's epileptic phenotype as well as cerebral atrophy.

Figure 1. 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.

Figure 2. 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 μM 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 μM 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.

Figure 3. 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 μM glycine in all solutions) (V_HOLD −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 μM glutamate in all solutions) (V_HOLD −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 μM 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 μM glutamate in all solutions) (V_HOLD −60 mV).

Figure 4. 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 μM 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.

Figure 5. 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 μM glycine. (e, f, g, h) show the composite glycine concentration-response curves determined in the presence of 100 μM glutamate. (i, j) show the composite glutamate (i, in the presence of 100 μM glycine) and glycine (j, in the presence of 100 μM glutamate) concentration-response curves of GluN2A-L812G, -L812S, -L812C, -L812A, -L812Q, -L812E constructs.