Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3.
ABSTRACT The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.
- SourceAvailable from: Tyler Mark Pierson[Show abstract] [Hide abstract]
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.Nature Communications 02/2014; 5:3251. · 10.74 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Abundant evidence supports a role for NMDA receptor inhibition in the behavioral actions of ethanol, but the underlying molecular mechanisms have not been fully elucidated. We recently found that clusters of five positions in the third and fourth membrane-associated domains (M3 and M4) at the intersubunit interfaces form putative sites of alcohol action. In the present study, we found that one of these positions, GluN2A(F636), can strongly regulate ethanol sensitivity, glutamate potency, and apparent desensitization: ethanol IC50 values, peak (Ip) and steady-state (Iss) glutamate EC50 values, and steady-state to peak current ratio (Iss:Ip) values differed significantly among the mutants tested. Changes in glutamate affinity among the various mutants were not attributable to agonist trapping due to desensitization, as glutamate peak EC50 values were correlated with values of both steady-state EC50 and Iss:Ip. Mean open times determined in selected mutants could be altered up to four-fold, but did not account for changes in ethanol sensitivity. Ethanol sensitivity was significantly correlated with glutamate EC50 and Iss:Ip values, but the changes in ethanol IC50 among mutants at this position do not appear to be secondary to changes in ion channel kinetics. Substitution of the isomeric amino acids leucine and isoleucine had markedly different effects on ethanol sensitivity, agonist potency, and desensitization, which is consistent with a stringent structural requirement for ion channel modulation by the side chain at this position. Our results indicate that GluN2A(F636) plays an important role in both channel function and ethanol inhibition in NMDA receptors.Molecular pharmacology 07/2013; · 4.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: N-methyl-D-aspartate (NMDA) receptors are obligate heterotetrameric ligand-gated ion channels that play critical roles in learning and memory. Here, using targeted molecular dynamics simulations, we developed an atomistic model for the gating of the GluN1/GluN2A NMDA receptor. Upon agonist binding, lobe closure of the ligand-binding domain produced outward pulling of the M3-D2 linkers, leading to outward movements of the C-termini of the pore-lining M3 helices and opening of the channel. The GluN2A subunits, similar to the distal (B/D) subunits in the homotetrameric GluA2 α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor, had greater M3 outward movements and thus contributed more to channel gating than the GluN1 subunits. Our gating model is validated by functional studies, including cysteine modification data indicating wider accessibility to the GluN1 M3 helices than to the GluN2A M3 helices from the lumen of the open channel, and reveals why the Lurcher mutation in GluN1 has a stronger ability in maintaining channel opening than the counterpart in GluN2A. The resulting structural model for the open state provides an explanation for the Ca(2+) permeability of NMDA receptors, and the structural differences between the closed and open states form the basis for drug design.Biophysical Journal 05/2013; 104(10):2170-2181. · 3.67 Impact Factor